FIELD OF THE INVENTION
The present invention relates to polynucleotides of human origin in substantially isolated form and gene products that are differentially expressed in cancer cells, and uses thereof.
BACKGROUND OF THE INVENTION
Cancer, like many diseases, is not the result of a single, well-defined cause, but rather can be viewed as several diseases, each caused by different aberrations in informational pathways, that ultimately result in apparently similar pathologic phenotypes. Identification of polynucleotides that correspond to genes that are differentially expressed in cancerous, pre-cancerous, or low metastatic potential cells relative to normal cells of the same tissue type, provides the basis for diagnostic tools, facilitates drug discovery by providing for targets for candidate agents, and further serves to identify therapeutic targets for cancer therapies that are more tailored for the type of cancer to be treated.
Identification of differentially expressed gene products also furthers the understanding of the progression and nature of complex diseases such as cancer, and is key to identifying the genetic factors that are responsible for the phenotypes associated with development of, for example, the metastatic phenotype. Identification of gene products that are differentially expressed at various stages, and in various types of cancers, can both provide for early diagnostic tests, and further serve as therapeutic targets. Additionally, the product of a differentially expressed gene can be the basis for screening assays to identify chemotherapeutic agents that modulate its activity (e.g. its expression, biological activity, and the like).
Early disease diagnosis is of central importance to halting disease progression, and reducing morbidity. Analysis of a patient's tumor to identify the gene products that are differentially expressed, and administration of therapeutic agent(s) designed to modulate the activity of those differentially expressed gene products, provides the basis for more specific, rational cancer therapy that may result in diminished adverse side effects relative to conventional therapies. Furthermore, confirmation that a tumor poses less risk to the patient (e.g., that the tumor is benign) can avoid unnecessary therapies. In short, identification of genes and the encoded gene products that are differentially expressed in cancerous cells can provide the basis of therapeutics, diagnostics, prognostics, therametrics, and the like.
For example, breast cancer is a leading cause of death among women. One of the priorities in breast cancer research is the discovery of new biochemical markers that can be used for diagnosis, prognosis and monitoring of breast cancer. The prognostic usefulness of these markers depends on the ability of the marker to distinguish between patients with breast cancer who require aggressive therapeutic treatment and patients who should be monitored.
While the pathogenesis of breast cancer is unclear, transformation of non-tumorigenic breast epithelium to a malignant phenotype may be the result of genetic factors, especially in women under 30 (Miki, et al., Science, 266: 66-71, 1994). However, it is likely that other, non-genetic factors are also significant in the etiology of the disease. Regardless of its origin, breast cancer morbidity increases significantly if a lesion is not detected early in its progression. Thus, considerable effort has focused on the elucidation of early cellular events surrounding transformation in breast tissue. Such effort has led to the identification of several potential breast cancer markers.
Thus, the identification of new markers associated with cancer, for example, breast cancer, and the identification of genes involved in transforming cells into the cancerous phenotype, remains a significant goal in the management of this disease. In exemplary aspects, the invention described herein provides cancer diagnostics, prognostics, therametrics, and therapeutics based upon polynucleotides and/or their encoded gene products.
SUMMARY OF THE INVENTION
The present invention provides methods and compositions useful in detection of cancerous cells, identification of agents that modulate the phenotype of cancerous cells, and identification of therapeutic targets for chemotherapy of cancerous cells. Cancerous, breast, colon and prostate cells are of particular interest in each of these aspects of the invention. More specifically, the invention provides polynucleotides in substantially isolated form, as well as polypeptides encoded thereby, that are differentially expressed in cancer cells. Also provided are antibodies that specifically bind the encoded polypeptides. These polynucleotides, polypeptides and antibodies are thus useful in a variety of diagnostic, therapeutic, and drug discovery methods. In some embodiments, a polynucleotide that is differentially expressed in cancer cells can be used in diagnostic assays to detect cancer cells. In other embodiments, a polynucleotide that is differentially expressed in cancer cells, and/or a polypeptide encoded thereby, is itself a target for therapeutic intervention.
Accordingly, the invention features an isolated polynucleotide comprising a nucleotide sequence having at least 90% sequence identity to an identifying sequence of any one of the sequences set forth herein or a degenerate variant thereof. In related aspects, the invention features recombinant host cells and vectors comprising the polynucleotides of the invention, as well as isolated polypeptides encoded by the polynucleotides of the invention and antibodies that specifically bind such polypeptides.
In other aspects, the invention provides a method for detecting a cancerous cell. In general, the method involves contacting a test sample obtained from a cell that is suspected of being a cancer cell with a probe for detecting a gene product differentially expressed in cancer. Many embodiments of the invention involve a gene identifiable by or comprising a sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11-13, 15, 16, 18, 20, 22, 24, 26, 27, 29 and 128-1618, contacting the probe and the gene product for a time sufficient for binding of the probe to the gene product; and comparing a level of binding of the probe to the sample with a level of probe binding to a control sample obtained from a control cell of known cancerous state. A modulated (i.e. increased or decreased) level of binding of the probe in the test cell sample relative to the level of binding in a control sample is indicative of the cancerous state of the test cell. In certain embodiments, the level of binding of the probe in the test cell sample, usually in relation to at least one control gene, is similar to binding of the probe to a cancerous cell sample. In certain other embodiments, the level of binding of the probe in the test cell sample, usually in relation to at least one control gene, is different, i.e. opposite, to binding of the probe to a non-cancerous cell sample. In specific embodiments, the probe is a polynucleotide probe and the gene product is nucleic acid. In other specific embodiments, the gene product is a polypeptide. In further embodiments, the gene product or the probe is immobilized on an array.
In another aspect, the invention provides a method for assessing the cancerous phenotype (e.g., metastasis, metastatic potential, aberrant cellular proliferation, and the like) of a cell comprising detecting expression of a gene product in a test cell sample, wherein the gene comprises or is identifiable using a sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11-13, 15, 16, 18, 20, 22, 24, 26, 27, 29 and 128-1618; and comparing a level of expression of the gene product in the test cell sample with a level of expression of the gene in a control cell sample. Comparison of the level of expression of the gene in the test cell sample relative to the level of expression in the control cell sample is indicative of the cancerous phenotype of the test cell sample. In specific embodiments, detection of gene expression is by detecting a level of an RNA transcript in the test cell sample. In other specific embodiments detection of expression of the gene is by detecting a level of a polypeptide in a test sample.
In another aspect, the invention provides a method for suppressing or inhibiting a cancerous phenotype of a cancerous cell, the method comprising introducing into a mammalian cell an expression modulatory agent (e.g. an antisense molecule, small molecule, antibody, neutralizing antibody, inhibitory RNA molecule, etc.) to inhibit expression of a gene identified by a sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11-13, 15, 16, 18, 20, 22, 24, 26, 27, 29 and 128-1618. Inhibition of expression of the gene inhibits development of a cancerous phenotype in the cell. In specific embodiments, the cancerous phenotype is metastasis, aberrant cellular proliferation relative to a normal cell, or loss of contact inhibition of cell growth. In the context of this invention “expression” of a gene is intended to encompass the expression of an activity of a gene product, and, as such, inhibiting expression of a gene includes inhibiting the activity of a product of the gene.
In another aspect, the invention provides a method for assessing the tumor burden of a subject, the method comprising detecting a level of a differentially expressed gene product in a test sample from a subject suspected of or having a tumor, the differentially expressed gene product identified by or comprising a sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11-13, 15, 16, 18, 20, 22, 24, 26, 27, 29 and 128-1618. Detection of the level of the gene product in the test sample is indicative of the tumor burden in the subject.
In another aspect, the invention provides a method for identifying agents that modulate (i.e. increase or decrease) the biological activity of a gene product differentially expressed in a cancerous cell, the method comprising contacting a candidate agent with a differentially expressed gene product, the differentially expressed gene product corresponding to a sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11-13, 15, 16, 18, 20, 22, 24, 26, 27, 29 and 128-1618; and detecting a modulation in a biological activity of the gene product relative to a level of biological activity of the gene product in the absence of the candidate agent. In specific embodiments, the detecting is by identifying an increase or decrease in expression of the differentially expressed gene product. In other specific embodiments, the gene product is mRNA or cDNA prepared from the mRNA gene product. In further embodiments, the gene product is a polypeptide.
In another aspect, the invention provides a method of inhibiting growth of a tumor cell by modulating expression of a gene product, where the gene product is encoded by a gene identified by a sequence selected from the group consisting of: SEQ ID NOS: 1, 3, 5, 7, 9, 11-13, 15, 16, 18, 20, 22, 24, 26, 27, 29 and 128-1618.
These and other objects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the invention as more fully described below.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a graph showing the message levels of the gene corresponding to SK2 (c9083, SEQ ID NO:3) in the indicated cell lines.
FIG. 2 is a graph showing the effect of SK2 (9083) antisense oligonucleotides upon message levels for the gene corresponding to SK2 (SEQ ID NO:3).
FIG. 3 is a graph showing the effect of SK2 (9083) antisense oligonucleotides upon proliferation of SW620 cells.
FIG. 4 is a graph showing the effect of SK2 (9083) antisense oligonucleotides upon proliferation of a non-colon cell line, HT1080.
FIG. 5 is a graph showing the effect of antisense oligonucleotides to the gene corresponding to cluster 378805 upon growth of SW620 cells (31-4 as: antisense; 31-4rc: reverse control; WT: wild type control (no oligo)).
FIG. 6 is a graph showing the results of proliferation assay with SW620 assays to examine the effects of expression of K-Ras (control).
FIG. 7 is a graph showing the results of proliferation assay with SW620 assays to examine the effects of expression of, the gene corresponding to c3376 (CHIR11-4).
FIG. 8 is a graph showing the results of proliferation assay with SW620 assays to examine the effects of expression of the gene corresponding to 402380 (CHIR33-4).
FIG. 9 is a graph showing the effects of expression of genes corresponding to K-Ras (control) and to 402380 (CHIR33-4) upon colon formation of SW620 cells in soft agar (values normalized to WST1).
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides polynucleotides, as well as polypeptides encoded thereby, that are differentially expressed in cancer cells. Methods are provided in which these polynucleotides and polypeptides are used for detecting and reducing the growth of cancer cells. Also provided are methods in which the polynucleotides and polypeptides of the invention are used in a variety of diagnostic and therapeutic applications for cancer. The invention finds use in the prevention, treatment, detection or research into any cancer, including prostrate, pancreas, colon, brain, lung, breast, bone, skin cancers. For example, the invention finds use in the prevention, treatment, detection of or research into endocrine system cancers, such as cancers of the thyroid, pituitary, and adrenal glands and the pancreatic islets; gastrointestinal cancers, such as cancer of the anus, colon, esophagus, gallbladder, stomach, liver, and rectum; genitourinary cancers such as cancer of the penis, prostate and testes; gynecological cancers, such as cancer of the ovaries, cervix, endometrium, uterus, fallopian tubes, vagina, and vulva; head and neck cancers, such as hypopharyngeal, laryngeal, oropharyngeal cancers, lip, mouth and oral cancers, cancer of the salivary gland, cancer of the digestive tract and sinus cancer; leukemia; lymphomas including Hodgkin's and non-Hodgkin's lymphoma; metastatic cancer; myelomas; sarcomas; skin cancer; urinary tract cancers including bladder, kidney and urethral cancers; and pediatric cancers, such as pediatric brain tumors, leukemia, lymphomas, sarcomas, liver cancer and neuroblastoma and retinoblastoma.
Before the present invention is described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications and patent applications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
It must be noted that as used herein and in the appended claims, the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a polynucleotide” includes a plurality of such polynucleotides and reference to “the cancer cell” includes reference to one or more cells and equivalents thereof known to those skilled in the art, and so forth.
The publications and applications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
Definitions
The terms “polynucleotide” and “nucleic acid”, used interchangeably herein, refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxynucleotides. Thus, these terms include, but are not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. These terms further include, but are not limited to, mRNA or cDNA that comprise intronic sequences (see, e.g., Niwa et al. (1999) Cell 99(7):691-702). The backbone of the polynucleotide can comprise sugars and phosphate groups (as may typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of the polynucleotide can comprise a polymer of synthetic subunits such as phosphoramidites and thus can be an oligodeoxynucleoside phosphoramidate or a mixed phosphoramidate-phosphodiester oligomer. Peyrottes et al. (1996) Nucl. Acids Res. 24:1841-1848; Chaturvedi et al. (1996) Nucl. Acids Res. 24:2318-2323. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars, and linking groups such as fluororibose and thioate, and nucleotide branches. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications included in this definition are caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides, or a solid support. The term “polynucleotide” also encompasses peptidic nucleic acids (Pooga et al Curr Cancer Drug Targets. (2001) 1:231-9).
A “gene product” is a biopolymeric product that is expressed or produced by a gene. A gene product may be, for example, an unspliced RNA, an mRNA, a splice variant mRNA, a polypeptide, a post-translationally modified polypeptide, a splice variant polypeptide etc. Also encompassed by this term is biopolymeric products that are made using an RNA gene product as a template (i.e. cDNA of the RNA). A gene product may be made enzymatically, recombinantly, chemically, or within a cell to which the gene is native. In many embodiments, if the gene product is proteinaceous, it exhibits a biological activity. In many embodiments, if the gene product is a nucleic acid, it can be translated into a proteinaceous gene product that exhibits a biological activity.
A composition (e.g. a polynucleotide, polypeptide, antibody, or host cell) that is “isolated” or “in substantially isolated form” refers to a composition that is in an environment different from that in which the composition naturally occurs. For example, a polynucleotide that is in substantially isolated form is outside of the host cell in which the polynucleotide naturally occurs, and could be a purified fragment of DNA, could be part of a heterologous vector, or could be contained within a host cell that is not a host cell from which the polynucleotide naturally occurs. The term “isolated” does not refer to a genomic or cDNA library, whole cell total protein or mRNA preparation, genomic DNA preparation, or an isolated human chromosome. A composition which is in substantially isolated form is usually substantially purified.
As used herein, the term “substantially purified” refers to a compound (e.g., a polynucleotide, a polypeptide or an antibody, etc.) that is removed from its natural environment and is usually at least 60% free, preferably 75% free, and most preferably 90% free from other components with which it is naturally associated. Thus, for example, a composition containing A is “substantially free of” B when at least 85% by weight of the total A+B in the composition is A. Preferably, A comprises at least about 90% by weight of the total of A+B in the composition, more preferably at least about 95% or even 99% by weight. In the case of polynucleotides, “A” and “B” may be two different genes positioned on different chromosomes or adjacently on the same chromosome, or two isolated cDNA species, for example.
The terms “polypeptide” and “protein”, interchangeably used herein, refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like.
“Heterologous” refers to materials that are derived from different sources (e.g., from different genes, different species, etc.).
As used herein, the terms “a gene that is differentially expressed in a cancer cell,” and “a polynucleotide that is differentially expressed in a cancer cell” are used interchangeably herein, and generally refer to a polynucleotide that represents or corresponds to a gene that is differentially expressed in a cancerous cell when compared with a cell of the same cell type that is not cancerous, e.g., mRNA is found at levels at least about 25%, at least about 50% to about 75%, at least about 90%, at least about 1.5-fold, at least about 2-fold, at least about 5-fold, at least about 10-fold, or at least about 50-fold or more, different (e.g., higher or lower). The comparison can be made in tissue, for example, if one is using in situ hybridization or another assay method that allows some degree of discrimination among cell types in the tissue. The comparison may also or alternatively be made between cells removed from their tissue source.
“Differentially expressed polynucleotide” as used herein refers to a nucleic acid molecule (RNA or DNA) comprising a sequence that represents a differentially expressed gene, e.g., the differentially expressed polynucleotide comprises a sequence (e.g., an open reading frame encoding a gene product; a non-coding sequence) that uniquely identifies a differentially expressed gene so that detection of the differentially expressed polynucleotide in a sample is correlated with the presence of a differentially expressed gene in a sample. “Differentially expressed polynucleotides” is also meant to encompass fragments of the disclosed polynucleotides, e.g., fragments retaining biological activity, as well as nucleic acids homologous, substantially similar, or substantially identical (e.g., having about 90% sequence identity) to the disclosed polynucleotides.
“Corresponds to” or “represents” when used in the context of, for example, a polynucleotide or sequence that “corresponds to” or “represents” a gene means that at least a portion of a sequence of the polynucleotide is present in the gene or in the nucleic acid gene product (e.g., mRNA or cDNA). A subject nucleic acid may also be “identified” by a polynucleotide if the polynucleotide corresponds to or represents the gene. Genes identified by a polynucleotide may have all or a portion of the identifying sequence wholly present within an exon of a genomic sequence of the gene, or different portions of the sequence of the polynucleotide may be present in different exons (e.g., such that the contiguous polynucleotide sequence is present in an mRNA, either pre- or post-splicing, that is an expression product of the gene). In some embodiments, the polynucleotide may represent or correspond to a gene that is modified in a cancerous cell relative to a normal cell. The gene in the cancerous cell may contain a deletion, insertion, substitution, or translocation relative to the polynucleotide and may have altered regulatory sequences, or may encode a splice variant gene product, for example. The gene in the cancerous cell may be modified by insertion of an endogenous retrovirus, a transposable element, or other naturally occurring or non-naturally occurring nucleic acid. In most cases, a polynucleotide corresponds to or represents a gene if the sequence of the polynucleotide is most identical to the sequence of a gene or its product (e.g. mRNA or cDNA) as compared to other genes or their products. In most embodiments, the most identical gene is determined using a sequence comparison of a polynucleotide to a database of polynucleotides (e.g. GenBank) using the BLAST program at default settings For example, if the most similar gene in the human genome to an exemplary polynucleotide is the protein kinase C gene, the exemplary polynucleotide corresponds to protein kinase C. In most cases, the sequence of a fragment of an exemplary polynucleotide is at least 95%, 96%, 97%, 98%, 99% or up to 100% identical to a sequence of at least 15, 20, 25, 30, 35, 40, 45, or 50 contiguous nucleotides of a corresponding gene or its product (mRNA or cDNA), when nucleotides that are “N” represent G, A, T or C.
An “identifying sequence” is a minimal fragment of a sequence of contiguous nucleotides that uniquely identifies or defines a polynucleotide sequence or its complement. In many embodiments, a fragment of a polynucleotide uniquely identifies or defines a polynucleotide sequence or its complement. In some embodiments, the entire contiguous sequence of a gene, cDNA, EST, or other provided sequence is an identifying sequence.
“Diagnosis” as used herein generally includes determination of a subject's susceptibility to a disease or disorder, determination as to whether a subject is presently affected by a disease or disorder, prognosis of a subject affected by a disease or disorder (e.g., identification of pre-metastatic or metastatic cancerous states, stages of cancer, or responsiveness of cancer to therapy), and use of therametrics (e.g., monitoring a subject's condition to provide information as to the effect or efficacy of therapy).
As used herein, the term “a polypeptide associated with cancer” refers to a polypeptide encoded by a polynucleotide that is differentially expressed in a cancer cell.
The term “biological sample” encompasses a variety of sample types obtained from an organism and can be used in a diagnostic or monitoring assay. The term encompasses blood and other liquid samples of biological origin, solid tissue samples, such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. The term encompasses samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components. The term encompasses a clinical sample, and also includes cells in cell culture, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples.
The terms “treatment”, “treating”, “treat” and the like are used herein to generally refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and/or adverse effect attributable to the disease. “Treatment” as used herein covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease or symptom from occurring in a subject which may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease symptom, i.e., arresting its development; or (c) relieving the disease symptom, i.e., causing regression of the disease or symptom.
The terms “individual,” “subject,” “host,” and “patient,” used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. Other subjects may include cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and the like.
A “host cell”, as used herein, refers to a microorganism or a eukaryotic cell or cell line cultured as a unicellular entity which can be, or has been, used as a recipient for a recombinant vector or other transfer polynucleotides, and include the progeny of the original cell which has been transfected. It is understood that the progeny of a single cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation.
The terms “cancer”, “neoplasm”, “tumor”, and “carcinoma”, are used interchangeably herein to refer to cells which exhibit relatively autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation. In general, cells of interest for detection or treatment in the present application include precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and non-metastatic cells. Detection of cancerous cells is of particular interest.
The term “normal” as used in the context of “normal cell,” is meant to refer to a cell of an untransformed phenotype or exhibiting a morphology of a non-transformed cell of the tissue type being examined.
“Cancerous phenotype” generally refers to any of a variety of biological phenomena that are characteristic of a cancerous cell, which phenomena can vary with the type of cancer. The cancerous phenotype is generally identified by abnormalities in, for example, cell growth or proliferation (e.g., uncontrolled growth or proliferation), regulation of the cell cycle, cell mobility, cell-cell interaction, or metastasis, etc.
“Therapeutic target” generally refers to a gene or gene product that, upon modulation of its activity (e.g., by modulation of expression, biological activity, and the like), can provide for modulation of the cancerous phenotype.
As used throughout, “modulation” is meant to refer to an increase or a decrease in the indicated phenomenon (e.g., modulation of a biological activity refers to an increase in a biological activity or a decrease in a biological activity).
Polynucleotide Compositions
The present invention provides isolated polynucleotides that contain nucleic acids that are differentially expressed in cancer cells. The polynucleotides, as well as any polypeptides encoded thereby, find use in a variety of therapeutic and diagnostic methods.
The scope of the invention with respect to compositions containing the isolated polynucleotides useful in the methods described herein includes, but is not necessarily limited to, polynucleotides having (i.e., comprising) a sequence set forth in any one of the polynucleotide sequences provided herein, or fragment thereof; polynucleotides obtained from the biological materials described herein or other biological sources (particularly human sources) by hybridization under stringent conditions (particularly conditions of high stringency); genes corresponding to the provided polynucleotides; cDNAs corresponding to the provided polynucleotides; variants of the provided polynucleotides and their corresponding genes, particularly those variants that retain a biological activity of the encoded gene product (e.g., a biological activity ascribed to a gene product corresponding to the provided polynucleotides as a result of the assignment of the gene product to a protein family(ies) and/or identification of a functional domain present in the gene product). Other nucleic acid compositions contemplated by and within the scope of the present invention will be readily apparent to one of ordinary skill in the art when provided with the disclosure here. “Polynucleotide” and “nucleic acid” as used herein with reference to nucleic acids of the composition is not intended to be limiting as to the length or structure of the nucleic acid unless specifically indicated.
The invention features polynucleotides that represent genes that are expressed in human tissue, specifically polynucleotides that are differentially expressed in tissues containing cancerous cells. Nucleic acid compositions described herein of particular interest are at least about 15 bp in length, at least about 30 bp in length, at least about 50 bp in length, at least about 100 bp, at least about 200 bp in length, at least about 300 bp in length, at least about 500 bp in length, at least about 800 bp in length, at least about 1 kb in length, at least about 2.0 kb in length, at least about 3.0 kb in length, at least about 5 kb in length, at least about 10 kb in length, at least about 50 kb in length and are usually less than about 200 kb in length. These polynucleotides (or polynucleotide fragments) have uses that include, but are not limited to, diagnostic probes and primers as starting materials for probes and primers, as discussed herein.
The subject polynucleotides usually comprise a sequence set forth in any one of the polynucleotide sequences provided herein, for example, in the sequence listing, incorporated by reference in a table (e.g. by an NCBI accession number), a cDNA deposited at the A.T.C.C., or a fragment or variant thereof. A “fragment” or “portion” of a polynucleotide is a contiguous sequence of residues at least about 10 nt to about 12 nt, 15 nt, 16 nt, 18 nt or 20 nt in length, usually at least about 22 nt, 24 nt, 25 nt, 30 nt, 40 nt, 50 nt, 60 nt, 70 nt, 80 nt, 90 nt, 100 nt to at least about 150 nt, 200 nt, 250 nt, 300 nt, 350 nt, 400 nt, 500 nt, 800 nt or up to about 1000 nt, 1500 or 2000 nt in length. In some embodiments, a fragment of a polynucleotide is the coding sequence of a polynucleotide. A fragment of a polynucleotide may start at position 1 (i.e. the first nucleotide) of a nucleotide sequence provided herein, or may start at about position 10, 20, 30, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500 or 2000, or an ATG translational initiation codon of a nucleotide sequence provided herein. In this context “about” includes the particularly recited value or a value larger or smaller by several (5, 4, 3, 2, or 1) nucleotides. The described polynucleotides and fragments thereof find use as hybridization probes, PCR primers, BLAST probes, or as an identifying sequence, for example.
The subject nucleic acids may be variants or degenerate variants of a sequence provided herein. In general, a variants of a polynucleotide provided herein have a fragment of sequence identity that is greater than at least about 65%, greater than at least about 70%, greater than at least about 75%, greater than at least about 80%, greater than at least about 85%, or greater than at least about 90%, 95%, 96%, 97%, 98%, 99% or more (i.e. 100%) as compared to an identically sized fragment of a provided sequence. as determined by the Smith-Waterman homology search algorithm as implemented in MPSRCH program (Oxford Molecular). For the purposes of this invention, a preferred method of calculating percent identity is the Smith-Waterman algorithm. Global DNA sequence identity should be greater than 65% as determined by the Smith-Waterman homology search algorithm as implemented in MPSRCH program (Oxford Molecular) using an gap search with the following search parameters: gap open penalty, 12; and gap extension penalty, 1.
The subject nucleic acid compositions include full-length cDNAs or mRNAs that encompass an identifying sequence of contiguous nucleotides from any one of the polynucleotide sequences provided herein.
As discussed above, the polynucleotides useful in the methods described herein also include polynucleotide variants having sequence similarity or sequence identity. Nucleic acids having sequence similarity are detected by hybridization under low stringency conditions, for example, at 50° C. and 10×SSC (0.9 M saline/0.09 M sodium citrate) and remain bound when subjected to washing at 55° C. in 1×SSC. Sequence identity can be determined by hybridization under high stringency conditions, for example, at 50° C. or higher and 0.1×SSC (9 mM saline/0.9 mM sodium citrate). Hybridization methods and conditions are well known in the art, see, e.g., U.S. Pat. No. 5,707,829. Nucleic acids that are substantially identical to the provided polynucleotide sequences, e.g. allelic variants, genetically altered versions of the gene, etc., bind to the provided polynucleotide sequences under stringent hybridization conditions. By using probes, particularly labeled probes of DNA sequences, one can isolate homologous or related genes. The source of homologous genes can be any species, e.g. primate species, particularly human; rodents, such as rats and mice; canines, felines, bovines, ovines, equines, yeast, nematodes, etc.
In one embodiment, hybridization is performed using a fragment of at least 15 contiguous nucleotides (nt) of at least one of the polynucleotide sequences provided herein. That is, when at least 15 contiguous nt of one of the disclosed polynucleotide sequences is used as a probe, the probe will preferentially hybridize with a nucleic acid comprising the complementary sequence, allowing the identification and retrieval of the nucleic acids that uniquely hybridize to the selected probe. Probes from more than one polynucleotide sequence provided herein can hybridize with the same nucleic acid if the cDNA from which they were derived corresponds to one mRNA.
Polynucleotides contemplated for use in the invention also include those having a sequence of naturally occurring variants of the nucleotide sequences (e.g., degenerate variants (e.g., sequences that encode the same polypeptides but, due to the degenerate nature of the genetic code, different in nucleotide sequence), allelic variants, etc.). Variants of the polynucleotides contemplated by the invention are identified by hybridization of putative variants with nucleotide sequences disclosed herein, preferably by hybridization under stringent conditions. For example, by using appropriate wash conditions, variants of the polynucleotides described herein can be identified where the allelic variant exhibits at most about 25-30% base pair (bp) mismatches relative to the selected polynucleotide probe. In general, allelic variants contain 15-25% bp mismatches, and can contain as little as even 5-15%, or 2-5%, or 1-2% bp mismatches, as well as a single bp mismatch.
The invention also encompasses homologs corresponding to any one of the polynucleotide sequences provided herein, where the source of homologous genes can be any mammalian species, e.g., primate species, particularly human; rodents, such as rats; canines, felines, bovines, ovines, equines, yeast, nematodes, etc. Between mammalian species, e.g., human and mouse, homologs generally have substantial sequence similarity, e.g., at least 75% sequence identity, usually at least 80%%, at least 85, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% identity between nucleotide sequences. Sequence similarity is calculated based on a reference sequence, which may be a subset of a larger sequence, such as a conserved motif, coding region, flanking region, etc. A reference sequence will usually be at least about a fragment of a polynucleotide sequence and may extend to the complete sequence that is being compared. Algorithms for sequence analysis are known in the art, such as gapped BLAST, described in Altschul, et al. Nucleic Acids Res. (1997) 25:3389-3402, or TeraBLAST available from TimeLogic Corp. (Crystal Bay, Nev.).
The subject nucleic acids can be cDNAs or genomic DNAs, as well as fragments thereof, particularly fragments that encode a biologically active gene product and/or are useful in the methods disclosed herein (e.g., in diagnosis, as a unique identifier of a differentially expressed gene of interest, etc.). The term “cDNA” as used herein is intended to include all nucleic acids that share the arrangement of sequence elements found in native mature mRNA species, where sequence elements are exons and 3′ and 5′ non-coding regions. Normally mRNA species have contiguous exons, with the intervening introns, when present, being removed by nuclear RNA splicing, to create a continuous open reading frame encoding a polypeptide. mRNA species can also exist with both exons and introns, where the introns may be removed by alternative splicing. Furthermore it should be noted that different species of mRNAs encoded by the same genomic sequence can exist at varying levels in a cell, and detection of these various levels of mRNA species can be indicative of differential expression of the encoded gene product in the cell.
A genomic sequence of interest comprises the nucleic acid present between the initiation codon and the stop codon, as defined in the listed sequences, including all of the introns that are normally present in a native chromosome. It can further include the 3′ and 5′ untranslated regions found in the mature mRNA. It can further include specific transcriptional and translational regulatory sequences, such as promoters, enhancers, etc., including about 1 kb, but possibly more, of flanking genomic DNA at either the 5′ and 3′ end of the transcribed region. The genomic DNA can be isolated as a fragment of 100 kbp or smaller; and substantially free of flanking chromosomal sequence. The genomic DNA flanking the coding region, either 3′ and 5′, or internal regulatory sequences as sometimes found in introns, contains sequences required for proper tissue, stage-specific, or disease-state specific expression.
The nucleic acid compositions of the subject invention can encode all or a part of the naturally-occurring polypeptides. Double or single stranded fragments can be obtained from the DNA sequence by chemically synthesizing oligonucleotides in accordance with conventional methods, by restriction enzyme digestion, by PCR amplification, etc.
Probes specific to the polynucleotides described herein can be generated using the polynucleotide sequences disclosed herein. The probes are usually a fragment of a polynucleotide sequences provided herein. The probes can be synthesized chemically or can be generated from longer polynucleotides using restriction enzymes. The probes can be labeled, for example, with a radioactive, biotinylated, or fluorescent tag. Preferably, probes are designed based upon an identifying sequence of any one of the polynucleotide sequences provided herein. More preferably, probes are designed based on a contiguous sequence of one of the subject polynucleotides that remain unmasked following application of a masking program for masking low complexity (e.g., XBLAST, RepeatMasker, etc.) to the sequence, i.e., one would select an unmasked region, as indicated by the polynucleotides outside the poly-n stretches of the masked sequence produced by the masking program.
The polynucleotides of interest in the subject invention are isolated and obtained in substantial purity, generally as other than an intact chromosome. Usually, the polynucleotides, either as DNA or RNA, will be obtained substantially free of other naturally-occurring nucleic acid sequences that they are usually associated with, generally being at least about 50%, usually at least about 90% pure and are typically “recombinant”, e.g., flanked by one or more nucleotides with which it is not normally associated on a naturally occurring chromosome.
The polynucleotides described herein can be provided as a linear molecule or within a circular molecule, and can be provided within autonomously replicating molecules (vectors) or within molecules without replication sequences. Expression of the polynucleotides can be regulated by their own or by other regulatory sequences known in the art. The polynucleotides can be introduced into suitable host cells using a variety of techniques available in the art, such as transferrin polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated DNA transfer, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, gene gun, calcium phosphate-mediated transfection, and the like.
The nucleic acid compositions described herein can be used to, for example, produce polypeptides, as probes for the detection of mRNA in biological samples (e.g., extracts of human cells) or cDNA produced from such samples, to generate additional copies of the polynucleotides, to generate ribozymes or antisense oligonucleotides, and as single stranded DNA probes or as triple-strand forming oligonucleotides. The probes described herein can be used to, for example, determine the presence or absence of any one of the polynucleotide provided herein or variants thereof in a sample. These and other uses are described in more detail below.
Polypeptides and Variants Thereof
The present invention further provides polypeptides encoded by polynucleotides that represent genes that are differentially expressed in cancer cells. Such polypeptides are referred to herein as “polypeptides associated with cancer.” The polypeptides can be used to generate antibodies specific for a polypeptide associated with cancer, which antibodies are in turn useful in diagnostic methods, prognostics methods, therametric methods, and the like as discussed in more detail herein. Polypeptides are also useful as targets for therapeutic intervention, as discussed in more detail herein.
The polypeptides contemplated by the invention include those encoded by the disclosed polynucleotides and the genes to which these polynucleotides correspond, as well as nucleic acids that, by virtue of the degeneracy of the genetic code, are not identical in sequence to the disclosed polynucleotides. Further polypeptides contemplated by the invention include polypeptides that are encoded by polynucleotides that hybridize to polynucleotide of the sequence listing. Thus, the invention includes within its scope a polypeptide encoded by a polynucleotide having the sequence of any one of the polynucleotide sequences provided herein, or a variant thereof.
In general, the term “polypeptide” as used herein refers to both the full length polypeptide encoded by the recited polynucleotide, the polypeptide encoded by the gene represented by the recited polynucleotide, as well as portions or fragments thereof. “Polypeptides” also includes variants of the naturally occurring proteins, where such variants are homologous or substantially similar to the naturally occurring protein, and can be of an origin of the same or different species as the naturally occurring protein (e.g., human, murine, or some other species that naturally expresses the recited polypeptide, usually a mammalian species). In general, variant polypeptides have a sequence that has at least about 80%, usually at least about 90%, and more usually at least about 98% sequence identity with a differentially expressed polypeptide described herein, as measured by BLAST 2.0 using the parameters described above. The variant polypeptides can be naturally or non-naturally glycosylated, i.e., the polypeptide has a glycosylation pattern that differs from the glycosylation pattern found in the corresponding naturally occurring protein.
The invention also encompasses homologs of the disclosed polypeptides (or fragments thereof) where the homologs are isolated from other species, i.e. other animal or plant species, where such homologs, usually mammalian species, e.g. rodents, such as mice, rats; domestic animals, e.g., horse, cow, dog, cat; and humans. By “homolog” is meant a polypeptide having at least about 35%, usually at least about 40% and more usually at least about 60% amino acid sequence identity to a particular differentially expressed protein as identified above, where sequence identity is determined using the BLAST 2.0 algorithm, with the parameters described supra.
In general, the polypeptides of interest in the subject invention are provided in a non-naturally occurring environment, e.g. are separated from their naturally occurring environment. In certain embodiments, the subject protein is present in a composition that is enriched for the protein as compared to a cell or extract of a cell that naturally produces the protein. As such, isolated polypeptide is provided, where by “isolated” or “in substantially isolated form” is meant that the protein is present in a composition that is substantially free of other polypeptides, where by substantially free is meant that less than 90%, usually less than 60% and more usually less than 50% of the composition is made up of other polypeptides of a cell that the protein is naturally found.
Also within the scope of the invention are variants; variants of polypeptides include mutants, fragments, and fusions. Mutants can include amino acid substitutions, additions or deletions. The amino acid substitutions can be conservative amino acid substitutions or substitutions to eliminate non-essential amino acids, such as to alter a glycosylation site, a phosphorylation site or an acetylation site, or to minimize misfolding by substitution or deletion of one or more cysteine residues that are not necessary for function. Conservative amino acid substitutions are those that preserve the general charge, hydrophobicity/hydrophilicity, and/or steric bulk of the amino acid substituted.
Variants can be designed so as to retain or have enhanced biological activity of a particular region of the protein (e.g., a functional domain and/or, where the polypeptide is a member of a protein family, a region associated with a consensus sequence). For example, muteins can be made which are optimized for increased antigenicity, i.e. amino acid variants of a polypeptide may be made that increase the antigenicity of the polypeptide. Selection of amino acid alterations for production of variants can be based upon the accessibility (interior vs. exterior) of the amino acid (see, e.g., Go et al, Int. J. Peptide Protein Res. (1980) 15:211), the thermostability of the variant polypeptide (see, e.g., Querol et al., Prot. Eng. (1996) 9:265), desired glycosylation sites (see, e.g., Olsen and Thomsen, J. Gen. Microbiol. (1991) 137:579), desired disulfide bridges (see, e.g., Clarke et al., Biochemistry (1993) 32:4322; and Wakarchuk et al., Protein Eng. (1994) 7:1379), desired metal binding sites (see, e.g., Toma et al., Biochemistry (1991) 30:97, and Haezerbrouck et al., Protein Eng. (1993) 6:643), and desired substitutions with in proline loops (see, e.g., Masul et al., Appl. Env. Microbiol. (1994) 60:3579). Cysteine-depleted muteins can be produced as disclosed in U.S. Pat. No. 4,959,314. Variants also include fragments of the polypeptides disclosed herein, particularly biologically active fragments and/or fragments corresponding to functional domains. Fragments of interest will typically be at least about 10 aa to at least about 15 aa in length, usually at least about 50 aa in length, and can be as long as 300 aa in length or longer, but will usually not exceed about 1000 aa in length, where the fragment will have a stretch of amino acids that is identical to a polypeptide encoded by a polynucleotide having a sequence of any one of the polynucleotide sequences provided herein, or a homolog thereof. The protein variants described herein are encoded by polynucleotides that are within the scope of the invention. The genetic code can be used to select the appropriate codons to construct the corresponding variants.
A fragment of a subject polypeptide is, for example, a polypeptide having an amino acid sequence which is a portion of a subject polypeptide e.g. a polypeptide encoded by a subject polynucleotide that is identified by any one of the sequence of SEQ ID NOS 1, 3, 5, 7, 9, 11-13, 15, 16, 18, 20, 22, 24, 26, 27, 29 and 128-1618 or its complement. The polypeptide fragments of the invention are preferably at least about 9 aa, at least about 15 aa, and more preferably at least about 20 aa, still more preferably at least about 30 aa, and even more preferably, at least about 40 aa, at least about 50 aa, at least about 75 aa, at least about 100 aa, at least about 125 aa or at least about 150 aa in length. A fragment “at least 20 aa in length,” for example, is intended to include 20 or more contiguous amino acids from, for example, the polypeptide encoded by a cDNA, in a cDNA clone contained in a deposited library, or a nucleotide sequence shown in SEQ ID NOS:1, 3, 5, 7, 9, 11-13, 15, 16, 18, 20, 22, 24, 26, 27, 29 and 128-1618 or the complementary stand thereof. In this context “about” includes the particularly recited value or a value larger or smaller by several (5, 4, 3, 2, or 1) amino acids. These polypeptide fragments have uses that include, but are not limited to, production of antibodies as discussed herein. Of course, larger fragments (e.g., at least 150, 175, 200, 250, 500, 600, 1000, or 2000 amino acids in length) are also encompassed by the invention.
Moreover, representative examples of polypeptides fragments of the invention (useful in, for example, as antigens for antibody production), include, for example, fragments comprising, or alternatively consisting of, a sequence from about amino acid number 1-10, 5-10, 10-20, 21-31, 31-40, 41-61, 61-81, 91-120, 121-140, 141-162, 162-200, 201-240, 241-280, 281-320, 321-360, 360-400, 400-450, 451-500, 500-600, 600-700, 700-800, 800-900 and the like. In this context “about” includes the particularly recited range or a range larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either terminus or at both termini. In some embodiments, these fragments has a functional activity (e.g., biological activity) whereas in other embodiments, these fragments may be used to make an antibody.
In one example, a polynucleotide having a sequence set forth in the sequence listing, containing no flanking sequences (i.e., consisting of the sequence set forth in the sequence listing), may be cloned into an expression vector having ATG and a stop codon (e.g. any one of the pET vector from Invitrogen, or other similar vectors from other manufactures), and used to express a polypeptide of interest encoded by the polynucleotide in a suitable cell, e.g., a bacterial cell. Accordingly, the polynucleotides may be used to produce polypeptides, and these polypeptides may be used to produce antibodies by known methods described above and below. In many embodiments, the sequence of the encoded polypeptide does not have to be known prior to its expression in a cell. However, if it desirable to know the sequence of the polypeptide, this may be derived from the sequence of the polynucleotide. Using the genetic code, the polynucleotide may be translated by hand, or by computer means. Suitable software for identifying open reading frames and translating them into polypeptide sequences are well know in the art, and include: Lasergene™ from DNAStar (Madison, Wis.), and Vector NTI™ from Informax (Frederick Md.), and the like.
The amino acid sequences of xemplary polypeptides of the invention are shown in SEQ ID NOS: 2, 4, 6, 8, 10, 14, 17, 19, 21, 23, 25, 28 and 1619-1675.
Further polypeptide variants may are described in PCT publications WO/00-55173, WO/01-07611 and WO/02-16429
Vectors, Host Cells and Protein Production
The present invention also relates to vectors containing the polynucleotide of the present invention, host cells, and the production of polypeptides by recombinant techniques. The vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.
The polynucleotides of the invention may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
The polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria.
Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNHSA, pNH16a, pNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRITS available from Pharmacia Biotech, Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Preferred expression vectors for use in yeast systems include, but are not limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, and PAO815 (all available from Invitrogen, Carload, Calif.). Other suitable vectors will be readily apparent to the skilled artisan.
Nucleic acids of interest may be cloned into a suitable vector by route methods. Suitable vectors include plasmids, cosmids, recombinant viral vectors e.g. retroviral vectors, YACs, BACs and the like, phage vectors.
Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector.
A polypeptide of this invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.
Polypeptides of the present invention can also be recovered from: products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast higher plant, insect, and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host mediated processes. Thus, it is well known in the art that the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.
Suitable methods and compositions for polypeptide expression may be found in PCT publications WO/00-55173, WO/01-07611 and WO/02-16429, and suitable methods and compositions for production of modified polypeptides may be found in PCT publications WO/00-55173, WO/01-07611 and WO/02-16429.
Antibodies and Other Polypeptide or Polynucleotide Binding Molecules
The present invention further provides antibodies, which may be isolated antibodies, that are specific for a polypeptide encoded by a polynucleotide described herein and/or a polypeptide of a gene that corresponds to a polynucleotide described herein. Antibodies can be provided in a composition comprising the antibody and a buffer and/or a pharmaceutically acceptable excipient. Antibodies specific for a polypeptide associated with cancer are useful in a variety of diagnostic and therapeutic methods, as discussed in detail herein.
Gene products, including polypeptides, mRNA (particularly mRNAs having distinct secondary and/or tertiary structures), cDNA, or complete gene, can be prepared and used for raising antibodies for experimental, diagnostic, and therapeutic purposes. Antibodies may be used to identify a gene corresponding to a polynucleotide. The polynucleotide or related cDNA is expressed as described above, and antibodies are prepared. These antibodies are specific to an epitope on the polypeptide encoded by the polynucleotide, and can precipitate or bind to the corresponding native protein in a cell or tissue preparation or in a cell-free extract of an in vitro expression system.
Antibodies
Further polypeptides of the invention relate to antibodies and T-cell antigen receptors (TCR) which immunospecifically bind a subject polypeptide, subject polypeptide fragment, or variant thereof, and/or an epitope thereof (as determined by immunoassays well known in the art for assaying specific antibody-antigen binding). Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′) fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above. The term “antibody,” as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.
Most preferably the antibodies are human antigen-binding antibody fragments of the present invention and include, but are not limited to, Fab. Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CH1, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable region(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodies of the invention may be from any animal origin including birds and mammals. Preferably, the antibodies are human, murine (e.g., mouse and rat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken. As used herein, “human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from, human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al.
The antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).
Antibodies of the present invention may be described or specified in terms of the epitope(s) or portion(s) of a polypeptide of the present invention which they recognize or specifically bind. The epitope(s) or polypeptide portion(s) may be specified as described herein, e.g., by N-terminal and C-terminal positions, or by size in contiguous amino acid residues. Antibodies which specifically bind any epitope or polypeptide of the present invention may also be excluded. Therefore, the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the same.
Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homolog of a polypeptide of the present invention are included. Antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In specific embodiments, antibodies of the present invention cross-react with murine, rat and/or rabbit homologs of human proteins and the corresponding epitopes thereof. Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In a specific embodiment, the above-described cross-reactivity is with respect to any single specific antigenic or immunogenic polypeptide, or combination(s) of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenic polypeptides disclosed herein. Further included in the present invention are antibodies which bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions (as described herein). Antibodies of the present invention may also be described or specified in terms of their binding affinity to a polypeptide of the invention. Preferred binding affinities include those with a dissociation constant or Kd less 5×10−5 M, 10−5 M, 5×10−6 M, 10−6 M, 5×10−7 M, 10−7 M, 5×10−8 M, 10−8 M, 5×10−9 M, 10−9M, 5×10−10 M, 10-10 M, etc.
The invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein. In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%.
Methods for making screening, assaying, humanizing, and modifying different types of antibody are well known in the art and may be found in PCT publications WO/00-55173, WO/01-07611 and WO/02-16429.
In addition, the invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof. The invention also encompasses polynucleotides that hybridize under stringent or alternatively, under lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides that encode an antibody, preferably, that specifically binds to a polypeptide of the invention, preferably, an antibody that binds to a subject polypeptide.
The antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques. Recombinant expression of an antibody of the invention, or fragment, derivative or analog thereof, (e.g., a heavy or light chain of an antibody of the invention or a single chain antibody of the invention), requires construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.
The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention. Thus, the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter. In preferred embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2 (1990)).
Antibodies production is well known in the art. Exemplary methods and compositions for making antibodies may be found in PCT publications WO/00-55173, WO/01-07611 and WO/02-16429.
Immunophenotyping
The antibodies of the invention may be utilized for immunophenotyping of cell lines and biological samples. The translation product of the gene of the present invention may be useful as a cell specific marker, or more specifically as a cellular marker that is differentially expressed at various stages of differentiation and/or maturation of particular cell types. Monoclonal antibodies directed against a specific epitope, or combination of epitopes, will allow for the screening of cellular populations expressing the marker. Various techniques can be utilized using monoclonal antibodies to screen for cellular populations expressing the marker(s), and include magnetic separation using antibody-coated magnetic beads, “panning” with antibody attached to a solid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No. 5,985,660; and Morrison et al. Cell, 96:737-49 (1999)).
These techniques allow for the screening of particular populations of cells, such as might be found with hematological malignancies (i.e. minimal residual disease (MRD) in acute leukemic patients) and “non-self cells in transplantations to prevent Graft-versus-Host Disease (GVHD). Alternatively, these techniques allow for the screening of hematopoietic stem and progenitor cells capable of undergoing proliferation and/or differentiation, as might be found in human umbilical cord blood.
Kits
Also provided by the subject invention are kits for practicing the subject methods, as described above. The subject kits include at least one or more of: a subject nucleic acid, isolated polypeptide or an antibody thereto. Other optional components of the kit include: restriction enzymes, control primers and plasmids; buffers, cells, carriers adjuvents etc. The nucleic acids of the kit may also have restrictions sites, multiple cloning sites, primer sites, etc to facilitate their ligation other plasmids. The various components of the kit may be present in separate containers or certain compatible components may be precombined into a single container, as desired. In many embodiments, kits with unit doses of the active agent, e.g. in oral or injectable doses, are provided. In certain embodiments, controls, such as samples from a cancerous or non-cancerous cell are provided by the invention. Further embodiments of the kit include an antibody for a subject polypeptide and a chemotherapeutic agent to be used in combination with the polypeptide as a treatment.
In addition to above-mentioned components, the subject kits typically further include instructions for using the components of the kit to practice the subject methods. The instructions for practicing the subject methods are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.
Computer-Related Embodiments
In general, a library of polynucleotides is a collection of sequence information, which information is provided in either biochemical form (e.g., as a collection of polynucleotide molecules), or in electronic form (e.g., as a collection of polynucleotide sequences stored in a computer-readable form, as in a computer system and/or as part of a computer program). The sequence information of the polynucleotides can be used in a variety of ways, e.g., as a resource for gene discovery, as a representation of sequences expressed in a selected cell type (e.g., cell type markers), and/or as markers of a given disease or disease state. For example, in the instant case, the sequences of polynucleotides and polypeptides corresponding to genes differentially expressed in cancer, as well as the nucleic acid and amino acid sequences of the genes themselves, can be provided in electronic form in a computer database.
In general, a disease marker is a representation of a gene product that is present in all cells affected by disease either at an increased or decreased level relative to a normal cell (e.g., a cell of the same or similar type that is not substantially affected by disease). For example, a polynucleotide sequence in a library can be a polynucleotide that represents an mRNA, polypeptide, or other gene product encoded by the polynucleotide, that is either overexpressed or underexpressed in a cancerous cell affected by cancer relative to a normal (i.e., substantially disease-free) cell.
The nucleotide sequence information of the library can be embodied in any suitable form, e.g., electronic or biochemical forms. For example, a library of sequence information embodied in electronic form comprises an accessible computer data file (or, in biochemical form, a collection of nucleic acid molecules) that contains the representative nucleotide sequences of genes that are differentially expressed (e.g., overexpressed or underexpressed) as between, for example, i) a cancerous cell and a normal cell; ii) a cancerous cell and a dysplastic cell; iii) a cancerous cell and a cell affected by a disease or condition other than cancer; iv) a metastatic cancerous cell and a normal cell and/or non-metastatic cancerous cell; v) a malignant cancerous cell and a non-malignant cancerous cell (or a normal cell) and/or vi) a dysplastic cell relative to a normal cell. Other combinations and comparisons of cells affected by various diseases or stages of disease will be readily apparent to the ordinarily skilled artisan. Biochemical embodiments of the library include a collection of nucleic acids that have the sequences of the genes in the library, where the nucleic acids can correspond to the entire gene in the library or to a fragment thereof, as described in greater detail below.
The polynucleotide libraries of the subject invention generally comprise sequence information of a plurality of polynucleotide sequences, where at least one of the polynucleotides has a sequence of any of sequence described herein. By plurality is meant at least 2, usually at least 3 and can include up to all of the sequences described herein. The length and number of polynucleotides in the library will vary with the nature of the library, e.g., if the library is an oligonucleotide array, a cDNA array, a computer database of the sequence information, etc.
Where the library is an electronic library, the nucleic acid sequence information can be present in a variety of media. “Media” refers to a manufacture, other than an isolated nucleic acid molecule, that contains the sequence information of the present invention. Such a manufacture provides the genome sequence or a subset thereof in a form that can be examined by means not directly applicable to the sequence as it exists in a nucleic acid. For example, the nucleotide sequence of the present invention, e.g. the nucleic acid sequences of any of the polynucleotides of the sequences described herein, can be recorded on computer readable media, e.g. any medium that can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as a floppy disc, a hard disc storage medium, and a magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media.
One of skill in the art can readily appreciate how any of the presently known computer readable mediums can be used to create a manufacture comprising a recording of the present sequence information. “Recorded” refers to a process for storing information on computer readable medium, using any such methods as known in the art. Any convenient data storage structure can be chosen, based on the means used to access the stored information. A variety of data processor programs and formats can be used for storage, e.g. word processing text file, database format, etc. In addition to the sequence information, electronic versions of libraries comprising one or more sequence described herein can be provided in conjunction or connection with other computer-readable information and/or other types of computer-readable files (e.g., searchable files, executable files, etc, including, but not limited to, for example, search program software, etc.).
By providing the nucleotide sequence in computer readable form, the information can be accessed for a variety of purposes. Computer software to access sequence information (e.g. the NCBI sequence database) is publicly available. For example, the gapped BLAST (Altschul et al., Nucleic Acids Res. (1997) 25:3389-3402) and BLAZE (Brutlag et al., Comp. Chem. (1993) 17:203) search algorithms on a Sybase system, or the TeraBLAST (TimeLogic, Crystal Bay, Nev.) program optionally running on a specialized computer platform available from TimeLogic, can be used to identify open reading frames (ORFs) within the genome that contain homology to ORFs from other organisms.
As used herein, “a computer-based system” refers to the hardware means, software means, and data storage means used to analyze the nucleotide sequence information of the present invention. The minimum hardware of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means. A skilled artisan can readily appreciate that any one of the currently available computer-based system are suitable for use in the present invention. The data storage means can comprise any manufacture comprising a recording of the present sequence information as described above, or a memory access means that can access such a manufacture.
“Search means” refers to one or more programs implemented on the computer-based system, to compare a target sequence or target structural motif, or expression levels of a polynucleotide in a sample, with the stored sequence information. Search means can be used to identify fragments or regions of the genome that match a particular target sequence or target motif. A variety of known algorithms are publicly known and commercially available, e.g. MacPattern (EMBL), TeraBLAST (TimeLogic), BLASTN and BLASTX (NCBI). A “target sequence” can be any polynucleotide or amino acid sequence of six or more contiguous nucleotides or two or more amino acids, preferably from about 10 to 100 amino acids or from about 30 to 300 nt. A variety of means for comparing nucleic acids or polypeptides may be used to compare accomplish a sequence comparison (e.g., to analyze target sequences, target motifs, or relative expression levels) with the data storage means. A skilled artisan can readily recognize that any one of the publicly available homology search programs can be used to search the computer based systems of the present invention to compare of target sequences and motifs. Computer programs to analyze expression levels in a sample and in controls are also known in the art.
A “target structural motif,” or “target motif,” refers to any rationally selected sequence or combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration that is formed upon the folding of the target motif, or on consensus sequences of regulatory or active sites. There are a variety of target motifs known in the art. Protein target motifs include, but are not limited to, enzyme active sites and signal sequences, kinase domains, receptor binding domains, SH2 domains, SH3 domains, phosphorylation sites, protein interaction domains, transmembrane domains, etc. Nucleic acid target motifs include, but are not limited to, hairpin structures, promoter sequences and other expression elements such as binding sites for transcription factors.
A variety of structural formats for the input and output means can be used to input and output the information in the computer-based systems of the present invention. One format for an output means ranks the relative expression levels of different polynucleotides. Such presentation provides a skilled artisan with a ranking of relative expression levels to determine a gene expression profile. A gene expression profile can be generated from, for example, a cDNA library prepared from mRNA isolated from a test cell suspected of being cancerous or pre-cancerous, comparing the sequences or partial sequences of the clones against the sequences in an electronic database, where the sequences of the electronic database represent genes differentially expressed in a cancerous cell, e.g., a cancerous breast cell. The number of clones having a sequence that has substantial similarity to a sequence that represents a gene differentially expressed in a cancerous cell is then determined, and the number of clones corresponding to each of such genes is determined. An increased number of clones that correspond to differentially expressed gene is present in the cDNA library of the test cell (relative to, for example, the number of clones expected in a cDNA of a normal cell) indicates that the test cell is cancerous.
As discussed above, the “library” as used herein also encompasses biochemical libraries of the polynucleotides of the sequences described herein, e.g., collections of nucleic acids representing the provided polynucleotides. The biochemical libraries can take a variety of forms, e.g., a solution of cDNAs, a pattern of probe nucleic acids stably associated with a surface of a solid support (i.e., an array) and the like. Of particular interest are nucleic acid arrays in which one or more of the genes described herein is represented by a sequence on the array. By array is meant an article of manufacture that has at least a substrate with at least two distinct nucleic acid targets on one of its surfaces, where the number of distinct nucleic acids can be considerably higher, typically being at least 10 nt, usually at least 20 nt and often at least 25 nt. A variety of different array formats have been developed and are known to those of skill in the art. The arrays of the subject invention find use in a variety of applications, including gene expression analysis, drug screening, mutation analysis and the like, as disclosed in the above-listed exemplary patent documents.
In addition to the above nucleic acid libraries, analogous libraries of polypeptides are also provided, where the polypeptides of the library will represent at least a portion of the polypeptides encoded by a gene corresponding to a sequence described herein.
Diagnostic and Other Methods Involving Detection of Differentially Expressed Genes
The present invention provides methods of using the polynucleotides described herein in, for example, diagnosis of cancer and classification of cancer cells according to expression profiles. In specific non-limiting embodiments, the methods are useful for detecting cancer cells, facilitating diagnosis of cancer and the severity of a cancer (e.g., tumor grade, tumor burden, and the like) in a subject, facilitating a determination of the prognosis of a subject, and assessing the responsiveness of the subject to therapy (e.g., by providing a measure of therapeutic effect through, for example, assessing tumor burden during or following a chemotherapeutic regimen). Detection can be based on detection of a polynucleotide that is differentially expressed in a cancer cell, and/or detection of a polypeptide encoded by a polynucleotide that is differentially expressed in a cancer cell (“a polypeptide associated with cancer”). The detection methods of the invention can be conducted in vitro or in vivo, on isolated cells, or in whole tissues or a bodily fluid, e.g., blood, plasma, serum, urine, and the like).
In general, methods of the invention involving detection of a gene product (e.g., mRNA, cDNA generated from such mRNA, and polypeptides) involve contacting a sample with a probe specific for the gene product of interest. “Probe” as used herein in such methods is meant to refer to a molecule that specifically binds a gene product of interest (e.g., the probe binds to the target gene product with a specificity sufficient to distinguish binding to target over non-specific binding to non-target (background) molecules). “Probes” include, but are not necessarily limited to, nucleic acid probes (e.g., DNA, RNA, modified nucleic acid, and the like), antibodies (e.g., antibodies, antibody fragments that retain binding to a target epitope, single chain antibodies, and the like), or other polypeptide, peptide, or molecule (e.g., receptor ligand) that specifically binds a target gene product of interest.
The probe and sample suspected of having the gene product of interest are contacted under conditions suitable for binding of the probe to the gene product. For example, contacting is generally for a time sufficient to allow binding of the probe to the gene product (e.g., from several minutes to a few hours), and at a temperature and conditions of osmolarity and the like that provide for binding of the probe to the gene product at a level that is sufficiently distinguishable from background binding of the probe (e.g., under conditions that minimize non-specific binding). Suitable conditions for probe-target gene product binding can be readily determined using controls and other techniques available and known to one of ordinary skill in the art.
In this embodiment, the probe can be an antibody or other polypeptide, peptide, or molecule (e.g., receptor ligand) that specifically binds a target polypeptide of interest.
The detection methods can be provided as part of a kit. Thus, the invention further provides kits for detecting the presence and/or a level of a polynucleotide that is differentially expressed in a cancer cell (e.g., by detection of an mRNA encoded by the differentially expressed gene of interest), and/or a polypeptide encoded thereby, in a biological sample. Procedures using these kits can be performed by clinical laboratories, experimental laboratories, medical practitioners, or private individuals. The kits of the invention for detecting a polypeptide encoded by a polynucleotide that is differentially expressed in a cancer cell comprise a moiety that specifically binds the polypeptide, which may be a specific antibody. The kits of the invention for detecting a polynucleotide that is differentially expressed in a cancer cell comprise a moiety that specifically hybridizes to such a polynucleotide. The kit may optionally provide additional components that are useful in the procedure, including, but not limited to, buffers, developing reagents, labels, reacting surfaces, means for detection, control samples, standards, instructions, and interpretive information.
Detecting a Polypeptide Encoded by a Polynucleotide that is Differentially Expressed in a Cancer Cell
In some embodiments, methods are provided for a detecting cancer cell by detecting in a cell, a polypeptide encoded by a gene differentially expressed in a cancer cell. Any of a variety of known methods can be used for detection, including, but not limited to, immunoassay, using an antibody specific for the encoded polypeptide, e.g., by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and the like; and functional assays for the encoded polypeptide, e.g., binding activity or enzymatic activity.
For example, an immunofluorescence assay can be easily performed on cells without first isolating the encoded polypeptide. The cells are first fixed onto a solid support, such as a microscope slide or microtiter well. This fixing step can permeabilize the cell membrane. The permeablization of the cell membrane permits the polypeptide-specific probe (e.g, antibody) to bind. Alternatively, where the polypeptide is secreted or membrane-bound, or is otherwise accessible at the cell-surface (e.g., receptors, and other molecule stably-associated with the outer cell membrane or otherwise stably associated with the cell membrane, such permeabilization may not be necessary.
Next, the fixed cells are exposed to an antibody specific for the encoded polypeptide. To increase the sensitivity of the assay, the fixed cells may be further exposed to a second antibody, which is labeled and binds to the first antibody, which is specific for the encoded polypeptide. Typically, the secondary antibody is detectably labeled, e.g., with a fluorescent marker. The cells which express the encoded polypeptide will be fluorescently labeled and easily visualized under the microscope. See, for example, Hashido et al. (1992) Biochem. Biophys. Res. Comm. 187:1241-1248.
As will be readily apparent to the ordinarily skilled artisan upon reading the present specification, the detection methods and other methods described herein can be varied. Such variations are within the intended scope of the invention. For example, in the above detection scheme, the probe for use in detection can be immobilized on a solid support, and the test sample contacted with the immobilized probe. Binding of the test sample to the probe can then be detected in a variety of ways, e.g., by detecting a detectable label bound to the test sample.
The present invention further provides methods for detecting the presence of and/or measuring a level of a polypeptide in a biological sample, which polypeptide is encoded by a polynucleotide that represents a gene differentially expressed in cancer, particularly in a polynucleotide that represents a gene differentially cancer cell, using a probe specific for the encoded polypeptide. In this embodiment, the probe can be a an antibody or other polypeptide, peptide, or molecule (e.g., receptor ligand) that specifically binds a target polypeptide of interest.
The methods generally comprise: a) contacting the sample with an antibody specific for a differentially expressed polypeptide in a test cell; and b) detecting binding between the antibody and molecules of the sample. The level of antibody binding (either qualitative or quantitative) indicates the cancerous state of the cell. For example, where the differentially expressed gene is increased in cancerous cells, detection of an increased level of antibody binding to the test sample relative to antibody binding level associated with a normal cell indicates that the test cell is cancerous.
Suitable controls include a sample known not to contain the encoded polypeptide; and a sample contacted with an antibody not specific for the encoded polypeptide, e.g., an anti-idiotype antibody. A variety of methods to detect specific antibody-antigen interactions are known in the art and can be used in the method, including, but not limited to, standard immunohistological methods, immunoprecipitation, an enzyme immunoassay, and a radioimmunoassay.
In general, the specific antibody will be detectably labeled, either directly or indirectly. Direct labels include radioisotopes; enzymes whose products are detectable (e.g., luciferase, β-galactosidase, and the like); fluorescent labels (e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin, and the like); fluorescence emitting metals, e.g., 152Eu, or others of the lanthanide series, attached to the antibody through metal chelating groups such as EDTA; chemiluminescent compounds, e.g., luminol, isoluminol, acridinium salts, and the like; bioluminescent compounds, e.g., luciferin, aequorin (green fluorescent protein), and the like.
The antibody may be attached (coupled) to an insoluble support, such as a polystyrene plate or a bead. Indirect labels include second antibodies specific for antibodies specific for the encoded polypeptide (“first specific antibody”), wherein the second antibody is labeled as described above; and members of specific binding pairs, e.g., biotin-avidin, and the like. The biological sample may be brought into contact with and immobilized on a solid support or carrier, such as nitrocellulose, that is capable of immobilizing cells, cell particles, or soluble proteins. The support may then be washed with suitable buffers, followed by contacting with a detectably-labeled first specific antibody. Detection methods are known in the art and will be chosen as appropriate to the signal emitted by the detectable label. Detection is generally accomplished in comparison to suitable controls, and to appropriate standards.
In some embodiments, the methods are adapted for use in vivo, e.g., to locate or identify sites where cancer cells are present. In these embodiments, a detectably-labeled moiety, e.g., an antibody, which is specific for a cancer-associated polypeptide is administered to an individual (e.g., by injection), and labeled cells are located using standard imaging techniques, including, but not limited to, magnetic resonance imaging, computed tomography scanning, and the like. In this manner, cancer cells are differentially labeled.
Detecting a Polynucleotide that Represents a Gene Differentially Expressed in a Cancer Cell
In some embodiments, methods are provided for detecting a cancer cell by detecting expression in the cell of a transcript or that is differentially expressed in a cancer cell. Any of a variety of known methods can be used for detection, including, but not limited to, detection of a transcript by hybridization with a polynucleotide that hybridizes to a polynucleotide that is differentially expressed in a cancer cell; detection of a transcript by a polymerase chain reaction using specific oligonucleotide primers; in situ hybridization of a cell using as a probe a polynucleotide that hybridizes to a gene that is differentially expressed in a cancer cell and the like.
In many embodiments, the levels of a subject gene product are measured. By measured is meant qualitatively or quantitatively estimating the level of the gene product in a first biological sample either directly (e.g. by determining or estimating absolute levels of gene product) or relatively by comparing the levels to a second control biological sample. In many embodiments the second control biological sample is obtained from an individual not having not having cancer. As will be appreciated in the art, once a standard control level of gene expression is known, it can be used repeatedly as a standard for comparison. Other control samples include samples of cancerous tissue.
The methods can be used to detect and/or measure mRNA levels of a gene that is differentially expressed in a cancer cell. In some embodiments, the methods comprise: a) contacting a sample with a polynucleotide that corresponds to a differentially expressed gene described herein under conditions that allow hybridization; and b) detecting hybridization, if any. Detection of differential hybridization, when compared to a suitable control, is an indication of the presence in the sample of a polynucleotide that is differentially expressed in a cancer cell. Appropriate controls include, for example, a sample that is known not to contain a polynucleotide that is differentially expressed in a cancer cell. Conditions that allow hybridization are known in the art, and have been described in more detail above.
Detection can also be accomplished by any known method, including, but not limited to, in situ hybridization, PCR (polymerase chain reaction), RT-PCR (reverse transcription-PCR), and “Northern” or RNA blotting, arrays, microarrays, etc, or combinations of such techniques, using a suitably labeled polynucleotide. A variety of labels and labeling methods for polynucleotides are known in the art and can be used in the assay methods of the invention. Specific hybridization can be determined by comparison to appropriate controls.
Polynucleotides described herein are used for a variety of purposes, such as probes for detection of and/or measurement of, transcription levels of a polynucleotide that is differentially expressed in a cancer cell. Additional disclosure about preferred regions of the disclosed polynucleotide sequences is found in the Examples. A probe that hybridizes specifically to a polynucleotide disclosed herein should provide a detection signal at least 2-, 5-, 10-, or 20-fold higher than the background hybridization provided with other unrelated sequences. It should be noted that “probe” as used in this context of detection of nucleic acid is meant to refer to a polynucleotide sequence used to detect a differentially expressed gene product in a test sample. As will be readily appreciated by the ordinarily skilled artisan, the probe can be detectably labeled and contacted with, for example, an array comprising immobilized polynucleotides obtained from a test sample (e.g., mRNA). Alternatively, the probe can be immobilized on an array and the test sample detectably labeled. These and other variations of the methods of the invention are well within the skill in the art and are within the scope of the invention.
Labeled nucleic acid probes may be used to detect expression of a gene corresponding to the provided polynucleotide. In Northern blots, mRNA is separated electrophoretically and contacted with a probe. A probe is detected as hybridizing to an mRNA species of a particular size. The amount of hybridization can be quantitated to determine relative amounts of expression, for example under a particular condition. Probes are used for in situ hybridization to cells to detect expression. Probes can also be used in vivo for diagnostic detection of hybridizing sequences. Probes are typically labeled with a radioactive isotope. Other types of detectable labels can be used such as chromophores, fluorophores, and enzymes. Other examples of nucleotide hybridization assays are described in WO92/02526 and U.S. Pat. No. 5,124,246.
PCR is another means for detecting small amounts of target nucleic acids, methods for which may be found in Sambrook, et al. Molecular Cloning: A Laboratory Manual, CSH Press 1989, pp. 14.2-14.33.
A detectable label may be included in the amplification reaction. Suitable detectable labels include fluorochromes, (e.g. fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin, 6-carboxyfluorescein (6-FAM), 2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein, 6-carboxy-X-rhodamine (ROX), 6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein (5-FAM) or N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA)), radioactive labels, (e.g. 32P, 35S, 3H, etc.), and the like. The label may be a two stage system, where the polynucleotides is conjugated to biotin, haptens, etc. having a high affinity binding partner, e.g. avidin, specific antibodies, etc., where the binding partner is conjugated to a detectable label. The label may be conjugated to one or both of the primers. Alternatively, the pool of nucleotides used in the amplification is labeled, so as to incorporate the label into the amplification product.
Arrays
Polynucleotide arrays provide a high throughput technique that can assay a large number of polynucleotides or polypeptides in a sample. This technology can be used as a tool to test for differential expression.
A variety of methods of producing arrays, as well as variations of these methods, are known in the art and contemplated for use in the invention. For example, arrays can be created by spotting polynucleotide probes onto a substrate (e.g., glass, nitrocellulose, etc.) in a two-dimensional matrix or array having bound probes. The probes can be bound to the substrate by either covalent bonds or by non-specific interactions, such as hydrophobic interactions.
Samples of polynucleotides can be detectably labeled (e.g., using radioactive or fluorescent labels) and then hybridized to the probes. Double stranded polynucleotides, comprising the labeled sample polynucleotides bound to probe polynucleotides, can be detected once the unbound portion of the sample is washed away. Alternatively, the polynucleotides of the test sample can be immobilized on the array, and the probes detectably labeled. Techniques for constructing arrays and methods of using these arrays are described in, for example, Schena et al. (1996) Proc Natl Acad Sci USA. 93(20):10614-9; Schena et al. (1995) Science 270(5235):467-70; Shalon et al. (1996) Genome Res. 6(7):639-45, U.S. Pat. No. 5,807,522, EP 799 897; WO 97/29212; WO 97/27317; EP 785 280; WO 97/02357; U.S. Pat. No. 5,593,839; U.S. Pat. No. 5,578,832; EP 728 520; U.S. Pat. No. 5,599,695; EP 721 016; U.S. Pat. No. 5,556,752; WO 95/22058; and U.S. Pat. No. 5,631,734. In most embodiments, the “probe” is detectably labeled. In other embodiments, the probe is immobilized on the array and not detectably labeled.
Arrays can be used, for example, to examine differential expression of genes and can be used to determine gene function. For example, arrays can be used to detect differential expression of a gene corresponding to a polynucleotide described herein, where expression is compared between a test cell and control cell (e.g., cancer cells and normal cells). For example, high expression of a particular message in a cancer cell, which is not observed in a corresponding normal cell, can indicate a cancer specific gene product. Exemplary uses of arrays are further described in, for example, Pappalarado et al., Sem. Radiation Oncol. (1998) 8:217; and Ramsay, Nature Biotechnol. (1998) 16:40. Furthermore, many variations on methods of detection using arrays are well within the skill in the art and within the scope of the present invention. For example, rather than immobilizing the probe to a solid support, the test sample can be immobilized on a solid support which is then contacted with the probe.
Diagnosis, Prognosis, Assessment of Therapy (Therametrics), and Management of Cancer
The polynucleotides described herein, as well as their gene products and corresponding genes and gene products, are of particular interest as genetic or biochemical markers (e.g., in blood or tissues) that will detect the earliest changes along the carcinogenesis pathway and/or to monitor the efficacy of various therapies and preventive interventions.
For example, the level of expression of certain polynucleotides can be indicative of a poorer prognosis, and therefore warrant more aggressive chemo- or radio-therapy for a patient or vice versa. The correlation of novel surrogate tumor specific features with response to treatment and outcome in patients can define prognostic indicators that allow the design of tailored therapy based on the molecular profile of the tumor. These therapies include antibody targeting, antagonists (e.g., small molecules), and gene therapy.
Determining expression of certain polynucleotides and comparison of a patient's profile with known expression in normal tissue and variants of the disease allows a determination of the best possible treatment for a patient, both in terms of specificity of treatment and in terms of comfort level of the patient. Surrogate tumor markers, such as polynucleotide expression, can also be used to better classify, and thus diagnose and treat, different forms and disease states of cancer. Two classifications widely used in oncology that can benefit from identification of the expression levels of the genes corresponding to the polynucleotides described herein are staging of the cancerous disorder, and grading the nature of the cancerous tissue.
The polynucleotides that correspond to differentially expressed genes, as well as their encoded gene products, can be useful to monitor patients having or susceptible to cancer to detect potentially malignant events at a molecular level before they are detectable at a gross morphological level. In addition, the polynucleotides described herein, as well as the genes corresponding to such polynucleotides, can be useful as therametrics, e.g., to assess the effectiveness of therapy by using the polynucleotides or their encoded gene products, to assess, for example, tumor burden in the patient before, during, and after therapy.
Furthermore, a polynucleotide identified as corresponding to a gene that is differentially expressed in, and thus is important for, one type of cancer can also have implications for development or risk of development of other types of cancer, e.g., where a polynucleotide represents a gene differentially expressed across various cancer types. Thus, for example, expression of a polynucleotide corresponding to a gene that has clinical implications for cancer can also have clinical implications for metastatic breast cancer, colon cancer, or ovarian cancer, etc.
Staging. Staging is a process used by physicians to describe how advanced the cancerous state is in a patient. Staging assists the physician in determining a prognosis, planning treatment and evaluating the results of such treatment. Staging systems vary with the types of cancer, but generally involve the following “TNM” system: the type of tumor, indicated by T; whether the cancer has metastasized to nearby lymph nodes, indicated by N; and whether the cancer has metastasized to more distant parts of the body, indicated by M. Generally, if a cancer is only detectable in the area of the primary lesion without having spread to any lymph nodes it is called Stage I. If it has spread only to the closest lymph nodes, it is called Stage II. In Stage III, the cancer has generally spread to the lymph nodes in near proximity to the site of the primary lesion. Cancers that have spread to a distant part of the body, such as the liver, bone, brain or other site, are Stage IV, the most advanced stage.
The polynucleotides and corresponding genes and gene products described herein can facilitate fine-tuning of the staging process by identifying markers for the aggressiveness of a cancer, e.g. the metastatic potential, as well as the presence in different areas of the body. Thus, a Stage II cancer with a polynucleotide signifying a high metastatic potential cancer can be used to change a borderline Stage II tumor to a Stage III tumor, justifying more aggressive therapy. Conversely, the presence of a polynucleotide signifying a lower metastatic potential allows more conservative staging of a tumor.
One type of breast cancer is ductal carcinoma in situ (DCIS): DCIS is when the breast cancer cells are completely contained within the breast ducts (the channels in the breast that carry milk to the nipple), and have not spread into the surrounding breast tissue. This may also be referred to as non-invasive or intraductal cancer, as the cancer cells have not yet spread into the surrounding breast tissue and so usually have not spread into any other part of the body.
Lobular carcinoma in situ breast cancer (LCIS) means that cell changes are found in the lining of the lobules of the breast. It can be present in both breasts. It is also referred to as non-invasive cancer as it has not spread into the surrounding breast tissue.
Invasive breast cancer can be staged as follows: Stage 1 tumours: these measure less than two centimetres. The lymph glands in the armpit are not affected and there are no signs that the cancer has spread elsewhere in the body; Stage 2 tumours: these measure between two and five centimetres, or the lymph glands in the armpit are affected, or both. However, there are no signs that the cancer has spread further; Stage 3 tumours: these are larger than five centimetres and may be attached to surrounding structures such as the muscle or skin. The lymph glands are usually affected, but there are no signs that the cancer has spread beyond the breast or the lymph glands in the armpit; Stage 4 tumours: these are of any size, but the lymph glands are usually affected and the cancer has spread to other parts of the body. This is secondary breast cancer.
Grading of cancers. Grade is a term used to describe how closely a tumor resembles normal tissue of its same type. The microscopic appearance of a tumor is used to identify tumor grade based on parameters such as cell morphology, cellular organization, and other markers of differentiation. As a general rule, the grade of a tumor corresponds to its rate of growth or aggressiveness, with undifferentiated or high-grade tumors generally being more aggressive than well-differentiated or low-grade tumors.
The polynucleotides of the Sequence Listing, and their corresponding genes and gene products, can be especially valuable in determining the grade of the tumor, as they not only can aid in determining the differentiation status of the cells of a tumor, they can also identify factors other than differentiation that are valuable in determining the aggressiveness of a tumor, such as metastatic potential.
Low grade means that the cancer cells look very like the normal cells. They are usually slowly growing and are less likely to spread. In high grade tumors the cells look very abnormal. They are likely to grow more quickly and are more likely to spread.
Assessment of proliferation of cells in tumor. The differential expression level of the polynucleotides described herein can facilitate assessment of the rate of proliferation of tumor cells, and thus provide an indicator of the aggressiveness of the rate of tumor growth. For example, assessment of the relative expression levels of genes involved in cell cycle can provide an indication of cellular proliferation, and thus serve as a marker of proliferation.
Detection of Cancer.
The polynucleotides corresponding to genes that exhibit the appropriate expression pattern can be used to detect cancer in a subject. The expression of appropriate polynucleotides can be used in the diagnosis, prognosis and management of cancer. Detection of cancer can be determined using expression levels of any of these sequences alone or in combination with the levels of expression of other known cancer genes. Determination of the aggressive nature and/or the metastatic potential of a cancer can be determined by comparing levels of one or more gene products of the genes corresponding to the polynucleotides described herein, and comparing total levels of another sequence known to vary in cancerous tissue, e.g., expression of p53, DCC, ras, FAP (see, e.g., Fearon E R, et al., Cell (1990) 61(5):759; Hamilton S R et al., Cancer (1993) 72:957; Bodmer W, et al., Nat Genet. (1994) 4(3):217; Fearon E R, Ann N Y Acad Sci. (1995) 768:101). For example, development of cancer can be detected by examining the level of expression of a gene corresponding to a polynucleotides described herein to the levels of oncogenes (e.g. ras) or tumor suppressor genes (e.g. FAP or p53). Thus expression of specific marker polynucleotides can be used to discriminate between normal and cancerous tissue, to discriminate between cancers with different cells of origin, to discriminate between cancers with different potential metastatic rates, etc. For a review of other markers of cancer, see, e.g., Hanahan et al. (2000) Cell 100:57-70.
Treatment of Cancer
The invention further provides methods for reducing growth of cancer cells. The methods provide for decreasing the expression of a gene that is differentially expressed in a cancer cell or decreasing the level of and/or decreasing an activity of a cancer-associated polypeptide. In general, the methods comprise contacting a cancer cell with a substance that modulates (1) expression of a gene that is differentially expressed in cancer; or (2) a level of and/or an activity of a cancer-associated polypeptide.
“Reducing growth of cancer cells” includes, but is not limited to, reducing proliferation of cancer cells, and reducing the incidence of a non-cancerous cell becoming a cancerous cell. Whether a reduction in cancer cell growth has been achieved can be readily determined using any known assay, including, but not limited to, [3H]-thymidine incorporation; counting cell number over a period of time; detecting and/or measuring a marker associated with breast cancer (e.g., PSA).
The present invention provides methods for treating cancer, generally comprising administering to an individual in need thereof a substance that reduces cancer cell growth, in an amount sufficient to reduce cancer cell growth and treat the cancer. Whether a substance, or a specific amount of the substance, is effective in treating cancer can be assessed using any of a variety of known diagnostic assays for cancer, including, but not limited to, proctoscopy, rectal examination, biopsy, contrast radiographic studies, CAT scan, and detection of a tumor marker associated with cancer in the blood of the individual (e.g., PSA (breast-specific antigen)). The substance can be administered systemically or locally. Thus, in some embodiments, the substance is administered locally, and cancer growth is decreased at the site of administration. Local administration may be useful in treating, e.g., a solid tumor.
A substance that reduces cancer cell growth can be targeted to a cancer cell. Thus, in some embodiments, the invention provides a method of delivering a drug to a cancer cell, comprising administering a drug-antibody complex to a subject, wherein the antibody is specific for a cancer-associated polypeptide, and the drug is one that reduces cancer cell growth, a variety of which are known in the art. Targeting can be accomplished by coupling (e.g., linking, directly or via a linker molecule, either covalently or non-covalently, so as to form a drug-antibody complex) a drug to an antibody specific for a cancer-associated polypeptide. Methods of coupling a drug to an antibody are well known in the art and need not be elaborated upon herein.
Tumor Classification and Patient Stratification
The invention further provides for methods of classifying tumors, and thus grouping or “stratifying” patients, according to the expression profile of selected differentially expressed genes in a tumor. Differentially expressed genes can be analyzed for correlation with other differentially expressed genes in a single tumor type or across tumor types. Genes that demonstrate consistent correlation in expression profile in a given cancer cell type (e.g., in a cancer cell or type of cancer) can be grouped together, e.g., when one gene is overexpressed in a tumor, a second gene is also usually overexpressed. Tumors can then be classified according to the expression profile of one or more genes selected from one or more groups.
The tumor of each patient in a pool of potential patients can be classified as described above. Patients having similarly classified tumors can then be selected for participation in an investigative or clinical trial of a cancer therapeutic where a homogeneous population is desired. The tumor classification of a patient can also be used in assessing the efficacy of a cancer therapeutic in a heterogeneous patient population. In addition, therapy for a patient having a tumor of a given expression profile can then be selected accordingly.
In another embodiment, differentially expressed gene products (e.g., polypeptides or polynucleotides encoding such polypeptides) may be effectively used in treatment through vaccination. The growth of cancer cells is naturally limited in part due to immune surveillance. Stimulation of the immune system using a particular tumor-specific antigen enhances the effect towards the tumor expressing the antigen. An active vaccine comprising a polypeptide encoded by the cDNA of this invention would be appropriately administered to subjects having an alteration, e.g., overabundance, of the corresponding RNA, or those predisposed for developing cancer cells with an alteration of the same RNA. Polypeptide antigens are typically combined with an adjuvant as part of a vaccine composition. The vaccine is preferably administered first as a priming dose, and then again as a boosting dose, usually at least four weeks later. Further boosting doses may be given to enhance the effect. The dose and its timing are usually determined by the person responsible for the treatment.
The invention also encompasses the selection of a therapeutic regimen based upon the expression profile of differentially expressed genes in the patient's tumor. For example, a tumor can be analyzed for its expression profile of the genes corresponding to SEQ ID NOS:1, 3, 5, 7, 9, 11-13, 15, 16, 18, 20, 22, 24, 26, 27, 29 and 128-1618 as described herein, e.g., the tumor is analyzed to determine which genes are expressed at elevated levels or at decreased levels relative to normal cells of the same tissue type. The expression patterns of the tumor are then compared to the expression patterns of tumors that respond to a selected therapy. Where the expression profiles of the test tumor cell and the expression profile of a tumor cell of known drug responsivity at least substantially match (e.g., selected sets of genes at elevated levels in the tumor of known drug responsivity and are also at elevated levels in the test tumor cell), then the therapeutic agent selected for therapy is the drug to which tumors with that expression pattern respond.
Pattern Matching in Diagnosis Using Arrays
In another embodiment, the diagnostic and/or prognostic methods of the invention involve detection of expression of a selected set of genes in a test sample to produce a test expression pattern (TEP). The TEP is compared to a reference expression pattern (REP), which is generated by detection of expression of the selected set of genes in a reference sample (e.g., a positive or negative control sample). The selected set of genes includes at least one of the genes of the invention, which genes correspond to the polynucleotide sequences described herein. Of particular interest is a selected set of genes that includes gene differentially expressed in the disease for which the test sample is to be screened.
Identification of Therapeutic Targets and Anti-Cancer Therapeutic Agents
The present invention also encompasses methods for identification of agents having the ability to modulate activity of a differentially expressed gene product, as well as methods for identifying a differentially expressed gene product as a therapeutic target for treatment of cancer.
Identification of compounds that modulate activity of a differentially expressed gene product can be accomplished using any of a variety of drug screening techniques. Such agents are candidates for development of cancer therapies. Of particular interest are screening assays for agents that have tolerable toxicity for normal, non-cancerous human cells. The screening assays of the invention are generally based upon the ability of the agent to modulate an activity of a differentially expressed gene product and/or to inhibit or suppress phenomenon associated with cancer (e.g., cell proliferation, colony formation, cell cycle arrest, metastasis, and the like).
Screening of Candidate Agents
Screening assays can be based upon any of a variety of techniques readily available and known to one of ordinary skill in the art. In general, the screening assays involve contacting a cancerous cell with a candidate agent, and assessing the effect upon biological activity of a differentially expressed gene product. The effect upon a biological activity can be detected by, for example, detection of expression of a gene product of a differentially expressed gene (e.g., a decrease in mRNA or polypeptide levels, would in turn cause a decrease in biological activity of the gene product). Alternatively or in addition, the effect of the candidate agent can be assessed by examining the effect of the candidate agent in a functional assay. For example, where the differentially expressed gene product is an enzyme, then the effect upon biological activity can be assessed by detecting a level of enzymatic activity associated with the differentially expressed gene product. The functional assay will be selected according to the differentially expressed gene product. In general, where the differentially expressed gene is increased in expression in a cancerous cell, agents of interest are those that decrease activity of the differentially expressed gene product.
Assays described infra can be readily adapted in the screening assay embodiments of the invention. Exemplary assays useful in screening candidate agents include, but are not limited to, hybridization-based assays (e.g., use of nucleic acid probes or primers to assess expression levels), antibody-based assays (e.g., to assess levels of polypeptide gene products), binding assays (e.g., to detect interaction of a candidate agent with a differentially expressed polypeptide, which assays may be competitive assays where a natural or synthetic ligand for the polypeptide is available), and the like. Additional exemplary assays include, but are not necessarily limited to, cell proliferation assays, antisense knockout assays, assays to detect inhibition of cell cycle, assays of induction of cell death/apoptosis, and the like. Generally such assays are conducted in vitro, but many assays can be adapted for in vivo analyses, e.g., in an animal model of the cancer.
Identification of Therapeutic Targets
In another embodiment, the invention contemplates identification of differentially expressed genes and gene products as therapeutic targets. In some respects, this is the converse of the assays described above for identification of agents having activity in modulating (e.g., decreasing or increasing) activity of a differentially expressed gene product.
In this embodiment, therapeutic targets are identified by examining the effect(s) of an agent that can be demonstrated or has been demonstrated to modulate a cancerous phenotype (e.g., inhibit or suppress or prevent development of a cancerous phenotype). Such agents are generally referred to herein as an “anti-cancer agent”, which agents encompass chemotherapeutic agents. For example, the agent can be an antisense oligonucleotide that is specific for a selected gene transcript. For example, the antisense oligonucleotide may have a sequence corresponding to a sequence of a differentially expressed gene described herein, e.g., a sequence of one of SEQ ID NOS:1, 3, 5, 7, 9, 11-13, 15, 16, 18, 20, 22, 24, 26, 27, 29 and 128-1618.
Assays for identification of therapeutic targets can be conducted in a variety of ways using methods that are well known to one of ordinary skill in the art. For example, a test cancerous cell that expresses or overexpresses a differentially expressed gene is contacted with an anti-cancer agent, the effect upon a cancerous phenotype and a biological activity of the candidate gene product assessed. The biological activity of the candidate gene product can be assayed be examining, for example, modulation of expression of a gene encoding the candidate gene product (e.g., as detected by, for example, an increase or decrease in transcript levels or polypeptide levels), or modulation of an enzymatic or other activity of the gene product. The cancerous phenotype can be, for example, cellular proliferation, loss of contact inhibition of growth (e.g., colony formation), tumor growth (in vitro or in vivo), and the like. Alternatively or in addition, the effect of modulation of a biological activity of the candidate target gene upon cell death/apoptosis or cell cycle regulation can be assessed.
Inhibition or suppression of a cancerous phenotype, or an increase in cell death or apoptosis as a result of modulation of biological activity of a candidate gene product indicates that the candidate gene product is a suitable target for cancer therapy. Assays described infra can be readily adapted for assays for identification of therapeutic targets. Generally such assays are conducted in vitro, but many assays can be adapted for in vivo analyses, e.g., in an appropriate, art-accepted animal model of the cancer.
Candidate Agents
The term “agent” as used herein describes any molecule, e.g. protein or pharmaceutical, with the capability of modulating a biological activity of a gene product of a differentially expressed gene. Generally a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection.
Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including, but not limited to: peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts (including extracts from human tissue to identify endogenous factors affecting differentially expressed gene products) are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.
Exemplary candidate agents of particular interest include, but are not limited to, antisense and RNAi polynucleotides, and antibodies, soluble receptors, and the like. Antibodies and soluble receptors are of particular interest as candidate agents where the target differentially expressed gene product is secreted or accessible at the cell-surface (e.g., receptors and other molecule stably-associated with the outer cell membrane).
For method that involve RNAi (RNA interference), a double stranded RNA (dsRNA) molecule is usually used. The dsRNA is prepared to be substantially identical to at least a segment of a subject polynucleotide (e.g. a cDNA or gene). In general, the dsRNA is selected to have at least 70%, 75%, 80%, 85% or 90% sequence identity with the subject polynucleotide over at least a segment of the candidate gene. In other instances, the sequence identity is even higher, such as 95%, 97% or 99%, and in still other instances, there is 100% sequence identity with the subject polynucleotide over at least a segment of the subject polynucleotide. The size of the segment over which there is sequence identity can vary depending upon the size of the subject polynucleotide. In general, however, there is substantial sequence identity over at least 15, 20, 25, 30, 35, 40 or 50 nucleotides. In other instances, there is substantial sequence identity over at least 100, 200, 300, 400, 500 or 1000 nucleotides; in still other instances, there is substantial sequence identity over the entire length of the subject polynucleotide, i.e., the coding and non-coding region of the candidate gene.
Because only substantial sequence similarity between the subject polynucleotide and the dsRNA is necessary, sequence variations between these two species arising from genetic mutations, evolutionary divergence and polymorphisms can be tolerated. Moreover, as described further infra, the dsRNA can include various modified or nucleotide analogs.
Usually the dsRNA consists of two separate complementary RNA strands. However, in some instances, the dsRNA may be formed by a single strand of RNA that is self-complementary, such that the strand loops back upon itself to form a hairpin loop. Regardless of form, RNA duplex formation can occur inside or outside of a cell.
The size of the dsRNA that is utilized varies according to the size of the subject polynucleotide whose expression is to be suppressed and is sufficiently long to be effective in reducing expression of the subject polynucleotide in a cell. Generally, the dsRNA is at least 10-15 nucleotides long. In certain applications, the dsRNA is less than 20, 21, 22, 23, 24 or 25 nucleotides in length. In other instances, the dsRNA is at least 50, 100, 150 or 200 nucleotides in length. The dsRNA can be longer still in certain other applications, such as at least 300, 400, 500 or 600 nucleotides. Typically, the dsRNA is not longer than 3000 nucleotides. The optimal size for any particular subject polynucleotide can be determined by one of ordinary skill in the art without undue experimentation by varying the size of the dsRNA in a systematic fashion and determining whether the size selected is effective in interfering with expression of the subject polynucleotide.
dsRNA can be prepared according to any of a number of methods that are known in the art, including in vitro and in vivo methods, as well as by synthetic chemistry approaches.
In vitro methods. Certain methods generally involve inserting the segment corresponding to the candidate gene that is to be transcribed between a promoter or pair of promoters that are oriented to drive transcription of the inserted segment and then utilizing an appropriate RNA polymerase to carry out transcription. One such arrangement involves positioning a DNA fragment corresponding to the candidate gene or segment thereof into a vector such that it is flanked by two opposable polymerase-specific promoters that can be same or different. Transcription from such promoters produces two complementary RNA strands that can subsequently anneal to form the desired dsRNA. Exemplary plasmids for use in such systems include the plasmid (PCR 4.0 TOPO) (available from Invitrogen). Another example is the vector pGEM-T (Promega, Madison, Wis.) in which the oppositely oriented promoters are T7 and SP6; the T3 promoter can also be utilized.
In a second arrangement, DNA fragments corresponding to the segment of the subject polynucleotide that is to be transcribed is inserted both in the sense and antisense orientation downstream of a single promoter. In this system, the sense and antisense fragments are cotranscribed to generate a single RNA strand that is self-complementary and thus can form dsRNA.
Various other in vitro methods have been described. Examples of such methods include, but are not limited to, the methods described by Sadher et al. (Biochem. Int. 14:1015, 1987); by Bhattacharyya (Nature 343:484, 1990); and by Livache, et al. (U.S. Pat. No. 5,795,715), each of which is incorporated herein by reference in its entirety.
Single-stranded RNA can also be produced using a combination of enzymatic and organic synthesis or by total organic synthesis. The use of synthetic chemical methods enable one to introduce desired modified nucleotides or nucleotide analogs into the dsRNA.
In vivo methods. dsRNA can also be prepared in vivo according to a number of established methods (see, e.g., Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed.; Transcription and Translation (B. D. Hames, and S. J. Higgins, Eds., 1984); DNA Cloning, volumes I and II (D. N. Glover, Ed., 1985); and Oligonucleotide Synthesis (M. J. Gait, Ed., 1984, each of which is incorporated herein by reference in its entirety).
Once the single-stranded RNA has been formed, the complementary strands are allowed to anneal to form duplex RNA. Transcripts are typically treated with DNAase and further purified according to established protocols to remove proteins. Usually such purification methods are not conducted with phenol:chloroform. The resulting purified transcripts are subsequently dissolved in RNAase free water or a buffer of suitable composition.
dsRNA is generated by annealing the sense and anti-sense RNA in vitro. Generally, the strands are initially denatured to keep the strands separate and to avoid self-annealing. During the annealing process, typically certain ratios of the sense and antisense strands are combined to facilitate the annealing process. In some instances, a molar ratio of sense to antisense strands of 3:7 is used; in other instances, a ratio of 4:6 is utilized; and in still other instances, the ratio is 1:1.
The buffer composition utilized during the annealing process can in some instances affect the efficacy of the annealing process and subsequent transfection procedure. While some have indicated that the buffered solution used to carry out the annealing process should include a potassium salt such as potassium chloride (e.g. at a concentration of about 80 mM). In some embodiments, the buffer is substantially postassium free. Once single-stranded RNA has annealed to form duplex RNA, typically any single-strand overhangs are removed using an enzyme that specifically cleaves such overhangs (e.g., RNAase A or RNAase T).
Once the dsRNA has been formed, it is introduced into a reference cell, which can include an individual cell or a population of cells (e.g., a tissue, an embryo and an entire organism). The cell can be from essentially any source, including animal, plant, viral, bacterial, fungal and other sources. If a tissue, the tissue can include dividing or nondividing and differentiated or undifferentiated cells. Further, the tissue can include germ line cells and somatic cells. Examples of differentiated cells that can be utilized include, but are not limited to, neurons, glial cells, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, basophils, mast cells, leukocytes, granulocytes, keratinocytes, adipocytes, osteoblasts, osteoclasts, hepatocytes, cells of the endocrine or exocrine glands, fibroblasts, myocytes, cardiomyocytes, and endothelial cells. The cell can be an individual cell of an embryo, and can be a blastocyte or an oocyte.
Certain methods are conducted using model systems for particular cellular states (e.g., a disease). For instance, certain methods provided herein are conducted with a cancer cell lines that serves as a model system for investigating genes that are correlated with various cancers.
A number of options can be utilized to deliver the dsRNA into a cell or population of cells such as in a cell culture, tissue or embryo. For instance, RNA can be directly introduced intracellularly. Various physical methods are generally utilized in such instances, such as administration by microinjection (see, e.g., Zernicka-Goetz, et al. (1997) Development 124:1133-1137; and Wianny, et al. (1998) Chromosoma 107: 430-439).
Other options for cellular delivery include permeabilizing the cell membrane and electroporation in the presence of the dsRNA, liposome-mediated transfection, or transfection using chemicals such as calcium phosphate. A number of established gene therapy techniques can also be utilized to introduce the dsRNA into a cell. By introducing a viral construct within a viral particle, for instance, one can achieve efficient introduction of an expression construct into the cell and transcription of the RNA encoded by the construct.
If the dsRNA is to be introduced into an organism or tissue, gene gun technology is an option that can be employed. This generally involves immobilizing the dsRNA on a gold particle which is subsequently fired into the desired tissue. Research has also shown that mammalian cells have transport mechanisms for taking in dsRNA (see, e.g., Asher, et al. (1969) Nature 223:715-717). Consequently, another delivery option is to administer the dsRNA extracellularly into a body cavity, interstitial space or into the blood system of the mammal for subsequent uptake by such transport processes. The blood and lymph systems and the cerebrospinal fluid are potential sites for injecting dsRNA. Oral, topical, parenteral, rectal and intraperitoneal administration are also possible modes of administration.
The composition introduced can also include various other agents in addition to the dsRNA. Examples of such agents include, but are not limited to, those that stabilize the dsRNA, enhance cellular uptake and/or increase the extent of interference. Typically, the dsRNA is introduced in a buffer that is compatible with the composition of the cell into which the RNA is introduced to prevent the cell from being shocked. The minimum size of the dsRNA that effectively achieves gene silencing can also influence the choice of delivery system and solution composition.
Sufficient dsRNA is introduced into the tissue to cause a detectable change in expression of a taget gene (assuming the candidate gene is in fact being expressed in the cell into which the dsRNA is introduced) using available detection methodologies. Thus, in some instances, sufficient dsRNA is introduced to achieve at least a 5-10% reduction in candidate gene expression as compared to a cell in which the dsRNA is not introduced. In other instances, inhibition is at least 20, 30, 40 or 50%. In still other instances, the inhibition is at least 60, 70, 80, 90 or 95%. Expression in some instances is essentially completely inhibited to undetectable levels.
The amount of dsRNA introduced depends upon various factors such as the mode of administration utilized, the size of the dsRNA, the number of cells into which dsRNA is administered, and the age and size of an animal if dsRNA is introduced into an animal. An appropriate amount can be determined by those of ordinary skill in the art by initially administering dsRNA at several different concentrations for example, for example. In certain instances when dsRNA is introduced into a cell culture, the amount of dsRNA introduced into the cells varies from about 0.5 to 3 μg per 106 cells.
A number of options are available to detect interference of candidate gene expression (i.e., to detect candidate gene silencing). In general, inhibition in expression is detected by detecting a decrease in the level of the protein encoded by the candidate gene, determining the level of mRNA transcribed from the gene and/or detecting a change in phenotype associated with candidate gene expression.
Use of Polypeptides to Screen for Peptide Analogs and Antagonists
Polypeptides encoded by differentially expressed genes identified herein can be used to screen peptide libraries to identify binding partners, such as receptors, from among the encoded polypeptides. Peptide libraries can be synthesized according to methods known in the art (see, e.g., U.S. Pat. No. 5,010,175 and WO 91/17823).
Agonists or antagonists of the polypeptides of the invention can be screened using any available method known in the art, such as signal transduction, antibody binding, receptor binding, mitogenic assays, chemotaxis assays, etc. The assay conditions ideally should resemble the conditions under which the native activity is exhibited in vivo, that is, under physiologic pH, temperature, and ionic strength. Suitable agonists or antagonists will exhibit strong inhibition or enhancement of the native activity at concentrations that do not cause toxic side effects in the subject. Agonists or antagonists that compete for binding to the native polypeptide can require concentrations equal to or greater than the native concentration, while inhibitors capable of binding irreversibly to the polypeptide can be added in concentrations on the order of the native concentration.
Such screening and experimentation can lead to identification of a polypeptide binding partner, such as a receptor, encoded by a gene or a cDNA corresponding to a polynucleotide described herein, and at least one peptide agonist or antagonist of the binding partner. Such agonists and antagonists can be used to modulate, enhance, or inhibit receptor function in cells to which the receptor is native, or in cells that possess the receptor as a result of genetic engineering. Further, if the receptor shares biologically important characteristics with a known receptor, information about agonist/antagonist binding can facilitate development of improved agonists/antagonists of the known receptor.
Vaccines and Uses
The differentially expressed nucleic acids and polypeptides produced by the nucleic acids of the invention can also be used to modulate primary immune response to prevent or treat cancer. Every immune response is a complex and intricately regulated sequence of events involving several cell types. It is triggered when an antigen enters the body and encounters a specialized class of cells called antigen-presenting cells (APCs). These APCs capture a minute amount of the antigen and display it in a form that can be recognized by antigen-specific helper T lymphocytes. The helper (Th) cells become activated and, in turn, promote the activation of other classes of lymphocytes, such as B cells or cytotoxic T cells. The activated lymphocytes then proliferate and carry out their specific effector functions, which in many cases successfully activate or eliminate the antigen. Thus, activating the immune response to a particular antigen associated with a cancer cell can protect the patient from developing cancer or result in lymphocytes eliminating cancer cells expressing the antigen.
Gene products, including polypeptides, mRNA (particularly mRNAs having distinct secondary and/or tertiary structures), cDNA, or complete gene, can be prepared and used in vaccines for the treatment or prevention of hyperproliferative disorders and cancers. The nucleic acids and polypeptides can be utilized to enhance the immune response, prevent tumor progression, prevent hyperproliferative cell growth, and the like. Methods for selecting nucleic acids and polypeptides that are capable of enhancing the immune response are known in the art. Preferably, the gene products for use in a vaccine are gene products which are present on the surface of a cell and are recognizable by lymphocytes and antibodies.
The gene products may be formulated with pharmaceutically acceptable carriers into pharmaceutical compositions by methods known in the art. The composition is useful as a vaccine to prevent or treat cancer. The composition may further comprise at least one co-immunostimulatory molecule, including but not limited to one or more major histocompatibility complex (MHC) molecules, such as a class I or class II molecule, preferably a class I molecule. The composition may further comprise other stimulator molecules including B7.1, B7.2, ICAM-1, ICAM-2, LFA-1, LFA-3, CD72 and the like, immunostimulatory polynucleotides (which comprise an 5′-CG-3′ wherein the cytosine is unmethylated), and cytokines which include but are not limited to IL-1 through IL-15, TNF-α, IFN-γ, RANTES, G-CSF, M-CSF, IFN-α, CTAP III, ENA-78, GRO, I-309, PF-4, IP-10, LD-78, MGSA, MIP-1α, MIP-1β, or combination thereof, and the like for immunopotentiation. In one embodiment, the immunopotentiators of particular interest are those that facilitate a Th1 immune response.
The gene products may also be prepared with a carrier that will protect the gene products against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, and the like. Methods for preparation of such formulations are known in the art.
In the methods of preventing or treating cancer, the gene products may be administered via one of several routes including but not limited to transdermal, transmucosal, intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal, intrathecal, intrapleural, intrauterine, rectal, vaginal, topical, intratumor, and the like. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, administration bile salts and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration may be by nasal sprays or suppositories. For oral administration, the gene products are formulated into conventional oral administration form such as capsules, tablets, elixirs and the like.
The gene product is administered to a patient in an amount effective to prevent or treat cancer. In general, it is desirable to provide the patient with a dosage of gene product of at least about 1 pg per Kg body weight, preferably at least about 1 ng per Kg body weight, more preferably at least about 1 μg or greater per Kg body weight of the recipient. A range of from about 1 ng per Kg body weight to about 100 mg per Kg body weight is preferred although a lower or higher dose may be administered. The dose is effective to prime, stimulate and/or cause the clonal expansion of antigen-specific T lymphocytes, preferably cytotoxic T lymphocytes, which in turn are capable of preventing or treating cancer in the recipient. The dose is administered at least once and may be provided as a bolus or a continuous administration. Multiple administrations of the dose over a period of several weeks to months may be preferable. Subsequent doses may be administered as indicated.
In another method of treatment, autologous cytotoxic lymphocytes or tumor infiltrating lymphocytes may be obtained from a patient with cancer. The lymphocytes are grown in culture, and antigen-specific lymphocytes are expanded by culturing in the presence of the specific gene products alone or in combination with at least one co-immunostimulatory molecule with cytokines. The antigen-specific lymphocytes are then infused back into the patient in an amount effective to reduce or eliminate the tumors in the patient. Cancer vaccines and their uses are further described in U.S. Pat. No. 5,961,978; U.S. Pat. No. 5,993,829; U.S. Pat. No. 6,132,980; and WO 00/38706.
Pharmaceutical Compositions and Uses
Pharmaceutical compositions can comprise polypeptides, receptors that specifically bind a polypeptide produced by a differentially expressed gene (e.g., antibodies, or polynucleotides (including antisense nucleotides and ribozymes) of the claimed invention in a therapeutically effective amount. The compositions can be used to treat primary tumors as well as metastases of primary tumors. In addition, the pharmaceutical compositions can be used in conjunction with conventional methods of cancer treatment, e.g., to sensitize tumors to radiation or conventional chemotherapy.
Where the pharmaceutical composition comprises a receptor (such as an antibody) that specifically binds to a gene product encoded by a differentially expressed gene, the receptor can be coupled to a drug for delivery to a treatment site or coupled to a detectable label to facilitate imaging of a site comprising cancer cells. Methods for coupling antibodies to drugs and detectable labels are well known in the art, as are methods for imaging using detectable labels.
The term “therapeutically effective amount” as used herein refers to an amount of a therapeutic agent to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect. The effect can be detected by, for example, chemical markers or antigen levels. Therapeutic effects also include reduction in physical symptoms, such as decreased body temperature.
The precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration. Thus, it is not useful to specify an exact effective amount in advance. However, the effective amount for a given situation is determined by routine experimentation and is within the judgment of the clinician. For purposes of the present invention, an effective dose will generally be from about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNA constructs in the individual to which it is administered.
A pharmaceutical composition can also contain a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which can be administered without undue toxicity. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in therapeutic compositions can include liquids such as water, saline, glycerol and ethanol. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, can also be present in such vehicles.
Typically, the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. Liposomes are included within the definition of a pharmaceutically acceptable carrier. Pharmaceutically acceptable salts can also be present in the pharmaceutical composition, e.g., mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in Remington: The Science and Practice of Pharmacy (1995) Alfonso Gennaro, Lippincott, Williams, & Wilkins.
Delivery Methods
Once formulated, the compositions contemplated by the invention can be (1) administered directly to the subject (e.g., as polynucleotide, polypeptides, small molecule agonists or antagonists, and the like); or (2) delivered ex vivo, to cells derived from the subject (e.g., as in ex vivo gene therapy). Direct delivery of the compositions will generally be accomplished by parenteral injection, e.g., subcutaneously, intraperitoneally, intravenously or intramuscularly, intratumoral or to the interstitial space of a tissue. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal applications, needles, and gene guns or hyposprays. Dosage treatment can be a single dose schedule or a multiple dose schedule.
Methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and described in e.g., International Publication No. WO 93/14778. Examples of cells useful in ex vivo applications include, for example, stem cells, particularly hematopoetic, lymph cells, macrophages, dendritic cells, or tumor cells. Generally, delivery of nucleic acids for both ex vivo and in vitro applications can be accomplished by, for example, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei, all well known in the art.
Once differential expression of a gene corresponding to a polynucleotide described herein has been found to correlate with a proliferative disorder, such as neoplasia, dysplasia, and hyperplasia, the disorder can be amenable to treatment by administration of a therapeutic agent based on the provided polynucleotide, corresponding polypeptide or other corresponding molecule (e.g., antisense, ribozyme, etc.). In other embodiments, the disorder can be amenable to treatment by administration of a small molecule drug that, for example, serves as an inhibitor (antagonist) of the function of the encoded gene product of a gene having increased expression in cancerous cells relative to normal cells or as an agonist for gene products that are decreased in expression in cancerous cells (e.g., to promote the activity of gene products that act as tumor suppressors).
The dose and the means of administration of the inventive pharmaceutical compositions are determined based on the specific qualities of the therapeutic composition, the condition, age, and weight of the patient, the progression of the disease, and other relevant factors. For example, administration of polynucleotide therapeutic composition agents includes local or systemic administration, including injection, oral administration, particle gun or catheterized administration, and topical administration. In general, the therapeutic polynucleotide composition contains an expression construct comprising a promoter operably linked to a polynucleotide of at least 12, 22, 25, 30, or 35 contiguous nt of the polynucleotide disclosed herein. Various methods can be used to administer the therapeutic composition directly to a specific site in the body. For example, a small metastatic lesion is located and the therapeutic composition injected several times in several different locations within the body of the tumor. Alternatively, arteries which serve a tumor are identified, and the therapeutic composition injected into such an artery, in order to deliver the composition directly into the tumor. A tumor that has a necrotic center is aspirated and the composition injected directly into the now empty center of the tumor. The antisense composition is directly administered to the surface of the tumor, for example, by topical application of the composition. X-ray imaging is used to assist in certain of the above delivery methods.
Targeted delivery of therapeutic compositions containing an antisense polynucleotide, subgenomic polynucleotides, or antibodies to specific tissues can also be used. Receptor-mediated DNA delivery techniques are described in, for example, Findeis et al., Trends Biotechnol. (1993) 11:202; Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer (J. A. Wolff, ed.) (1994); Wu et al., J. Biol. Chem. (1988) 263:621; Wu et al., J. Biol. Chem. (1994) 269:542; Zenke et al., Proc. Natl. Acad. Sci. (USA) (1990) 87:3655; Wu et al., J. Biol. Chem. (1991) 266:338. Therapeutic compositions containing a polynucleotide are administered in a range of about 100 ng to about 200 mg of DNA for local administration in a gene therapy protocol. Concentration ranges of about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA can also be used during a gene therapy protocol. Factors such as method of action (e.g., for enhancing or inhibiting levels of the encoded gene product) and efficacy of transformation and expression are considerations that will affect the dosage required for ultimate efficacy of the antisense subgenomic polynucleotides.
The therapeutic polynucleotides and polypeptides of the present invention can be delivered using gene delivery vehicles. The gene delivery vehicle can be of viral or non-viral origin (see generally, Jolly, Cancer Gene Therapy (1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995) 1:185; and Kaplitt, Nature Genetics (1994) 6:148). Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence can be either constitutive or regulated.
Viral-based vectors for delivery of a desired polynucleotide and expression in a desired cell are well known in the art. Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (see, e.g., WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; U.S. Pat. No. 5,219,740; WO 93/11230; WO 93/10218; U.S. Pat. No. 4,777,127; GB Patent No. 2,200,651; EP 0 345 242; and WO 91/02805), alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532), and adeno-associated virus (AAV) vectors (see, e.g., WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655). Administration of DNA linked to killed adenovirus as described in Curiel, Hum. Gene Ther. (1992) 3:147 can also be employed.
Non-viral delivery vehicles and methods can also be employed, including, but not limited to, polycationic condensed DNA linked or unlinked to killed adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992) 3:147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989) 264:16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S. Pat. No. 5,814,482; WO 95/07994; WO 96/17072; WO 95/30763; and WO 97/42338) and nucleic charge neutralization or fusion with cell membranes. Naked DNA can also be employed. Exemplary naked DNA introduction methods are described in WO 90/11092 and U.S. Pat. No. 5,580,859. Liposomes that can act as gene delivery vehicles are described in U.S. Pat. No. 5,422,120; WO 95/13796; WO 94/23697; WO 91/14445; and EP 0524968. Additional approaches are described in Philip, Mol. Cell Biol. (1994) 14:2411, and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.
The sequences disclosed in this patent application were disclosed in several earlier patent applications. The relationship between the SEQ ID NOS in those earlier application and the SEQ ID NOS disclosed herein is shown in Tables 26 and 27.
TABLE 26
|
|
relationship between SEQ ID NOs. this patent application
and SEQ ID NOs of parent patent applications
corresponding
parentSEQ IDs in
parentapplicationSEQ IDs inthis patent
caseno.filing dateparent caseapplication
|
166309/883,152Jun. 15, 20011-127 1-127
1552CON10/165,835Jun. 6, 20021-6128-133
18178WOUS03/15465May 16, 20031-1548134-1681
|
The disclosures of all prior U.S. applications to which the present application claims priority, which includes those U.S. applications referenced in the table above as well as their respective priority applications, are each incorporated herein by referenced in their entireties for all purposes, including the disclosures found in the Sequence Listings, tables, figures and Examples.
TABLE 27
|
|
Lookup table showing corresponding SEQ ID NOS in this
application and parent applications
corresponding
parentparentSEQ ID NO in
SEQ ID NO inapplicationapplicationparent
this applicationdocket noserial noapplication
|
12300-166309/883,1521
22300-166309/883,1522
32300-166309/883,1523
42300-166309/883,1524
52300-166309/883,1525
62300-166309/883,1526
72300-166309/883,1527
82300-166309/883,1528
92300-166309/883,1529
102300-166309/883,15210
112300-166309/883,15211
122300-166309/883,15212
132300-166309/883,15213
142300-166309/883,15214
152300-166309/883,15215
162300-166309/883,15216
172300-166309/883,15217
182300-166309/883,15218
192300-166309/883,15219
202300-166309/883,15220
212300-166309/883,15221
222300-166309/883,15222
232300-166309/883,15223
242300-166309/883,15224
252300-166309/883,15225
262300-166309/883,15226
272300-166309/883,15227
282300-166309/883,15228
292300-166309/883,15229
302300-166309/883,15230
312300-166309/883,15231
322300-166309/883,15232
332300-166309/883,15233
342300-166309/883,15234
352300-166309/883,15235
362300-166309/883,15236
372300-166309/883,15237
382300-166309/883,15238
392300-166309/883,15239
402300-166309/883,15240
412300-166309/883,15241
422300-166309/883,15242
432300-166309/883,15243
442300-166309/883,15244
452300-166309/883,15245
462300-166309/883,15246
472300-166309/883,15247
482300-166309/883,15248
492300-166309/883,15249
502300-166309/883,15250
512300-166309/883,15251
522300-166309/883,15252
532300-166309/883,15253
542300-166309/883,15254
552300-166309/883,15255
562300-166309/883,15256
572300-166309/883,15257
582300-166309/883,15258
592300-166309/883,15259
602300-166309/883,15260
612300-166309/883,15261
622300-166309/883,15262
632300-166309/883,15263
642300-166309/883,15264
652300-166309/883,15265
662300-166309/883,15266
672300-166309/883,15267
682300-166309/883,15268
692300-166309/883,15269
702300-166309/883,15270
712300-166309/883,15271
722300-166309/883,15272
732300-166309/883,15273
742300-166309/883,15274
752300-166309/883,15275
762300-166309/883,15276
772300-166309/883,15277
782300-166309/883,15278
792300-166309/883,15279
802300-166309/883,15280
812300-166309/883,15281
822300-166309/883,15282
832300-166309/883,15283
842300-166309/883,15284
852300-166309/883,15285
862300-166309/883,15286
872300-166309/883,15287
882300-166309/883,15288
892300-166309/883,15289
902300-166309/883,15290
912300-166309/883,15291
922300-166309/883,15292
932300-166309/883,15293
942300-166309/883,15294
952300-166309/883,15295
962300-166309/883,15296
972300-166309/883,15297
982300-166309/883,15298
992300-166309/883,15299
1002300-166309/883,152100
1012300-166309/883,152101
1022300-166309/883,152102
1032300-166309/883,152103
1042300-166309/883,152104
1052300-166309/883,152105
1062300-166309/883,152106
1072300-166309/883,152107
1082300-166309/883,152108
1092300-166309/883,152109
1102300-166309/883,152110
1112300-166309/883,152111
1122300-166309/883,152112
1132300-166309/883,152113
1142300-166309/883,152114
1152300-166309/883,152115
1162300-166309/883,152116
1172300-166309/883,152117
1182300-166309/883,152118
1192300-166309/883,152119
1202300-166309/883,152120
1212300-166309/883,152121
1222300-166309/883,152122
1232300-166309/883,152123
1242300-166309/883,152124
1252300-166309/883,152125
1262300-166309/883,152126
1272300-166309/883,152127
1282300-1552CON10/165,8351
1292300-1552CON10/165,8352
1302300-1552CON10/165,8353
1312300-1552CON10/165,8354
1322300-1552CON10/165,8355
1332300-1552CON10/165,8356
1342300-18178WOUS03/154651
1352300-18178WOUS03/154652
1362300-18178WOUS03/154653
1372300-18178WOUS03/154654
1382300-18178WOUS03/154655
1392300-18178WOUS03/154656
1402300-18178WOUS03/154657
1412300-18178WOUS03/154658
1422300-18178WOUS03/154659
1432300-18178WOUS03/1546510
1442300-18178WOUS03/1546511
1452300-18178WOUS03/1546512
1462300-18178WOUS03/1546513
1472300-18178WOUS03/1546514
1482300-18178WOUS03/1546515
1492300-18178WOUS03/1546516
1502300-18178WOUS03/1546517
1512300-18178WOUS03/1546518
1522300-18178WOUS03/1546519
1532300-18178WOUS03/1546520
1542300-18178WOUS03/1546521
1552300-18178WOUS03/1546522
1562300-18178WOUS03/1546523
1572300-18178WOUS03/1546524
1582300-18178WOUS03/1546525
1592300-18178WOUS03/1546526
1602300-18178WOUS03/1546527
1612300-18178WOUS03/1546528
1622300-18178WOUS03/1546529
1632300-18178WOUS03/1546530
1642300-18178WOUS03/1546531
1652300-18178WOUS03/1546532
1662300-18178WOUS03/1546533
1672300-18178WOUS03/1546534
1682300-18178WOUS03/1546535
1692300-18178WOUS03/1546536
1702300-18178WOUS03/1546537
1712300-18178WOUS03/1546538
1722300-18178WOUS03/1546539
1732300-18178WOUS03/1546540
1742300-18178WOUS03/1546541
1752300-18178WOUS03/1546542
1762300-18178WOUS03/1546543
1772300-18178WOUS03/1546544
1782300-18178WOUS03/1546545
1792300-18178WOUS03/1546546
1802300-18178WOUS03/1546547
1812300-18178WOUS03/1546548
1822300-18178WOUS03/1546549
1832300-18178WOUS03/1546550
1842300-18178WOUS03/1546551
1852300-18178WOUS03/1546552
1862300-18178WOUS03/1546553
1872300-18178WOUS03/1546554
1882300-18178WOUS03/1546555
1892300-18178WOUS03/1546556
1902300-18178WOUS03/1546557
1912300-18178WOUS03/1546558
1922300-18178WOUS03/1546559
1932300-18178WOUS03/1546560
1942300-18178WOUS03/1546561
1952300-18178WOUS03/1546562
1962300-18178WOUS03/1546563
1972300-18178WOUS03/1546564
1982300-18178WOUS03/1546565
1992300-18178WOUS03/1546566
2002300-18178WOUS03/1546567
2012300-18178WOUS03/1546568
2022300-18178WOUS03/1546569
2032300-18178WOUS03/1546570
2042300-18178WOUS03/1546571
2052300-18178WOUS03/1546572
2062300-18178WOUS03/1546573
2072300-18178WOUS03/1546574
2082300-18178WOUS03/1546575
2092300-18178WOUS03/1546576
2102300-18178WOUS03/1546577
2112300-18178WOUS03/1546578
2122300-18178WOUS03/1546579
2132300-18178WOUS03/1546580
2142300-18178WOUS03/1546581
2152300-18178WOUS03/1546582
2162300-18178WOUS03/1546583
2172300-18178WOUS03/1546584
2182300-18178WOUS03/1546585
2192300-18178WOUS03/1546586
2202300-18178WOUS03/1546587
2212300-18178WOUS03/1546588
2222300-18178WOUS03/1546589
2232300-18178WOUS03/1546590
2242300-18178WOUS03/1546591
2252300-18178WOUS03/1546592
2262300-18178WOUS03/1546593
2272300-18178WOUS03/1546594
2282300-18178WOUS03/1546595
2292300-18178WOUS03/1546596
2302300-18178WOUS03/1546597
2312300-18178WOUS03/1546598
2322300-18178WOUS03/1546599
2332300-18178WOUS03/15465100
2342300-18178WOUS03/15465101
2352300-18178WOUS03/15465102
2362300-18178WOUS03/15465103
2372300-18178WOUS03/15465104
2382300-18178WOUS03/15465105
2392300-18178WOUS03/15465106
2402300-18178WOUS03/15465107
2412300-18178WOUS03/15465108
2422300-18178WOUS03/15465109
2432300-18178WOUS03/15465110
2442300-18178WOUS03/15465111
2452300-18178WOUS03/15465112
2462300-18178WOUS03/15465113
2472300-18178WOUS03/15465114
2482300-18178WOUS03/15465115
2492300-18178WOUS03/15465116
2502300-18178WOUS03/15465117
2512300-18178WOUS03/15465118
2522300-18178WOUS03/15465119
2532300-18178WOUS03/15465120
2542300-18178WOUS03/15465121
2552300-18178WOUS03/15465122
2562300-18178WOUS03/15465123
2572300-18178WOUS03/15465124
2582300-18178WOUS03/15465125
2592300-18178WOUS03/15465126
2602300-18178WOUS03/15465127
2612300-18178WOUS03/15465128
2622300-18178WOUS03/15465129
2632300-18178WOUS03/15465130
2642300-18178WOUS03/15465131
2652300-18178WOUS03/15465132
2662300-18178WOUS03/15465133
2672300-18178WOUS03/15465134
2682300-18178WOUS03/15465135
2692300-18178WOUS03/15465136
2702300-18178WOUS03/15465137
2712300-18178WOUS03/15465138
2722300-18178WOUS03/15465139
2732300-18178WOUS03/15465140
2742300-18178WOUS03/15465141
2752300-18178WOUS03/15465142
2762300-18178WOUS03/15465143
2772300-18178WOUS03/15465144
2782300-18178WOUS03/15465145
2792300-18178WOUS03/15465146
2802300-18178WOUS03/15465147
2812300-18178WOUS03/15465148
2822300-18178WOUS03/15465149
2832300-18178WOUS03/15465150
2842300-18178WOUS03/15465151
2852300-18178WOUS03/15465152
2862300-18178WOUS03/15465153
2872300-18178WOUS03/15465154
2882300-18178WOUS03/15465155
2892300-18178WOUS03/15465156
2902300-18178WOUS03/15465157
2912300-18178WOUS03/15465158
2922300-18178WOUS03/15465159
2932300-18178WOUS03/15465160
2942300-18178WOUS03/15465161
2952300-18178WOUS03/15465162
2962300-18178WOUS03/15465163
2972300-18178WOUS03/15465164
2982300-18178WOUS03/15465165
2992300-18178WOUS03/15465166
3002300-18178WOUS03/15465167
3012300-18178WOUS03/15465168
3022300-18178WOUS03/15465169
3032300-18178WOUS03/15465170
3042300-18178WOUS03/15465171
3052300-18178WOUS03/15465172
3062300-18178WOUS03/15465173
3072300-18178WOUS03/15465174
3082300-18178WOUS03/15465175
3092300-18178WOUS03/15465176
3102300-18178WOUS03/15465177
3112300-18178WOUS03/15465178
3122300-18178WOUS03/15465179
3132300-18178WOUS03/15465180
3142300-18178WOUS03/15465181
3152300-18178WOUS03/15465182
3162300-18178WOUS03/15465183
3172300-18178WOUS03/15465184
3182300-18178WOUS03/15465185
3192300-18178WOUS03/15465186
3202300-18178WOUS03/15465187
3212300-18178WOUS03/15465188
3222300-18178WOUS03/15465189
3232300-18178WOUS03/15465190
3242300-18178WOUS03/15465191
3252300-18178WOUS03/15465192
3262300-18178WOUS03/15465193
3272300-18178WOUS03/15465194
3282300-18178WOUS03/15465195
3292300-18178WOUS03/15465196
3302300-18178WOUS03/15465197
3312300-18178WOUS03/15465198
3322300-18178WOUS03/15465199
3332300-18178WOUS03/15465200
3342300-18178WOUS03/15465201
3352300-18178WOUS03/15465202
3362300-18178WOUS03/15465203
3372300-18178WOUS03/15465204
3382300-18178WOUS03/15465205
3392300-18178WOUS03/15465206
3402300-18178WOUS03/15465207
3412300-18178WOUS03/15465208
3422300-18178WOUS03/15465209
3432300-18178WOUS03/15465210
3442300-18178WOUS03/15465211
3452300-18178WOUS03/15465212
3462300-18178WOUS03/15465213
3472300-18178WOUS03/15465214
3482300-18178WOUS03/15465215
3492300-18178WOUS03/15465216
3502300-18178WOUS03/15465217
3512300-18178WOUS03/15465218
3522300-18178WOUS03/15465219
3532300-18178WOUS03/15465220
3542300-18178WOUS03/15465221
3552300-18178WOUS03/15465222
3562300-18178WOUS03/15465223
3572300-18178WOUS03/15465224
3582300-18178WOUS03/15465225
3592300-18178WOUS03/15465226
3602300-18178WOUS03/15465227
3612300-18178WOUS03/15465228
3622300-18178WOUS03/15465229
3632300-18178WOUS03/15465230
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14122300-18178WOUS03/154651279
14132300-18178WOUS03/154651280
14142300-18178WOUS03/154651281
14152300-18178WOUS03/154651282
14162300-18178WOUS03/154651283
14172300-18178WOUS03/154651284
14182300-18178WOUS03/154651285
14192300-18178WOUS03/154651286
14202300-18178WOUS03/154651287
14212300-18178WOUS03/154651288
14222300-18178WOUS03/154651289
14232300-18178WOUS03/154651290
14242300-18178WOUS03/154651291
14252300-18178WOUS03/154651292
14262300-18178WOUS03/154651293
14272300-18178WOUS03/154651294
14282300-18178WOUS03/154651295
14292300-18178WOUS03/154651296
14302300-18178WOUS03/154651297
14312300-18178WOUS03/154651298
14322300-18178WOUS03/154651299
14332300-18178WOUS03/154651300
14342300-18178WOUS03/154651301
14352300-18178WOUS03/154651302
14362300-18178WOUS03/154651303
14372300-18178WOUS03/154651304
14382300-18178WOUS03/154651305
14392300-18178WOUS03/154651306
14402300-18178WOUS03/154651307
14412300-18178WOUS03/154651308
14422300-18178WOUS03/154651309
14432300-18178WOUS03/154651310
14442300-18178WOUS03/154651311
14452300-18178WOUS03/154651312
14462300-18178WOUS03/154651313
14472300-18178WOUS03/154651314
14482300-18178WOUS03/154651315
14492300-18178WOUS03/154651316
14502300-18178WOUS03/154651317
14512300-18178WOUS03/154651318
14522300-18178WOUS03/154651319
14532300-18178WOUS03/154651320
14542300-18178WOUS03/154651321
14552300-18178WOUS03/154651322
14562300-18178WOUS03/154651323
14572300-18178WOUS03/154651324
14582300-18178WOUS03/154651325
14592300-18178WOUS03/154651326
14602300-18178WOUS03/154651327
14612300-18178WOUS03/154651328
14622300-18178WOUS03/154651329
14632300-18178WOUS03/154651330
14642300-18178WOUS03/154651331
14652300-18178WOUS03/154651332
14662300-18178WOUS03/154651333
14672300-18178WOUS03/154651334
14682300-18178WOUS03/154651335
14692300-18178WOUS03/154651336
14702300-18178WOUS03/154651337
14712300-18178WOUS03/154651338
14722300-18178WOUS03/154651339
14732300-18178WOUS03/154651340
14742300-18178WOUS03/154651341
14752300-18178WOUS03/154651342
14762300-18178WOUS03/154651343
14772300-18178WOUS03/154651344
14782300-18178WOUS03/154651345
14792300-18178WOUS03/154651346
14802300-18178WOUS03/154651347
14812300-18178WOUS03/154651348
14822300-18178WOUS03/154651349
14832300-18178WOUS03/154651350
14842300-18178WOUS03/154651351
14852300-18178WOUS03/154651352
14862300-18178WOUS03/154651353
14872300-18178WOUS03/154651354
14882300-18178WOUS03/154651355
14892300-18178WOUS03/154651356
14902300-18178WOUS03/154651357
14912300-18178WOUS03/154651358
14922300-18178WOUS03/154651359
14932300-18178WOUS03/154651360
14942300-18178WOUS03/154651361
14952300-18178WOUS03/154651362
14962300-18178WOUS03/154651363
14972300-18178WOUS03/154651364
14982300-18178WOUS03/154651365
14992300-18178WOUS03/154651366
15002300-18178WOUS03/154651367
15012300-18178WOUS03/154651368
15022300-18178WOUS03/154651369
15032300-18178WOUS03/154651370
15042300-18178WOUS03/154651371
15052300-18178WOUS03/154651372
15062300-18178WOUS03/154651373
15072300-18178WOUS03/154651374
15082300-18178WOUS03/154651375
15092300-18178WOUS03/154651376
15102300-18178WOUS03/154651377
15112300-18178WOUS03/154651378
15122300-18178WOUS03/154651379
15132300-18178WOUS03/154651380
15142300-18178WOUS03/154651381
15152300-18178WOUS03/154651382
15162300-18178WOUS03/154651383
15172300-18178WOUS03/154651384
15182300-18178WOUS03/154651385
15192300-18178WOUS03/154651386
15202300-18178WOUS03/154651387
15212300-18178WOUS03/154651388
15222300-18178WOUS03/154651389
15232300-18178WOUS03/154651390
15242300-18178WOUS03/154651391
15252300-18178WOUS03/154651392
15262300-18178WOUS03/154651393
15272300-18178WOUS03/154651394
15282300-18178WOUS03/154651395
15292300-18178WOUS03/154651396
15302300-18178WOUS03/154651397
15312300-18178WOUS03/154651398
15322300-18178WOUS03/154651399
15332300-18178WOUS03/154651400
15342300-18178WOUS03/154651401
15352300-18178WOUS03/154651402
15362300-18178WOUS03/154651403
15372300-18178WOUS03/154651404
15382300-18178WOUS03/154651405
15392300-18178WOUS03/154651406
15402300-18178WOUS03/154651407
15412300-18178WOUS03/154651408
15422300-18178WOUS03/154651409
15432300-18178WOUS03/154651410
15442300-18178WOUS03/154651411
15452300-18178WOUS03/154651412
15462300-18178WOUS03/154651413
15472300-18178WOUS03/154651414
15482300-18178WOUS03/154651415
15492300-18178WOUS03/154651416
15502300-18178WOUS03/154651417
15512300-18178WOUS03/154651418
15522300-18178WOUS03/154651419
15532300-18178WOUS03/154651420
15542300-18178WOUS03/154651421
15552300-18178WOUS03/154651422
15562300-18178WOUS03/154651423
15572300-18178WOUS03/154651424
15582300-18178WOUS03/154651425
15592300-18178WOUS03/154651426
15602300-18178WOUS03/154651427
15612300-18178WOUS03/154651428
15622300-18178WOUS03/154651429
15632300-18178WOUS03/154651430
15642300-18178WOUS03/154651431
15652300-18178WOUS03/154651432
15662300-18178WOUS03/154651433
15672300-18178WOUS03/154651434
15682300-18178WOUS03/154651435
15692300-18178WOUS03/154651436
15702300-18178WOUS03/154651437
15712300-18178WOUS03/154651438
15722300-18178WOUS03/154651439
15732300-18178WOUS03/154651440
15742300-18178WOUS03/154651441
15752300-18178WOUS03/154651442
15762300-18178WOUS03/154651443
15772300-18178WOUS03/154651444
15782300-18178WOUS03/154651445
15792300-18178WOUS03/154651446
15802300-18178WOUS03/154651447
15812300-18178WOUS03/154651448
15822300-18178WOUS03/154651449
15832300-18178WOUS03/154651450
15842300-18178WOUS03/154651451
15852300-18178WOUS03/154651452
15862300-18178WOUS03/154651453
15872300-18178WOUS03/154651454
15882300-18178WOUS03/154651455
15892300-18178WOUS03/154651456
15902300-18178WOUS03/154651457
15912300-18178WOUS03/154651458
15922300-18178WOUS03/154651459
15932300-18178WOUS03/154651460
15942300-18178WOUS03/154651461
15952300-18178WOUS03/154651462
15962300-18178WOUS03/154651463
15972300-18178WOUS03/154651464
15982300-18178WOUS03/154651465
15992300-18178WOUS03/154651466
16002300-18178WOUS03/154651467
16012300-18178WOUS03/154651468
16022300-18178WOUS03/154651469
16032300-18178WOUS03/154651470
16042300-18178WOUS03/154651471
16052300-18178WOUS03/154651472
16062300-18178WOUS03/154651473
16072300-18178WOUS03/154651474
16082300-18178WOUS03/154651475
16092300-18178WOUS03/154651476
16102300-18178WOUS03/154651477
16112300-18178WOUS03/154651478
16122300-18178WOUS03/154651479
16132300-18178WOUS03/154651480
16142300-18178WOUS03/154651481
16152300-18178WOUS03/154651482
16162300-18178WOUS03/154651483
16172300-18178WOUS03/154651484
16182300-18178WOUS03/154651485
16192300-18178WOUS03/154651486
16202300-18178WOUS03/154651487
16212300-18178WOUS03/154651488
16222300-18178WOUS03/154651489
16232300-18178WOUS03/154651490
16242300-18178WOUS03/154651491
16252300-18178WOUS03/154651492
16262300-18178WOUS03/154651493
16272300-18178WOUS03/154651494
16282300-18178WOUS03/154651495
16292300-18178WOUS03/154651496
16302300-18178WOUS03/154651497
16312300-18178WOUS03/154651498
16322300-18178WOUS03/154651499
16332300-18178WOUS03/154651500
16342300-18178WOUS03/154651501
16352300-18178WOUS03/154651502
16362300-18178WOUS03/154651503
16372300-18178WOUS03/154651504
16382300-18178WOUS03/154651505
16392300-18178WOUS03/154651506
16402300-18178WOUS03/154651507
16412300-18178WOUS03/154651508
16422300-18178WOUS03/154651509
16432300-18178WOUS03/154651510
16442300-18178WOUS03/154651511
16452300-18178WOUS03/154651512
16462300-18178WOUS03/154651513
16472300-18178WOUS03/154651514
16482300-18178WOUS03/154651515
16492300-18178WOUS03/154651516
16502300-18178WOUS03/154651517
16512300-18178WOUS03/154651518
16522300-18178WOUS03/154651519
16532300-18178WOUS03/154651520
16542300-18178WOUS03/154651521
16552300-18178WOUS03/154651522
16562300-18178WOUS03/154651523
16572300-18178WOUS03/154651524
16582300-18178WOUS03/154651525
16592300-18178WOUS03/154651526
16602300-18178WOUS03/154651527
16612300-18178WOUS03/154651528
16622300-18178WOUS03/154651529
16632300-18178WOUS03/154651530
16642300-18178WOUS03/154651531
16652300-18178WOUS03/154651532
16662300-18178WOUS03/154651533
16672300-18178WOUS03/154651534
16682300-18178WOUS03/154651535
16692300-18178WOUS03/154651536
16702300-18178WOUS03/154651537
16712300-18178WOUS03/154651538
16722300-18178WOUS03/154651539
16732300-18178WOUS03/154651540
16742300-18178WOUS03/154651541
16752300-18178WOUS03/154651542
16762300-18178WOUS03/154651543
16772300-18178WOUS03/154651544
16782300-18178WOUS03/154651545
16792300-18178WOUS03/154651546
16802300-18178WOUS03/154651547
16812300-18178WOUS03/154651548
|
EXAMPLES
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
Example 1
Source of Biological Materials and Overview of Polynucleotides Expressed by the Biological Materials
In order to identify genes that are differentially expressed in colon cancer, cDNA libraries were prepared from several different cell lines and tissue sources. Table 1 provides a summary of these libraries, including the shortened library name (used hereafter), the mRNA source used to prepared the cDNA library, the “nickname” of the library that is used in the tables below (in quotes), and the approximate number of clones in the library. cDNA libraries were prepared according to methods well known in the art, and the sequences of the cDNA inserts were determined using well known methods.
TABLE 1
|
|
Description of cDNA Libraries
Number
of
LibraryDescriptionClones
|
1Human Colon Cell Line Km12 L4: High Metastatic308731
Potential (derived from Km12C)
2Human Colon Cell Line Km12C: Low Metastatic284771
Potential
3Human Breast Cancer Cell Line MDA-MB-231: High326937
Metastatic Potential; micromets in lung
4Human Breast Cancer Cell Line MCF7: Non-318979
Metastatic
8Human Lung Cancer Cell Line MV-522: High223620
Metastatic Potential
9Human Lung Cancer Cell Line UCP-3: Low312503
Metastatic Potential
12Human microvascular endothelial cells (HMEC) -41938
UNTREATED (PCR (OligodT) cDNA library)
13Human microvascular endothelial cells (HMEC) -42100
bFGF TREATED (PCR (OligodT) cDNA library)
14Human microvascular endothelial cells (HMEC) -42825
VEGF TREATED (PCR (OligodT) cDNA library)
15Normal Colon - UC#2 Patient (MICRODISSECTED282718
PCR (OligodT) cDNA library)
16Colon Tumor - UC#2 Patient (MICRODISSECTED298829
PCR (OligodT) cDNA library)
17Liver Metastasis from Colon Tumor of UC#2 Patient303462
(MICRODISSECTED PCR (OligodT) cDNA library)
18Normal Colon - UC#3 Patient (MICRODISSECTED36216
PCR (OligodT) cDNA library)
19Colon Tumor - UC#3 Patient (MICRODISSECTED41388
PCR (OligodT) cDNA library)
20Liver Metastasis from Colon Tumor of UC#3 Patient30956
(MICRODISSECTED PCR (OligodT) cDNA library)
21GRRpz Cells derived from normal prostate epithelium164801
22WOca Cells derived from Gleason Grade 4 prostate162088
cancer epithelium
23Normal Lung Epithelium of Patient #1006306198
(MICRODISSECTED PCR (OligodT) cDNA library)
24Primary tumor, Large Cell Carcinoma of Patient309349
#1006 (MICRODISSECTED PCR (OligodT) cDNA
library)
25Normal Prostate Epithelium from Patient 1F97-26811279437
26Prostate Cancer Epithelium Gleason 3 + 3 Patient269366
IF97-26811
|
The KM12L4 cell line is derived from the KM12C cell line (Morikawa, et al., Cancer Research (1988) 48:6863). The KM12C cell line, which is poorly metastatic (low metastatic) was established in culture from a Dukes' stage B2 surgical specimen (Morikawa et al. Cancer Res. (1988) 48:6863). The KML4-A is a highly metastatic subline derived from KM12C (Yeatman et al. Nucl. Acids. Res. (1995) 23:4007; Bao-Ling et al. Proc. Annu. Meet. Am. Assoc. Cancer. Res. (1995) 21:3269). The KM12C and KM12C-derived cell lines (e.g., KM12L4, KM12L4-A, etc.) are well-recognized in the art as a model cell line for the study of colon cancer (see, e.g., Moriakawa et al., supra; Radinsky et al. Clin. Cancer Res. (1995) 1:19; Yeatman et al., (1995) supra; Yeatman et al. Clin. Exp. Metastasis (1996) 14:246).
The MDA-MB-231 cell line was originally isolated from pleural effusions (Cailleau, J. Natl. Cancer. Inst. (1974) 53:661), is of high metastatic potential, and forms poorly differentiated adenocarcinoma grade II in nude mice consistent with breast carcinoma. The MCF7 cell line was derived from a pleural effusion of a breast adenocarcinoma and is non-metastatic. These cell lines are well-recognized in the art as models for the study of human breast and lung cancer (see, e.g., Chandrasekaran et al., Cancer Res. (1979) 39:870; Gastpar et al., J Med Chem (1998) 41:4965; Ranson et al., Br J Cancer (1998) 77:1586; Kuang et al., Nucleic Acids Res (1998) 26:1116. The samples of libraries 15-20 are derived from two different patients (UC#2 and UC#3). The GRRpz and WOca cell lines were provided by Dr. Donna M. Peehl, Department of Medicine, Stanford University School of Medicine. GRRpz was derived from normal prostate epithelium. The WOca cell line is a Gleason Grade 4 cell line.
Each of the libraries is composed of a collection of cDNA clones that in turn are representative of the mRNAs expressed in the indicated mRNA source. In order to facilitate the analysis of the millions of sequences in each library, the sequences were assigned to clusters. The concept of “cluster of clones” is derived from a sorting/grouping of cDNA clones based on their hybridization pattern to a panel of roughly 300 7 bp oligonucleotide probes (see Drmanac et al., Genomics (1996) 37(1):29). Random cDNA clones from a tissue library are hybridized at moderate stringency to 300 7 bp oligonucleotides. Each oligonucleotide has some measure of specific hybridization to that specific clone. The combination of 300 of these measures of hybridization for 300 probes equals the “hybridization signature” for a specific clone. Clones with similar sequence will have similar hybridization signatures. By developing a sorting/grouping algorithm to analyze these signatures, groups of clones in a library can be identified and brought together computationally. These groups of clones are termed “clusters”.
Depending on the stringency of the selection in the algorithm (similar to the stringency of hybridization in a classic library cDNA screening protocol), the “purity” of each cluster can be controlled. For example, artifacts of clustering may occur in computational clustering just as artifacts can occur in “wet-lab” screening of a cDNA library with 400 bp cDNA fragments, at even the highest stringency. The stringency used in the implementation of cluster herein provides groups of clones that are in general from the same cDNA or closely related cDNAs. Closely related clones can be a result of different length clones of the same cDNA, closely related clones from highly related gene families, or splice variants of the same cDNA.
Differential expression for a selected cluster was assessed by first determining the number of cDNA clones corresponding to the selected cluster in the first library (Clones in 1st), and the determining the number of cDNA clones corresponding to the selected cluster in the second library (Clones in 2nd). Differential expression of the selected cluster in the first library relative to the second library is expressed as a “ratio” of percent expression between the two libraries. In general, the “ratio” is calculated by: 1) calculating the percent expression of the selected cluster in the first library by dividing the number of clones corresponding to a selected cluster in the first library by the total number of clones analyzed from the first library; 2) calculating the percent expression of the selected cluster in the second library by dividing the number of clones corresponding to a selected cluster in a second library by the total number of clones analyzed from the second library; 3) dividing the calculated percent expression from the first library by the calculated percent expression from the second library. If the “number of clones” corresponding to a selected cluster in a library is zero, the value is set at 1 to aid in calculation. The formula used in calculating the ratio takes into account the “depth” of each of the libraries being compared, i.e., the total number of clones analyzed in each library.
As a result of this library comparison, 17 polynucleotides, listed as SEQ ID NOS:1, 3, 5, 7, 9, 11-13, 15, 16, 18, 20, 22, 24, 26, 27 and 29 in the accompanying Sequence Listing and summarized in Table 2, were identified as corresponding to genes differentially expressed in colon cancer patient tissues. Table 2 provides: 1) the sequence identification number (“SEQ ID NO of polynucleotide”) assigned to each sequence for use in the present specification; 2) the cluster identification number (“CLUSTER”); 3) the Candidation Idnetification number; 4) ththe CHIR number (which serves as tha cross-reference to antisense oligos discussed below), with, for examplek CHIR7 having corresponding oligos CHIR7-2AS (antibsense) and CHIR7-RC (reverse control); 5) the sequence name (“SEQ NAME”) used as an internal identifier of the sequence; 6) the name assigned to the clone from which the sequence was isolated (“CLONE ID”); 7) the first nucleotide of the start and stop codons of identified open reading frames (“ORF start” and “ORF stop”); and 8) the sequence identification number (“SEQ ID NO of encoded polypeptide”) assigned to the encoded polypeptide, where appropriate. Because the provided polynucleotides represent partial mRNA transcripts, two or more polynucleotides of the invention may represent different regions of the same mRNA transcript and the same gene. Thus, if two or more sequences are identified as belonging to the same clone, then either sequence can be used to obtain the full-length mRNA or gene.
TABLE 2
|
|
Polynucleotide sequence identificaton and characterization
SEQ ID NO
SEQCandidateSEQORFof encoded
BID NOCLUSTERIDCHIRNAMEstartstoppolypeptide
|
1719196CHIR-7SK1213962
39083181CHIR-8SK22196934
5115762188CHIR-16SK5517606
71665195CHIR-91665 long786428
91665195CHIR-91665 short7923210
112334SK8 partial
122334SK8 full
length
133376118CHIR-11SK197937614
15376130Junc2181, 363,361, 542, 911
731
16402380202CHIR-33XD41653817
18726682198CHIR-43XD1255119
20552930174CHIR-42XD724058521
22454001161CHIR-29XD1053170023
24378805163CHIR-31XD111040025
26374641160CHIR-32374641 long33, 420183, 615
(Junc4)
27374641160CHIR-32374641 short32451928
(XD6)
29374641160CHIR-3237464140, 388190, 583
electronic
|
Table 3 summarizes polynucleotides that correspond to genes differentially expressed in colon tissue from a single patient.
TABLE 3
|
|
SEQNormalTumorHigh MetTumor/High Met/High Met/
ID(Lib15)(Lib16)(Lib17)NormalNormalTumor
NOCLUSTERClonesClonesClones(Lib16/Lib15)(Lib17/Lib15)(Lib17/Lib16)
|
|
17190202720271
390830101410141
5115762067671
71665414203.551
122334061610
13337632019761
1537613009159152
1640238001521520
1872668205205200
2055293011421420
2245400108138132
243788051121212121
263746419471295143
|
Example 2
Analysis and Characterization of Polynucleotides of the Invention
Several of the provided polynucleotides contain one or more putative open reading frames (ORFs) encoding a gene product. The start and stop sites for these ORFs are listed in Table 2.
SEQ ID NO:15 contains three ORFs. The first ORF extends from nucleotide 181 to nucleotide 361. The second ORF extends from nucleotide 363 to nucleotide 542. The third ORF extends from nucleotide 731 to nucleotide 911.
SEQ ID NO:26 contains a 39-nucleotide insertion sequence (from nucleotide 269 to nucleotide 307) and two ORFs. The first ORF extends from nucleotide 33 to nucleotide 183. The second ORF extends from nucleotide 420 to nucleotide 615.
SEQ ID NO:29 is an electronic sequence according to the 5′-RACE result and contains two ORFs. The first ORF extends from nucleotide 40 to nucleotide 190. The second ORF extends from nucleotide 388 to nucleotide 583.
Example 3
Members of Protein Families
Translations of the provided polynucleotides were aligned with amino acid profiles that define either protein families or common motifs. Several of the polynucleotides of the invention were found to encode polypeptides having characteristics of a polypeptide belonging to a known protein family (and thus represent new members of these protein families) and/or comprising a known functional domain. Similarity between a query sequence and a protein family or motif was determined by (a) comparing the query sequence against the profile and/or (b) aligning the query sequence with the members of the family or motif.
Each of the profile hits is described in more detail below. Table 4 provides the corresponding SEQ ID NO of the provided polynucleotides that encode gene products with similarity or identity to the profile sequences. Similarity (strong or weak) is also noted in Table 4. The acronyms for the profiles (provided in parentheses) are those used to identify the profile in the Pfam and Prosite databases. The Pfam database can be accessed through any of the following URLS: http://pfam.wustl.edu/index.html; http://www.sanger.ac.uk/Software/Pfam/; and http://www.cgr.ki.se/Pfam/. The Prosite database can be accessed at http://www.expasy.ch/prosite/. The public information available on the Pfam and Prosite databases regarding the various profiles, including but not limited to the activities, function, and consensus sequences of various proteinss families and protein domains, is incorporated herein by reference.
TABLE 4
|
|
Profile hits.
SEQ
ID NOCLUSTERProfileDescriptionSimilarity
|
1719Glycosyl hydrolaseweak
39083ANKAnkyrin repeatsstrong
51157627tm_17 transmembrane receptorweak
(rhodopsin family)
112334EFhandEF-handstrong
122334EfhandEF-handstrong
15376130Endogenous retrograde
protease/integrase
16402380RrmRNA recognition motif.
(aka RRM, RBD,
or RNP domain)
|
Glycosyl hydrolase family 5 (GLYCOSYL_HYDROL_F5; Pfam Accession No. PS00659; PDOC00565). SEQ ID NO:1 corresponds to a gene encoding a polypeptide having homology to polypeptides of the glycosyl hydrolase family 5 (Henrissat Biochem. J. (1991) 280:309-316) (also known as the cellulase family A (Henrissat et al. Gene (1989) 81:83-95)). The members of this family participate in the degradation of cellulose and xylans, and are generally found in bacteria, fungi, and yeast. The consensus pattern for members of this family is: [LIV]-[LIVMFYWGA](2)-[DNEQG]-[LIVMGST]-x-N-E-[PV]-[RHDNSTLIVFY] (where E is a putative active site residue).
SEQ ID NO:1 corresponds to a gene encoding a member of one of the families of glycosyl hydrolases (Henrissat et al. Biochem. J. (1993) 293:781-788). These enzymes contain at least one conserved glutamic acid residue (or aspartic acid residue) which has been shown to be directly involved in glycosidic bond cleavage by acting as a nucleophile.
Ank Repeats (ANK; Pfam Accession No. PF0023). SEQ ID NO:3 corresponds to a gene encoding an Ank repeat-containing protein. The ankyrin motif is a 33 amino acid sequence named after the protein ankyrin which has 24 tandem 33-amino-acid motifs. Ank repeats were originally identified in the cell-cycle-control protein cdc10 (Breeden et al., Nature (1987) 329:651). Proteins containing ankyrin repeats include ankyrin, myotropin, I-kappaB proteins, cell cycle protein cdc10, the Notch receptor (Matsuno et al., Development (1997) 124(21):4265); G9a (or BAT8) of the class III region of the major histocompatibility complex (Biochem J. 290:811-818, 1993), FABP, GABP, 53BP2, Lin12, glp-1, SW14, and SW16. The functions of the ankyrin repeats are compatible with a role in protein-protein interactions (Bork, Proteins (1993) 17(4):363; Lambert and Bennet, Eur. J. Biochem. (1993) 211:1; Kerr et al., Current Op. Cell Biol. (1992) 4:496; Bennet et al., J. Biol. Chem. (1980) 255:6424).
Seven Transmembrane Integral Membrane Proteins—Rhodopsin Family (7tm—1: Pfam Accession No. PF00001). SEQ ID NO:3 corresponds to a gene encoding a polypeptide that is a member of the seven transmembrane (7tm) receptor rhodopsin family. G-protein coupled receptors of the (7tm) rhodopsin family (also called R7G) are an extensive group of hormones, neurotransmitters, and light receptors which transduce extracellular signals by interaction with guanine nucleotide-binding (G) proteins (Strosberg A. D. Eur. J. Biochem. (1991) 196:1, Kerlavage A. R. Curr. Opin. Struct. Biol. (1991) 1:394, Probst, et al., DNA Cell Biol. (1992) 11:1, Savarese, et al., Biochem. J. (1992) 283:1, http://www.gcrdb.uthscsa.edu/, http://swift.embl-heidelberg.de/7tm/. The consensus pattern that contains the conserved triplet and that also spans the major part of the third transmembrane helix is used to detect this widespread family of proteins:
|
[GSTALIVMFYWC]-[GSTANCPDE]-{EDPKRH}-x(2)-
|
[LIVMNQGA]-x(2)-[LIVMFT]-[GSTANC]-[LIVMFYWSTAC]-
|
[DENH]-R-[FYWCSH]-x(2)-[LIVM].
[GSTALIVMFYWC]-[GSTANCPDE]-{EDPKRH}-x(2)-[LIVMNQGA]-x(2)-[LIVMFT]-[GSTANC]-[LIVMFYWSTAC]-[DENH]-R-[FYWCSH]-x(2)-[LIVM].
EF Hand (EFhand: Pfam Accession No. PF00036). SEQ ID NOS:11 and 12 correspond to genes encoding a protein in the family of EF-hand proteins. Many calcium-binding proteins belong to the same evolutionary family and share a type of calcium-binding domain known as the EF-hand (Kawasaki et al., Protein. Prof. (1995) 2:305-490). This type of domain consists of a twelve residue loop flanked on both sides by a twelve residue alpha-helical domain. In an EF-hand loop the calcium ion is coordinated in a pentagonal bipyramidal configuration. The six residues involved in the binding are in positions 1, 3, 5, 7, 9 and 12; these residues are denoted by X, Y, Z, —Y, —X and -Z. The invariant Glu or Asp at position 12 provides two oxygens for liganding Ca (bidentate ligand). The consensus pattern includes the complete EF-hand loop as well as the first residue which follows the loop and which seem to always be hydrophobic: D-x-[DNS]-{ILVFYW}-[DENSTG]-[DNQGHRK]-{GP}-[LIVMC]-[DENQSTAGC]-x(2)-[DE]-[LIVMFYW].
Endogenous retroviral protease/integrase. SEQ ID NO:15 corresponds to a gene encoding a polypeptide having a domain homologous to a human endogenous retrovirus protease/integrase domain of a retroviral pol protein.
RNA Recognition Motif (rrm: Pfam Accession No. PF00076). SEQ ID NO:16 corresponds to a gene encoding an RNA recognition motif, also known as an RRM, RBD, or RNP domain. This domain, which is about 90 amino acids long, is contained in eukaryotic proteins that bind single-stranded RNA (Bandziulis et al. Genes Dev. (1989) 3:431-437; Dreyfuss et al. Trends Biochem. Sci. (1988) 13:86-91). Two regions within the RNA-binding domain are highly conserved: the first is a hydrophobic segment of six residues (which is called the RNP-2 motif), the second is an octapeptide motif (which is called RNP-1 or RNP-CS). The consensus pattern is: [RK]-G-{EDRKHPCG}-[AGSCI]-[FY]-[LIVA]-x-[FYLM].
Example 4
Detection and Quantification of Polynucleotides of the Invention
The polynucleotides of the invention were detected and quantified in patient tissue samples by reverse transcriptase PCR (RT-PCR). Total RNA amplifications were performed using the LightCycler™ thermal cycling system (Roche Diagnostics) in a standard PCR reaction containing the provided primers and the dsDNA-binding dye SYBR Green I. PCR amplifacaiotn was monitored by fluroescence dye SYBR Green I, which fluroesces only when bound to double-stranded DNA. The specific of the products was verified by melting curve analysis.
Standard Preparation. 1 μg human placenta total RNA (Clontech, Palo Alto, Calif.) was reverse-transcribed at 42° C. for 1 hour then heated at 94° C. for 5 minutes in a total reaction volume of 20 μl (1st-Strand™ cDNA Synthesis Kit, Clontech). The reaction mix was used as 1× template standard. Serial dilutions from 1× template standard were then prepared: 10−1×, 10−2×, 10−3×, 10−4×, 10−5×, 10−6× template standards.
Total RNA Sample Preparation. The patient tissue samples were shipped in frozen TRIZOL reagent. The samples were homogenized in TRIZOL reagent. Chloroform was then added to isolate RNA, followed by RNA precipitation with isopropanol. The RNA precipitates were washed with 75% ethanol, dried in air, then dissolved in RNase-free distilled water. Before reverse-transcription, RNA samples were treated with DNase I (RNase-free) (2 U/μl, Ambion, Austin, Tex.) and cleaned up using RNeasy Mini Kit (Qiagen, Santa Clarita, Calif.).
RT-PCR. Total RNA samples were reverse-transcribed with oligo-dT18 primer (1st-Strand™ cDNA Synthesis Kit, Clontech). PCR was performed using the following gene-specific primers:
|
SK1:forward primer5′-AGGAGTTTCTGAGGACCATGCAC-3′(SEQ ID NO:30)
|
reverse primer5′-TCAAGGGTTGGGGATACACACG-3′(SEQ ID NO:31)
|
SK2:forward primer5′-CTTGCTTGCTTTCTTCTCTGGC-3′(SEQ ID NO:32)
|
reverse primer5′-AGTCTGGAAATCCACATGACCAAG-3′(SEQ ID NO:33)
|
SK5:forward primer5′-CCCAATGAGGAACCTAAAGTTGC-3′(SEQ ID NO:34)
|
reverse primer5′-GGTGCCAAATCTGGACTCTTGTC-3′(SEQ ID NO:35)
|
1665:forward primer5′-GATCCATTTTCAGCAGTGCTCTG-3′(SEQ ID NO:36)
|
reverse primer5′-CAGTGTTCACAGAAGGGGTACTCAC-3′(SEQ ID NO:37)
|
SK8:forward primer5′-ACGAGAGCGACACGGACAAG-3′(SEQ ID NO:38)
|
reverse primer5′-TCTGAGGCTGTGGCAGGTGC-3′(SEQ ID NO:39)
|
SK19:forward primer5′-CCAGTCTTTGCCAACTCGTGC-3′(SEQ ID NO:40)
|
reverse primer5′-TTCGATCTTCAAACTGTGCCTTG-3′(SEQ ID NO:41)
|
Junc2:forward primer5′-TTGGCAACCAGACCAGCATC-3′(SEQ ID NO:42)
|
reverse primer5′-TTTCCCATAGGTGTGAGTGGCG-3′(SEQ ID NO:43)
|
XD4:forward primer5′-GACTGGTGTTTTGTTCGGGGTC-3′(SEQ ID NO:44)
|
reverse primer5′-TTTGTCCAAGGCTGCATGGTC-3′(SEQ ID NO:45)
|
XD1:forward primer5′-TGCCCTGGTTAAGCCAGAAGTC-3′(SEQ ID NO:46)
|
reverse primer5′-AGCTTCACTTTGGTCTTGACGG-3′(SEQ ID NO:47)
|
XD7:forward primer5′-GGTCATCTGCATCAAGGTTGGC-3′(SEQ ID NO:48)
|
reverse primer5′-GGTTCGTAACCGTGACTTCAGG-3′(SEQ ID NO:49)
|
XD10:forward primer5′-GCATCCTTTTCCAGTCTTCCG-3′(SEQ ID NO:50)
|
reverse primer5′-TGCAGCAAACATGCCTGAGC-3′(SEQ ID NO:51)
|
XD11:forward primer5′-TGTTCCACGAGCAAAGCATGTG-3′(SEQ ID NO:52)
|
reverse primer5′-ATCCTTCTTCCACTCCCGCTTC-3′(SEQ ID NO:53)
|
37641:forward primer5′-TCGGCTTGACTACACTGTGTGG-3′(SEQ ID NO:54)
|
reverse primer5′-TACAAAGACCACTGGGAGGCTG-3′(SEQ ID NO:55)
|
β-actin:forward primer5′-CGGGAAATCGTGCGTGACATTAAG-3′(SEQ ID NO:56)
|
reverse primer5′-TGATCTCCTTCTGCATCCTGTCGG-3′(SEQ ID NO:57)
|
GAPDH:forward primer5′-TTTGGCTACAGCAACAGGGTG-3′(SEQ ID NO:58)
|
reverse primer5′-TGTGAGGAGGGGAGATTCAGTG-3′(SEQ ID NO:59)
β-actin and GAPDH were used as positive controls. All PCR products are 150-250 bp. The 20-μl PCR reaction mix in each LightCycler™ capillary contained 2 μl of 10×PCR buffer II, 3 mM MgCl2 (Perkin-Elmer, Foster City, Calif.), 140 μM dNTP, 1:50000 of SYBR Green I, 0.25 mg/ml BSA, 1 unit of Taq polymerase (Boehringer Mannheim, Indianapolis, Ind.), 0.175 μM each primer, 2 μl of RT reaction mix. The PCR amplification began with 20-second denaturation at 95° C., followed by 45 cycles of denaturation at 95° C. for 5 seconds, annealing at 60° C. for 1 second and extension at 72° C. for 30 seconds. At the end of final cycle, PCR products were annealed at 60° C. for 5 seconds, then slowly heated to 95° C. at 0.2° C./second, to measure melting curve of specific PCR products. All experiments were performed in duplicate.
Data analysis was performed using LightCycler™ software (Roche Diagnostics) with quantification and melting curve options. Fluorescence is normalized relative to positive and negative controls.
Overexpression of genes in colon cancer patient whole tissue. Results provided in the tables below include fluoresence data for polynucleotides isolated from colon tissue samples that were harvested directly, not microdissected (i.e., whole tissue), and amplified using the indicated primers. Normal, primary tumor and metastatic cell types are denoted as N, PT and Met, respectively. Overexpression was determined by comparing either metastatic cells or primary tumor cells, or both, to normal cells. The results for each gene corresponding to the indicated clusters in each patient sample are summarized in the tables below. All values are adjusted to levels relative to beta-actin control.
|
|
Cluster#719 (SK1): overexpression detected in
4 of 6 patients (67%)
PatientsNPTMET
|
UC#10.0220.1170.364
UC#20.1210.1090.142
UC#40.0830.0530.078
UC#70.0420.1990.145
UC#80.2150.5150.794
UC#90.2330.5850.613
|
|
|
Cluster#9083 (SK2): overexpression inf 3 or 4
|
patients (75%)
|
Patients
N
PT
MET
|
|
UC#1
0.0021
0.0013
0.0078
|
UC#2
0.008
0.012
0.014
|
UC#4
0.0021
0.0022
0.0026
|
UC#7
0.0009
0.0021
0.0039
|
|
|
|
Cluster#115762 (SK5): overexpression in
|
5 of 6 patients (83%)
|
Patients
N
PT
MET
|
|
UC#1
0.0053
0.0159
0.044
|
UC#2
0.0195
0.0174
0.0269
|
UC#4
0.022
0.033
0.034
|
UC#7
0.013
0.028
0.025
|
UC#8
0.0275
0.105
0.143
|
UC#9
0.0336
0.0595
0.0541
|
|
|
|
Cluster#1665: overexpression in 4 of 6
|
patients (67%)
|
Patients
N
PT
MET
|
|
UC#1
0.00006
0.0003
0.002
|
UC#2
0.0015
0.001
0.0012
|
UC#4
0.0016
0.0013
0.0016
|
UC#7
0.00003
0.0003
0.0012
|
UC#8
0.0016
0.0122
0.0154
|
UC#9
0.006
0.057
0.097
|
|
|
|
Cluster#2334 (SK8): overexpression in 4
|
of 6 patients (67%)
|
Patients
N
PT
MET
|
|
UC#1
0.011
0.022
0.017
|
UC#2
0.0266
0.0317
0.026
|
UC#4
0.02
0.006
0.01
|
UC#7
0.046
0.093
0.042
|
UC#8
0.042
0.168
0.472
|
UC#9
0.208
0.322
0.29
|
|
|
|
Cluster#3376 (SK19): overexpression in 4
|
of 6 patients (67%)
|
Patients
N
PT
MET
|
|
UC#1
0.00018
0.00042
0.0012
|
UC#2
0.002
0.0025
0.0016
|
UC#4
0.0013
0.0012
0.002
|
UC#7
0.00024
0.00055
0.00062
|
UC#8
0.0003
0.00127
0.0023
|
UC#9
0.001
0.0075
0.009
|
|
|
|
Cluster#376130 (Junc2): overexpression
|
in 3 of 4 patients (75%)
|
Patients
N
PT
MET
|
|
UC#1
0.00871
0.0111
0.0142
|
UC#2
0.000567
0.00663
0.0163
|
UC#4
0.000107
0.00048
0.000237
|
UC#7
0.0000401
0.000259
0.00159
|
|
|
|
Cluster#402380 (XD4): overexpression in
|
2 of 4 patients (50%)
|
Patients
N
PT
MET
|
|
UC#1
0.0763
0.123
0.2
|
UC#2
0.0867
0.0629
0.069
|
UC#4
0.0735
0.0672
0.0664
|
UC#7
0.0559
0.112
0.139
|
|
|
|
Cluster#726682 (XD1): overexpression
|
in 0 of 4 patients
|
Patients
N
PT
MET
|
|
UC#1
0.0679
0.0822
0.136
|
UC#2
0.175
0.124
0.147
|
UC#4
0.2
0.145
0.145
|
UC#7
0.108
0.144
0.114
|
|
|
|
Cluster#552930 (XD7): overexpression in
|
1 of 4 patients (25%)
|
Patients
N
PT
MET
|
|
UC#1
0.018
0.019
0.0902
|
UC#2
0.204
0.161
0.212
|
UC#4
0.299
0.25
0.238
|
UC#7
0.246
0.409
0.248
|
|
|
|
Cluster#454001 (XD10): overexpression
|
in 2 of 4 patients)
|
Patients
N
PT
MET
|
|
UC#1
0.0197
0.0363
0.0587
|
UC#2
0.0514
0.0451
0.069
|
UC#4
0.0587
0.0889
0.096
|
UC#7
0.0342
0.1
0.0705
|
|
|
|
Cluster#378805 (XD11): overexpression
|
in 1 of 4 patients)
|
Patients
N
PT
MET
|
|
UC#1
0.00117
0.00269
0.00697
|
UC#2
0.00864
0.00371
0.00672
|
UC#4
0.0098
0.00525
0.00497
|
UC#7
0.00912
0.00989
0.0127
|
|
|
|
Cluster#374641: overexpression in 3 of 4
|
patients (75%)
|
Patients
N
PT
MET
|
|
UC#1
0.0124
0.163
0.0947
|
UC#2
0.28
0.317
0.544
|
UC#4
0.685
1.809
1.996
|
UC#7
0.569
1.714
1.073
|
|
Overexpression of genes in colon cancer patient epithelium. Results provided in the tables below include fluorescence data for polynucleotides isolated from colon epithelial cells that were prepared by the epithelial shakeoff method to obtain >97% pure epithelium without stroma. Normal, precancerous (adenomatous polyp), and primary tumor cell types are denoted as N, polyp and PT, respectively. Overexpression was determined by comparing either primary tumor cells or precancerous cells, or both, to normal cells. All values are adjusted to levels relative to beta-actin control.
|
|
Cluster#719 (SK1): overexpression in 4
of 4 patients (100%)
PatientsNPolypPT
|
UW#170.09240.117N/A
UW#180.0864N/A0.327
UW#190.151N/A0.227
UW#200.06240.1620.164
|
|
|
Cluster#115762 (SK5): overexpression
|
in 4 of 4 patients (100%).
|
Patients
N
Polyp
PT
|
|
UW#17
0.00724
0.0122
N/A
|
UW#18
0.0156
N/A
0.111
|
UW#19
0.0158
N/A
0.0461
|
UW#20
0.00728
0.0187
0.0306
|
|
|
|
Cluster#1665: overexpression in 4 of 4
|
patients (100%)
|
Patients
N
Polyp
PT
|
|
UW#17
0.0041
0.0306
N/A
|
UW#18
0.0029
N/A
0.0357
|
UW#19
0.0045
N/A
0.0357
|
UW#20
0.0028
0.025
0.047
|
|
|
|
Cluster#2334 (SK8) overexpressed in 1
|
of 4 patients (25%)
|
Patients
N
Polyp
PT
|
|
UW#17
0.1835
0.041
N/A
|
|
|
|
Cluster#2334 (SK8) overexpressed in 1
|
of 4 patients (25%)
|
Patients
N
Polyp
PT
|
|
UW#18
0.0638
N/A
0.0927
|
UW#19
0.04
N/A
0.04
|
UW#20
0.2236
0.0576
0.0454
|
|
|
|
Cluster#3376 (SK19) overexpressed in 4 of 4 patients (100%)
|
Patients
N
Polyp
PT
|
|
UW#17
0.0053
0.012
N/A
|
UW#18
0.0028
N/A
0.0084
|
UW#19
0.003
N/A
0.0135
|
UW#20
0.0023
0.023
0.012
|
|
Example 5
Northern Blot Analysis
Differential gene expression in cancerous colon cells can be further confirmed by other techniques, such as Northern blot analysis. Northern analysis can be accomplished by methods well-known in the art. Briefly, rapid-Hyb buffer (Amersham Life Science, Little Chalfont, England) with 5 mg/ml denatured single stranded sperm DNA is pre-warmed to 65° C. and human colon tumor total RNA blots (Invitrogen, Carlsbad, Calif.) are pre-hybridized in the buffer with shaking at 65° C. for 30 minutes. Gene-specific DNA probes (50 ng per reaction) labeled with [α-32P]dCTP (3000 Ci/mmol, Amersham Pharmacia Biotech Inc., Piscataway, N.J.) (Prime-It RmT Kit, Stratagene, La Jolla, Calif.) and purified with ProbeQuant™ G-50 Micro Columns (Amersham Pharmacia Biotech Inc.) are added and hybridized to the blots with shaking at 65° C. for overnight. The blots are washed in 2×SSC, 0.1% (w/v) SDS at room temperature for 20 minutes, twice in 1×SSC, 0.1% (w/v) SDS at 65° C. for 15 minutes, then exposed to Hyperfilms (Amersham Life Science).
Example 6
Analysis of Expression of Gene Corresponding to SK2 (Cluster 9083 (c9083)) (SEQ ID NO:3) in Colorectal Carcinoma
The expression of the gene comprising the sequence of SK2, which clusters to cluster i.d. no. 9083, was examined by quantitative PCR in several cancer cell lines, including a number of colorectal carcinoma cell lines. The cells in which expression was tested are summarized below.
|
|
Cell LineTissue SourceCell LineTissue Source
|
MDA-MB-231Human breast; high metastaticCaco-2Human colorectal
potential (micromets in lung;adenocarcinoma
adenocarcinoma; pleural
effusion
MDA-MB-435Human breast, high metastaticSW620Human colorectal
potential (macrometastases inadenocarcinoma; from
lung)metastatic site (lymph node)
MCF-7Human breast; non-metastaticLS174THigh metastatic potential
human colorectal
adenocarcinoma
MDA-MB-468Human breast; adenocarcinomaLOVOHuman colorectal
adenocarcinoma; colon; from
metastatic site (colon)
AlabHuman breast, metastaticHT29Human colorectal
adenocarcinoma; colon
SKOV3Human ovarianSW480Human colorectal
adenocarcinomaadenocarcinoma; colon
OVCAR3Human ovarianHCT116Human colorectal carcinoma;
adenocarcinomacolon
KM12CHuman colon; low metastaticColoHuman colorectal
potential320DNadenocarcinoma; colon
KM12L4Human colon; high metastaticT84Human colorectal carcinoma;
potential (derived fromcolon; from metastatic site
Km12C)(lung)
DU 145Human prostate; carcinoma;HCT15Human colorectal
from metastatic site: brainadenocarcinoma; colon
HT1080Human sarcoma cell line;CCD112Human colorectal
adenocarcinoma, low
metastatic potential
HMVECPrimary human microvascularDLD1Human colon; colorectal
endothelial cellsadenocarcinoma
185B4normal breast epithelial cells;293kidney epithelial cells
chemically transformed
LNCAPprostate carcinoma; metastasisGRDPprimary prostate epithelium
to left supraclavicular lymph
U373MGglioblastoma cellIMR90primary lung fibroblast
WOCAprimary prostate epitheliumPC3prostate cancer; androgen
receptor negative
|
Quantitative real-time PCR was performed by first isolating RNA from cells using a Roche RNA Isolation kit according to manufacturer's directions. One microgram of RNA was used to synthesize a first-strand cDNA using MMLV reverse transcriptase (Ambion) using the manufacturers buffer and recommended concentrations of oligo dT, nucleotides, and Rnasin. This first-strand cDNA served as a template for quantitative real-time PCR using the Roche light-cycler as recommended in the machine manual. The gene corresponding to SK2 (C9083) (SEQ ID NO:3) was amplified with forward primer: 5′-cgctgacctcaaccag-3′ (SEQ ID NO:60) and reverse primer: 5′-ctgtttgcccgttcttattac-3′ (SEQ ID NO:61). Product was quantified based on the cycle at which the amplification entered the linear phase of amplification in comparison to an internal standard and using the software supplied by the manufacturer. Small differences in amounts or total template in the first-strand cDNA reaction were eliminated by normalizing to amount of actin amplified in a separate quantitative PCR reaction using the forward primer 5′-CGGGAAATCGTGCGTGACATTAAG-3′ (SEQ ID NO:56) and the reverse primer: 5′-TGATCTCCTTCTGCATCCTGTCGG-3′ (SEQ ID NO:57). The results are shown in FIG. 1
Example 7
Functional Analysis of Gene Corresponding to SK2 (c9083) (SEQ ID NO:3)
In order to further assess the role of the gene corresponding to SK2 (c9083) (SEQ ID NO:3), the functional information on the gene corresponding to this sequence was obtained using antisense knockout technology. In short, the cell type to be tested, SW620 or HT1080 cells which express the polypeptide encoded by the gene corresponding to c9083, were plated to approximately 60-80% confluency on 6-well or, for proliferation assays, 96-well dishes. Antisense or reverse control oligonucleotide was diluted to 2 μM in optimem and added to optimem into which the delivery vehicle, lipitoid 116-6 in the case of SW620 cells or 1:1 lipitoid 1:cholesteroid 1 in the case of HT1080 cells, had been diluted. The oligo/delivery vehicle mixture was then further diluted into medium with serum on the cells. The final concentration of oligonucleotide for all experiments was 300 nM, and the final ratio of oligo to delivery vehicle for all experiments was 1.5 nmol lipitoid/μg oligonucleotide. Cells were transfected overnight at 37 C and the transfection mixture was replaced with fresh medium the next morning.
The following antisense oligonucleotides were tested for the ability to deplete c9083 (SEQ ID NO:3) RNA:
|
|
Olig NameSequenceNucleotides
|
CHIR-8-4ASATTTGGGCATCACTGGCTACAAGCA25
C9083:P0463(SEQ ID NO:64)
CHIR-8-4RCACGAACATCGGTCACTACGGGTTTA25
C9083:P0463RC(SEQ ID NO:65)
CHIR-8-5A5CAGAGAGGTGAGACACTCGCCGCA24
C9083:P0157(SEQ ID NO:66)
CHIR-8-5RCACGCCGCTCACAGAGTGGAGAGAC24
C9083:POI57RC(SEQ ID NO:67)
|
RC: reverse control oligos (control oligos);
|
AS: antisense oligos (test)
|
The effect of the oligonucleotide on the cells was assessed by both quantitation of PCR levels as described above, and in proliferation assays using amount of DNA as quantified with the Stratagene Quantos™ kit to determine cell number.
The results of the mRNA level quantitation are shown in FIG. 2. The effects of the oligonucleotides upon proliferation over a four day period are shown in FIGS. 3 and 4. Cells without oligonucleotide treatment (WT) served as a control. The oligo CHIR-8-4AS was most effective in decreasing mRNA for the gene corresponding to 9083c. Transfection of these oligos into SW620 cells resulted in a decreased rate of proliferation relative to matched reverse control oligos, with CHIR-8-4 being somewhat more effective than CHIR-8-5 (FIG. 3). Significantly, the same antisense oligonucleotide had no effect on growth of a fibrosarcoma cell line, HT1080 (FIG. 4). This indicates that the functional role of the gene corresponding to c9083 is tissue-specific, and further that the gene corresponding to c9083 has a specific effect on growth.
The oligos were next tested for their effect on colony formation in a soft agar assay. Soft agar assays were conducted by first establishing a bottom layer of 2 ml of 0.6% agar in media plated fresh within a few hours of layering on the cells. The cell layer was formed on the bottom layer by removing cells transfected as described above (either an antisense k-Ras oligo as a positive control), CHIR-8-4, CHIR-8-5, CHIR-8-4RC, or CHIR-8-5RC) from plates using 0.05% trypsin and washing twice in media. The cells were counted in a Coulter counter, and resuspended to 106 per ml in media. 10 μl aliquots are placed with media in 96-well plates (to check counting with WST1), or diluted further for soft agar assay. 2000 cells are plated in 800 μl 0.4% agar in duplicate wells above 0.6% agar bottom layer. After the cell layer agar solidifies, 2 ml of media is dribbled on top and antisense or reverse control oligo is added without delivery vehicles. Fresh media and oligos are added every 3-4 days. Colonies are formed in 10 days to 3 weeks. Fields of colonies were counted by eye. WST-1 metabolism values can be used to compensate for small differences in starting cell number. Larger fields can be scanned for visual record of differences.
Both the CHIR-8-4 and CHIR-8-5 antisense oligos led to decreased colony size and number compared to the control CHIR-8-4RC and CHIR-8-5RC oligos. These results further validate the gene corresponding to c9083 (SEQ ID NO:3) as a target for therapeutic intervention.
Example 8
Effect of Antisense Oligonucleotides on Message Levels for Target Genes
The effect of antisense oligonucleotides upon message levels for the genes corresponding to the sequences and clusters described herein was analyzed using antisense knockout technology as described for c9083 in the Example above. Specifically, antisense oligos for genes corresponding to each of c719, c1665, c3376, c115762, c454001, c3788805, and c776682 were prepared as described above. Once synthesized and quantitated, the oligomers were screened for efficiency of a transcript knock-out in a panel of cancer cell lines. The efficiency of the knock-out was determined by analyzing mRNA levels using lightcycler quantification. The oligomers that resulted in the highest level of transcript knock-out, wherein the level was at least about 50%, preferably about 80-90%, up to 95% or more up to undetectable message, were selected for use in a cell-based proliferation assay, an anchorage independent growth assay, and an apoptosis assay.
SW620 cells, which express the polypeptide encoded by the corresponding genes to be analyzed, were plated to approximately 60-80% confluency on 6-well or, for proliferation assays, 96-well dishes. For each transfection mixture, a carrier molecule, preferably a lipitoid or cholesteroid, was prepared to a working concentration of 0.5 mM in water, sonicated to yield a uniform solution, and filtered through a 0.45 μm PVDF membrane. The antisense or control oligonucleotide was then prepared to a working concentration of 100 μM in sterile Millipore water. The oligonucleotide was further diluted in OptiMEM™ (Gibco/BRL), in a microfuge tube, to 2 μM, or approximately 20 μg oligo/ml of OptiMEM™. In a separate microfuge tube, lipitoid or cholesteroid, typically in the amount of about 1.5-2 mmol lipitoid/μg antisense oligonucleotide, was diluted into the same volume of OptiMEM™ used to dilute the oligonucleotide. The diluted antisense oligonucleotide was immediately added to the diluted lipitoid and mixed by pipetting up and down. Oligonucleotide was added to the cells to a final concentration of 30 nM.
The level of target mRNA that corresponds to a target gene of interest in the transfected cells was quantitated in the cancer cell lines using the Roche LightCycler™ real-time PCR machine. Values for the target mRNA were normalized versus an internal control (e.g., beta-actin). For each 20 μl reaction, extracted RNA (generally 0.2-1 μg total) was placed into a sterile 0.5 or 1.5 ml microcentrifuge tube, and water was added to a total volume of 12.5 μl. To each tube was added 7.5 μl of a buffer/enzyme mixture, prepared by mixing (in the order listed) 2.5 μl H2O, 2.0 μl 10× reaction buffer, 10 μl oligo dT (20 pmol), 1.0 μl dNTP mix (10 mM each), 0.5 μl RNAsin® (20 u) (Ambion, Inc., Hialeah, Fla.), and 0.5 μl MMLV reverse transcriptase (50 u) (Ambion, Inc.). The contents were mixed by pipetting up and down, and the reaction mixture was incubated at 42° C. for 1 hour. The contents of each tube were centrifuged prior to amplification.
An amplification mixture was prepared by mixing in the following order: 1×PCR buffer II, 3 mM MgCl2, 140 μM each dNTP, 0.175 pmol each oligo, 1:50,000 dil of SYBR® Green, 0.25 mg/ml BSA, 1 unit Taq polymerase, and H2O to 20 μl. (PCR buffer II is available in 10× concentration from Perkin-Elmer, Norwalk, Conn.). In 1× concentration it contains 10 mM Tris pH 8.3 and 50 mM KCl. SYBR® Green (Molecular Probes, Eugene, Oreg.) is a dye which fluoresces when bound to double stranded DNA. As double stranded PCR product is produced during amplification, the fluorescence from SYBR® Green increases. To each 20 μl aliquot of amplification mixture, 2 μl of template RT was added, and amplification was carried out according to standard protocols.
The following antisense oligonucleotides were tested for the ability to deplete the message levels of the gene corresponding to the indicated cluster. Target Gene: Oligo Location provides the name of the cluster to which the target gene is assigned and the name of the oligo used. AS indicates antisense; RC indicates reverse control. Data for the genes corresponding to c9083 are provided for comparison.
|
|
Target% KO of
Gene:Oligo LocationOligo SequenceSEQ ID NO:Message
|
c719:1-ASTTGGTGTCATTGGGTCAAGGGTTGG6885%
|
C719:1-RCGGTTGGGAACTGGGTTACTGTGGTT69
|
c719:2-ASACAGGGCAGATACGGACCTCGGTG7093%
|
c719:2-RCGTGGCTCCAGGCATAGACGGGACA71
|
c719:3-ASTTGTGGGTAAGCAGTTTCATGTCGC7267%
|
c719:3-RCCGCTGTACTTTGACGAATGGGTGTT73
|
c719:4-ASCCTGGATCAGACGCAAGTTATCGGC7485%
|
c719:4-RCCGGCTATTGAACGCAGACTAGGTCC75
|
C9083:4-ASATTTGGGCATCACTGGCTACAAGCA6483.0
|
C9083:4-RCACGAACATCGGTCACTACGGGTTTA65
|
C9083:5-ASCAGAGAGGTGAGACACTCGCCGCA6673.0
|
C9083:5-RCACGCCGCTCACAGAGTGGAGAGAC67
|
C1665:1-ASCTACTCCCCACACTTCATCGCCAGG7673.0
|
C1665:1-RCGGACCGCTACTTCACACCCCTCATC77
|
C1665:2-ASCTCTTGATACTCCAGCGGCAAACCA7881.0
|
C1665:2-RCACCAAACGGCGACCTCATAGTTCTC79
|
c3376:1-ASGCGCCCAAGCCGTTCGTTCTTAAG8078.0
|
c3376:1-RCGAATTCTTGCTTGCCGAACCCGCG81
|
c3376:2-ASCCAGGTAGGCACGAGTTGGCAAAGA8297.0
|
c3376:2-RCAGAAACGGTTGAGCACGGATGGACC83
|
c3376:3-ASGCCATTGAAGATGCCCAGATCCCAC8456.0
|
c3376:3-RCCACCCTAGACCCGTAGAAGTTACCG85
|
c3376:4-ASCCTGCGTTTGTCCCTCCAGCATCT8693.0
|
c3376:4-RCTCTACGACCTCCCTGTTTGCGTCC87
|
c3376:5-ASAAGTCACAGTCCCCGGATACCAGTC8888.0
|
c3376:5-RCCTGACCATAGGCCCCTGACACTGAA89
|
c115762:1-ASTTGTCGCTTTGGCAGGCATAAAACC9097.5
|
c115762:2-ASTCTGGTCATCAACTTGCTTTCCGTG9199.0
|
c115762:3-ASCAGTGTTTCGTGGTGTGCTCTGTGG9298.0
|
c115762:4-ASGCTCACCATCCGGGCACCAAGCA9397.0
|
c115762:5-ASTGAGAGACAGTGTTTCGTGGTGTGC9493.0
|
454001:1-ASTGCCTTCACACGCTTGGTTATCTTC950
|
454001:2-ASGACAACATCGGAGGCTTCAATCACC960
|
454001:3-ASGTTGAGGCTCTGAACACCACTGTTG970
|
454001:4-ASGTTTGGCAGCACCTTCAACATTTGG9887
|
454001:5-ASAGCAGTTTGGCAGCACCTTCAACA9992
|
454001:-1-RCCTTCTATTGGTTCGCACACTTCCGT100
|
454001:2-RCCCACTAACTTCGGAGGCTACAACAG101
|
454001:3-RCGTTGTCACCACAAGTCTCGGAGTTG102
|
454001:4-RCGGTTTACAACTTCCACGACGGTTTG103
|
454001:5-RCACAACTTCCACGACGGTTTGACGA104
|
378805:1-ASATCTGGCATGGACGGATGAGCGAA105 41.0
|
378805:2-ASGCTGGGTGGTTTCCGAACTCAACG106 97
|
378805:3-ASGTCCCAATCACCTTCCCCACAATCC107 65.0
|
378805:4-ASTCAGATCCTTCTTCCACTCCCGCTT108 100.0
|
378805:5-ASTGCTCGTGGAACAGGTAAAGCTCTG109 98
|
378805:1-RCAAGCGAGTAGGCAGGTACGGTCTA110
|
378805:2-RCGCAACTCAAGCCTTTGGTGGGTCG111
|
378805:3-RCCCTAACACCCCTTCCACTAACCCTG112
|
378805:4-RCTTCGCCCTCACCTTCTTCCTAGACT113
|
378805:5-RCGTCTCGAAATGGACAAGGTGCTCGT114
|
776682:1-ASAGCTTCACTTTGGTCTTGACGGCAT115 81
|
776682:2-ASCGGAGGGAAGTCAAGTCAGCCACA116 60
|
776682:3-ASCGGCATTCACCCTCTCCAGCACCT117 89
|
776682:4-ASCCTCCACCTGTTTGCGGGCTTCC118 61
|
776682:5-ASCCACATTGAGGGAGTCCTCTTGCAA119 80
|
776682:1-RCTACGGCAGTTCTGGTTTCACTTCGA120
|
776682:2-RCACACCGACTGAACTGAAGGGAGGC121
|
776682:3-RCTCCACGACCTCTCCCACTTACGGC122
|
776682:5-RCCCTTCGGGCGTTTGTCCACCTCC123
|
402380:P464:4-ASCCCCGAACAAAACACCAGTCAACG124 94
|
402380:P464:4-RCGCAACTGACCACAAAACAAGCCCC125
|
402380:P414:5 ASGGCCATTGAGTCCCTCCATAGCAGC126 92
|
402380:P414:5-RCCGACGATACCTCCCTGAGTTACCGG127
|
The effect of the oligonucleotide on the cells was assessed by quantitation of PCR levels. The results of the mRNA level quantitation are summarized in the table immediately above.
The effect of the loss of message for each gene above can be assessed in cell-based assays as described in Example 7 above. One such use of the antisense oligonucleotide described by SEQ ID NO:108 resulted in an inhibition of proliferation of SW620 cells when used as described in the transfection and proliferation assay protocols in Example 7 (FIG. 5).
Example 9
The Effect of Expression of Genes Corresponding to c3376 and 402380 Upon on Proliferation
The effect of expression of genes corresponding to c3376 (gene corresponding to SEQ ID NO:13) and 402380 (gene corresponding to SEQ ID NO:16) on the inhibition of cell proliferation was assessed in SW620 colon colorectal carcinoma cells.
Cells were plated to approximately 60-80% confluency in 96-well dishes. Antisense or reverse control oligonucleotide was diluted to 2 μM in OptiMEM™ and added to OptiMEM™ into which the delivery vehicle, lipitoid 116-6 in the case of SW620 cells or 1:1 lipitoid 1:cholesteroid 1 in the case of MDA-MB-231 cells, had been diluted. The oligo/delivery vehicle mixture was then further diluted into medium with serum on the cells. The final concentration of oligonucleotide for all experiments was 300 nM, and the final ratio of oligo to delivery vehicle for all experiments was 1.5 nmol lipitoid/μg oligonucleotide.
Antisense oligonucleotides were prepared as described above. Cells were transfected overnight at 37° C. and the transfection mixture was replaced with fresh medium the next morning. Transfection was carried out as described above in Example 8. Proliferaton was measured using the colormetric reagent WST-1 according to methods well known in the art. The results of the antisense experiments are shown in FIGS. 6-9. The values on the y-axis represent relative fluorescent units. Antisense and reverse control oligos to K-Ras served as a control to demonstrate the assay worked as expected (FIG. 6).
Example 10
Effect of Gene Expression on Colony Formation in Soft Agar
The effect of expression of the gene corresponding to 402380 (gene corresponding to SEQ ID NO:16) upon colony formation of SW620 cells was tested in a soft agar assay. Soft agar assays were conducted by first establishing a bottom layer of 2 ml of 0.6% agar in media plated fresh within a few hours of layering on the cells. The cell layer was formed on the bottom layer by removing cells transfected as described above from plates using 0.05% trypsin and washing twice in media. The cells were counted in a Coulter counter, and resuspended to 106 per ml in media. 10 μl aliquots were placed with media in 96-well plates (to check counting with WST-1), or diluted further for the soft agar assay. 2000 cells were plated in 800 μl 0.4% agar in duplicate wells above 0.6% agar bottom layer. After the cell layer agar solidified, 2 ml of media was dribbled on top and antisense or reverse control oligo (produced as described above) was added without delivery vehicles. Fresh media and oligos were added every 3-4 days. Colonies formed in 10 days to 3 weeks. Fields of colonies were counted by eye. Wst-1 metabolism values were used to compensate for small differences in starting cell number. Larger fields can be scanned for visual record of differences.
The results are shown in FIG. 9. The y-axis represents the number of cells per a defined sector, using WST-1 to facilitate cell count and normalized to a control. Antisense and reverse control oligos to K-Ras (kRAS 2576-as and kRAS 2576-rc) served as controls to demonstrate the assay worked as expected.
Example 11
Effect of Gene Expression Upon Cell Death
Effect of expression of the genes corresponding to cluster 719 (gene corresponding to SEQ ID NO:1, CHIR-7); cluster 9083 (gene corresponding to SEQ ID NO:3, CHIR-8); cluster 1665 (gene corresponding to SEQ ID NOS:7 and 9, CHIR-9); cluster 3376 (gene corresponding to SEQ ID NO:13, CHIR-11); cluster 115762 (gene corresponding to SEQ ID NO:5, CHIR-16); and cluster 402380 (gene corresponding to SEQ ID NO:16, CHIR-33) upon cell death in an lactatae dehydrobenase (LDH) cytotoxitity assay was examined in HT1080 cells (a human fibrosarcoma cell line), SW620 cells, and metastatic breast cancer cell lines (MDA-MB-231 (“231”)) cells. The lactate dehydrogenase (LDH) cytotoxicity assay essentially as follows:
The lactate dehydrogenase (LDH) cytotoxicity assay was performed essentially as follows:
Day 1: Cells were seeded in 4 separate 96 well plates, typically 5000 cells/well and incubated at 37° C. and 5% CO2.
Day 2: Cells were transfected with the anti-sense as well as the reverse complement controls, essentially as described in Example 4. One plate (day 0) was left untransfected as a seeding control.
The transfection was carried out using a lipid vehicle for delivery as described in WO 01/16306, hereby incorporated in its entirety. Briefly, the transfection used agents known as “lipitoids” and “cholesteroids”, described, for example, in PCT publications WO 01/16306, WO 98/06437 and WO 99/08711, based on U.S. Ser. Nos. 60/023,867, 60/054,743, and 09/132,808, which are also hereby incorporated by reference. These lipid-cationic peptoid conjugates are shown in these references to be effective reagents for the delivery of plasmid DNA to cells in vitro. Any of the carriers described in the above-referenced applications are suitable for use in transfection of the oligonucleotides described herein.
These compounds may be prepared by conventional solution or solid-phase synthesis. In one such procedure, as described in WO 99/08711, cited above, the N-terminus of a resin-bound peptoid is acylated with a spacer such as Fmocaminohexanoic acid or Fmoc-3-alanine. After removal of the Fmoc group, the primary amino group is reacted with cholesterol chloroformate to form a carbamate linkage. The product is then cleaved from the resin with trifluoroacetic acid and purified by reverse-phase HPLC. A fatty acid-derived lipid moiety, such as a phospholipid, may be used in place of the steroid moiety. The steroid or other lipid moiety may also be linked to the peptoid moiety by other linkages, of any effective length, readily available to the skilled practitioner.
Depending on the cell type, different lipid vehicles were used for different lengths of time for transfection. However, the transfection time did not exceed 24 hrs. The transfection was carried out in complete medium and the final anti-sense oligonucleotide concentration was 300 nM per well. In the wells with drug, the drug was added to the culture at the beginning of the transfection.
Starting on day 3: cells were recovered, 1 plate/day and release of LDH into the supernatant as well as LDH in intact cells was measured using a kit from Roche according to manufacturer's instructions (Roche Diagnostics, Basel, Switzerland) (data labeled as day 1, 2, 3).
For each sample, were analyzed by examining the relative level of released LDH compared to total LDH, wherein an increase as a portion of total LDH signifies increased cell death (due to a higher proportion of released LDH in the media). The data was assessed qualitatively by comparison to an untreated control (no oligo). This assay allowed a determination as to whether antisense-induced loss of message for a particular gene causes death of cells when used alone, or wheter this loss of message sensitizes cells to the effects of a drug.
The results are shown in the table immediately below.
|
|
HT1080SW620231
|
|
chir7-2negativenegative
chir8-4positiveweakly positive
chir9-5positive
chir11-2negative
chir16-4negative
chir33-4very weaklystrong positivevery weakly
positivepositive
|
Example 12
Detection of Differential Expression Using Arrays
mRNA isolated from samples of cancerous and normal colon tissue obtained from patients were analyzed to identify genes differentially expressed in cancerous and normal cells. Normal and cancerous cells collected from cryopreserved patient tissues were isolated using laser capture microdissection (LCM) techniques, which techniques are well known in the art (see, e.g., Ohyama et al. (2000) Biotechniques 29:530-6; Curran et al. (2000) Mol. Pathol. 53:64-8; Suarez-Quian et al. (1999) Biotechniques 26:328-35; Simone et al. (1998) Trends Genet 14:272-6; Conia et al. (1997) J. Clin. Lab. Anal. 11:28-38; Emmert-Buck et al. (1996) Science 274:998-1001).
Table 5 (inserted before the claims) provides information about each patient from which the samples were isolated, including: the “Patient ID” and “Path ReportID”, which are numbers assigned to the patient and the pathology reports for identification purposes; the “Group” to which the patients have been assigned; the anatomical location of the tumor (“Anatom Loc”); the “Primary Tumor Size”; the “Primary Tumor Grade”; the identification of the histopathological grade (“Histopath Grade”); a description of local sites to which the tumor had invaded (“Local Invasion”); the presence of lymph node metastases (“Lymph Node Met”); the incidence of lymph node metastases (provided as a number of lymph nodes positive for metastasis over the number of lymph nodes examined) (“Incidence Lymphnode Met”); the “Regional Lymphnode Grade”; the identification or detection of metastases to sites distant to the tumor and their location (“Distant Met & Loc”); a description of the distant metastases (“Descrip Distant Met”); the grade of distant metastasis (“Dist Met Grade”); and general comments about the patient or the tumor (“Comments”). Adenoma was not described in any of the patients; adenoma dysplasia (described as hyperplasia by the pathologist) was described in Patient ID No. 695. Extranodal extensions were described in two patients, Patient ID Nos. 784 and 791. Lymphovascular invasion was described in seven patients, Patient ID Nos. 128, 278, 517, 534, 784, 786, and 791. Crohn's-like infiltrates were described in seven patients, Patient ID Nos. 52, 264, 268, 392, 393, 784, and 791.
TABLE 5
|
|
PrimaryPrimaryIncidenceRegional LympDistantDescrip
PatientPath ReportAnatomTumorTumorHisto pathLocalLymph nodeLymphnodeMet &DistantDist Met
IDIDGroupLocSizeGradeGradeInvasionMetnode MetGradeLocMetGradeComment
|
1521IIIAscending4.0T3G2extendingpositive3/8 N1negativeMXinvasive
colonintoadenocarcinoma,
subserosalmoderately
adiposedifferentiated;
tissuefocal
perineural
invasion is
seen
5271IIAscending9.0T3G3Invasionnegative0/12N0negativeM0Hyperplastic
colonthroughpolypin
muscularisappendix.
propria,
subserosal
involvement;
ileocec.
valve
involvement
121140IISigmoid6T4G2Invasionnegative0/34N0negativeM0Perineural
ofInvasion;
muscularisdonut
propriaanastomos
intois
serosa,negative.
involvingOne
submucosatubulovillous
ofand
urinaryone
bladdertubular
adenoma
with no
high grade
dysplasia.
125144IICecum6T3G2Invasionnegative0/19N0negativeM0patient
throughhistory of
themetastatic
muscularismelanoma
propria
into
suserosal
adipose
tissue.
Ileocecal
junction.
128147IIITransverse5.0T3G2Invasionpositive1/5 N1negativeM0
colonof
muscularis
propria
into
percolonic
fat
130149Splenic5.5T3throughpositive10/24 N2negativeM1
flexurewall
and
into
surrounding
adipose
tissue
133152IIRectum5.0T3G2Invasionnegative0/9 N0negativeM0Small
throughseparate
muscularistubular
propriaadenoma
into(0.4 cm)
non-
peritonealized
pericolic
tissue;
gross
configuration
is
annular.
141160IVCecum5.5T3G2Invasionpositive7/21N2positiveadenocarcinomaM1Perineural
of(Liver)consistantinvasion
musculariswithidentified
propriaprimaryadjacent
intoto
pericolonicmetastatic
adiposeadenocarcinoma.
tissue,
but
not
through
serosa.
Arising
from
tubular
adenoma.
156175IIIHepatic3.8T3G2Invasionpositive2/13N1negativeM0Separate
flexurethroughtubolovillous
mucsularisand
propriatubular
intoadenomas
subserosa/
pericolic
adipose,
no
serosal
involvement.
Gross
configuration
annular.
228247IIIRectum5.8T3G2 toInvasionpositive1/8 N1negativeMXHyperplastic
G3throughpolyps
muscularis
propria
to
involve
subserosal,
perirectoal
adipose,
and
serosa
264283IIAscending5.5T3G2Invasionnegative0/10N0negativeM0Tubulovillous
colonthroughadenoma
musculariswith high
propriagrade
intodysplasia
subserosal
adipose
tissue.
266285IIITransverse9T3G2Invadesnegative0/15N1positive0.4 cm,MX
colonthrough(Mesentericmay
muscularisdeposit)represent
proprialymph
tonode
involvecompletely
pericolonicreplaced
adipose,by tumor
extends
to
serosa
268287ICecum6.5T2G2Invadesnegative0/12N0negativeM0
full
thickness
of
muscularis
propria,
but
mesenteric
adipose
free
of
malignancy
278297IIIRectum4T3G2Invasionpositive7/10N2negativeM0Descending
intocolon
perirectalpolyps, no
adiposeHGD or
tissue.carcinoma
identified.
295314IIAscending5.0T3G2Invasionnegative0/12N0negativeM0Melanosis
colonthroughcoli and
muscularisdiverticular
propriadisease.
into
percolic
adipose
tissue.
339358IIRectosigmoid6T3G2Extendsnegative0/6 N0negativeM01
intohyperplastic
perirectalpolyp
fatidentified
but
does
not
reach
serosa
341360IIAscending2 cmT3G2Invasionnegative0/4 N0negativeMX
coloninvasivethrough
muscularis
propria
to
involve
pericolonic
fat.
Arising
from
villous
adenoma.
356375IISigmoid6.5T3G2Throughnegative0/4 N0negativeM0
colon
wall
into
subserosal
adipose
tissue.
No
serosal
spread
seen.
360412IIIAscending4.3T3G2Invasionpositive1/5 N1negativeM0Two
colonthrumucosal
muscularispolyps
propria
to
pericolonic
fat
392444IVAscending2T3G2Invasionpositive1/6 N1positiveMacrovesicularM1Tumor
colonthrough(Liver)andarising at
muscularismicrovesicularprior
propriasteatosisileocolic
intosurgical
subserosalanastomosis.
adipose
tissue,
not
serosa.
393445IICecum6.0T3G2Cecum,negative0/21N0negativeM0
invades
through
muscularis
propria
to
involve
subserosal
adipose
tissue
but
not
serosa.
413465IVAscending4.8T3G2Invasivenegative0/7 N0positiveadenocarcinomaM1rediagnosis
colonthrough(Liver)inof
muscularismultipleoophorectomy
toslidespath
involveto
periserosalmetastatic
fat;colon
abuttingcancer.
ileocecal
junction.
505383IV7.5 cmT3G2Invasionpositive2/17N1positivemoderatelyM1Anatomical
maxthrough(Liver)differentiatedlocation
dimmuscularisadenocarcinoma,of primary
propriaconsistantnot
involvingwithnotated in
pericolicprimaryreport.
adipose,Evidence
serosalof chronic
surfacecolitis.
uninvolved
517395IVSigmoid3T3G2penetratespositive6/6 N2negativeM0No
muscularismention
propria,of distant
involvesmet in
pericolonicreport
fat.
534553IIAscending12T3G3Invasionnegative0/8 N0negativeM0Omentum
colonthroughwith
thefibrosis
muscularisand fat
proprianecrosis.
involvingSmall
pericolicbowel
fat.with acute
Serosaand
free ofchronic
tumor.serositis,
focal
abscess
and
adhesions.
546565IVAscending5.5T3G2Invasionpositive6/12N2positivemetastaticM1
colonthrough(Liver)adenocarcinoma
muscularis
propria
extensively
through
submucosal
and
extending
to
serosa.
577596IICecum11.5T3G2Invasionnegative0/58N0negativeM0Appendix
throughdilated
theand
bowelfibrotic,
wall,but not
intoinvolved
suberosalby tumor
adipose.
Serosal
surface
free
of
tumor.
695714IICecum14T3G2extendingnegative0/22N0negativeMXtubular
throughadenoma
boweland
wallhyperplstic
intopolyps
serosalpresent,
fatmoderately
differentiated
adenoma
with
mucinous
diferentiation
(% not
stated)
784803IVAscending3.5T3G3throughpositive5/17N2positiveM1invasive
colonmuscularis(Liver)poorly
propriadifferentiated
intoadenosquamous
pericoliccarcinoma
soft
tissues
786805IVDescending9.5T3G2throughnegative0/12N0positiveM1moderately
colonmuscularis(Liver)differentiated
propriainvasive
intoadenocarcinoma
pericolic
fat,
but
not at
serosal
surface
791810IVAscending5.8T3G3throughpositive13/25 N2positiveM1poorly
colonthe(Liver)differentiated
muscularisinvasive
propriacolonic
intoadenocarcinoma
pericolic
fat
888908IVAscending2.0T2G1intopositive3/21N0positiveM1well-to
colonmuscularis(Liver)moderately-
propriadifferentiated
adenocarcinoma;
this
patient has
tumors of
the
ascending
colon and
the
sigmoid
colon
889909IVCecum4.8T3G2throughpositive1/4 N1positiveM1moderately
muscularis(Liver)differentiated
propriaadenocarcinoma
int
subserosal
tissue
|
Identification of Differentially Expressed Genes
cDNA probes were prepared from total RNA isolated from the patient cells described above. Since LCM provides for the isolation of specific cell types to provide a substantially homogenous cell sample, this provided for a similarly pure RNA sample.
Total RNA was first reverse transcribed into cDNA using a primer containing a T7 RNA polymerase promoter, followed by second strand DNA synthesis. cDNA was then transcribed in vitro to produce antisense RNA using the T7 promoter-mediated expression (see, e.g., Luo et al. (1999) Nature Med 5:117-122), and the antisense RNA was then converted into cDNA. The second set of cDNAs were again transcribed in vitro, using the T7 promoter, to provide antisense RNA. Optionally, the RNA was again converted into cDNA, allowing for up to a third round of T7-mediated amplification to produce more antisense RNA. Thus the procedure provided for two or three rounds of in vitro transcription to produce the final RNA used for fluorescent labeling.
Fluorescent probes were generated by first adding control RNA to the antisense RNA mix, and producing fluorescently labeled cDNA from the RNA starting material. Fluorescently labeled cDNAs prepared from the tumor RNA sample were compared to fluorescently labeled cDNAs prepared from normal cell RNA sample. For example, the cDNA probes from the normal cells were labeled with Cy3 fluorescent dye (green) and the cDNA probes prepared from the tumor cells were labeled with Cy5 fluorescent dye (red), and vice versa.
Each array used had an identical spatial layout and control spot set. Each microarray was divided into two areas, each area having an array with, on each half, twelve groupings of 32×12 spots, for a total of about 9,216 spots on each array. The two areas are spotted identically which provide for at least two duplicates of each clone per array.
Polynucleotides corresponding to the differentially expressed genes described herein for use on the arrays were obtained from both publicly available sources and from cDNA libraries generated from selected cell lines and patient tissues. PCR products of from about 0.5 kb to 2.0 kb amplified from these sources were spotted onto the array using a Molecular Dynamics Gen III spotter according to the manufacturer's recommendations. The first row of each of the 24 regions on the array had about 32 control spots, including 4 negative control spots and 8 test polynucleotides. The test polynucleotides were spiked into each sample before the labeling reaction with a range of concentrations from 2-600 pg/slide and ratios of 1:1. For each array design, two slides were hybridized with the test samples reverse-labeled in the labeling reaction. This provided for about four duplicate measurements for each clone, two of one color and two of the other, for each sample.
The differential expression assay was performed by mixing equal amounts of probes from tumor cells and normal cells of the same patient. The arrays were prehybridized by incubation for about 2 hrs at 60° C. in 5×SSC/0.2% SDS/1 mM EDTA, and then washed three times in water and twice in isopropanol. Following prehybridization of the array, the probe mixture was then hybridized to the array under conditions of high stringency (overnight at 42° C. in 50% formamide, 5×SSC, and 0.2% SDS. After hybridization, the array was washed at 55° C. three times as follows: 1) first wash in 1×SSC/0.2% SDS; 2) second wash in 0.1×SSC/0.2% SDS; and 3) third wash in 0.1×SSC.
The arrays were then scanned for green and red fluorescence using a Molecular Dynamics Generation III dual color laser-scanner/detector. The images were processed using BioDiscovery Autogene software, and the data from each scan set normalized to provide for a ratio of expression relative to normal. Data from the microarray experiments was analyzed according to the algorithms described in U.S. application Ser. No. 60/252,358, filed Nov. 20, 2000, by E. J. Moler, M. A. Boyle, and F. M. Randazzo, and entitled “Precision and accuracy in cDNA microarray data,” which application is specifically incorporated herein by reference.
The experiment was repeated, this time labeling the two probes with the opposite color in order to perform the assay in both “color directions.” Each experiment was sometimes repeated with two more slides (one in each color direction). The level fluorescence for each sequence on the array expressed as a ratio of the geometric mean of 8 replicate spots/genes from the four arrays or 4 replicate spots/gene from 2 arrays or some other permutation. The data were normalized using the spiked positive controls present in each duplicated area, and the precision of this normalization was included in the final determination of the significance of each differential. The fluorescent intensity of each spot was also compared to the negative controls in each duplicated area to determine which spots have detected significant expression levels in each sample.
A statistical analysis of the fluorescent intensities was applied to each set of duplicate spots to assess the precision and significance of each differential measurement, resulting in a p-value testing the null hypothesis that there is no differential in the expression level between the tumor and normal samples of each patient. During initial analysis of the microarrays, the hypothesis was accepted if p>10−3, and the differential ratio was set to 1.000 for those spots. All other spots have a significant difference in expression between the tumor and normal sample. If the tumor sample has detectable expression and the normal does not, the ratio is truncated at 1000 since the value for expression in the normal sample would be zero, and the ratio would not be a mathematically useful value (e.g., infinity). If the normal sample has detectable expression and the tumor does not, the ratio is truncated to 0.001, since the value for expression in the tumor sample would be zero and the ratio would not be a mathematically useful value. These latter two situations are referred to herein as “on/off.” Database tables were populated using a 95% confidence level (p>0.05).
The results are provided in Table 6 below. The table includes: 1) the SEQ ID NO; 2) the sample identification (Sample ID); 3) the spot identification number (“SpotID”); and 4) the percentage of patients tested in which expression levels of the gene was at least 2-fold greater in cancerous tissue than in matched normal tissue (“ColonPatients pvalcorrected 95—>=2×”). The ratios of differential expression is expressed as a normalized hybridization signal associated with the tumor probe divided by the normalized hybridization signal with the normal probe. Thus, a ratio greater than 1 indicates that the gene product is increased in expression in cancerous cells relative to normal cells, while a ratio of less than 1 indicates the opposite.
TABLE 6
|
|
ColonPatients
Chippvalcorrected
SEQ ID NOSampleIDSpot Id95_ >= 2x
|
|
1RG:727787:Order7TM31:E072991282.14
7M00055209C:B072429730.30
9M00056908A:H052154442.42
13M00057000D:E082159230.30
27RG:1418951:Order7TM11:D123362378.57
29RG:1418951:Order7TM11:D123362378.57
22M00001346C:A0524355
22M00054893C:D032195230
|
These data provide evidence that the genes represented by the polynucleotides having the indicated sequences are differentially expressed in colon cancer.
Those skilled in the art will recognize, or be able to ascertain, using not more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such specific embodiments and equivalents are intended to be encompassed by the following claims.
All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
Deposit Information. A deposit of biologically pure cultures of the following viruses was made with the American Type Culture Collection, 10801 University Blvd., Manassa, Va. 20110-2209, under the provisions of the Budapest Treaty, on or before the filing date of the present application. The accession number indicated was assigned after successful viability testing, and the requisite fees were paid. Access to said cultures will be available during pendency of the patent application to one determined by the Commissioner to be entitled to such under 37 C.F.R. §1.14 and 35 U.S.C. §122. All restriction on availability of said cultures to the public will be irrevocably removed upon the granting of a patent based upon the application. Moreover, the designated deposits will be maintained for a period of thirty (30) years from the date of deposit, or for five (5) years after the last request for the deposit; or for the enforceable life of the U.S. patent, whichever is longer. Should a culture become nonviable or be inadvertently destroyed, or, in the case of plasmid-containing strains, lose its plasmid, it will be replaced with a viable culture(s) of the same taxonomic description.
These deposits are provided merely as a convenience to those of skill in the art, and are not an admission that a deposit is required. The nucleic acid sequences of these plasmids, as well as the amino sequences of the polypeptides encoded thereby, are controlling in the event of any conflict with the description herein. A license may be required to make, use, or sell the deposited materials, and no such license is hereby granted.
In addition, pools of selected clones, as well as libraries containing specific clones, were assigned an “ES” number (internal reference) and deposited with the ATCC. Table 7 below provides the ATCC Accession Nos. of the deposited clones, all of which were deposited on or before the filing date of the application.
TABLE 7
|
|
Pools of Clones and Libraries Deposited with the ATCC
Sequence NameClonesCMCCATCC
|
SK1SK-15162PTA-1360
SK2SK-25163PTA-1361
SK5SK-55164PTA-1362
1665 short1665 short5165PTA-1363
1665 long1665 long5166PTA-1363
sk19SK-195167PTA-1364
Junc2Junc2-65168PTA-1365
XD4XD4b5169PTA-1366
XD1XD1b5170PTA-1367
XD7XD7c5171PTA-1368
XD10XD10b5172PTA-1369
XD11XD11b5173PTA-1370
Junc4Junc4-25174PTA-1371
|
CMCC refers to applicant's internal reference number.
|
Retrieval of Individual Clones from Deposit of Pooled Clones. Where the ATCC deposit is composed of a pool of cDNA clones or a library of cDNA clones, the deposit was prepared by first transfecting each of the clones into separate bacterial cells. The clones in the pool or library were then deposited as a pool of equal mixtures in the composite deposit. Particular clones can be obtained from the composite deposit using methods well known in the art. For example, a bacterial cell containing a particular clone can be identified by isolating single colonies, and identifying colonies containing the specific clone through standard colony hybridization techniques, using an oligonucleotide probe or probes designed to specifically hybridize to a sequence of the clone insert (e.g., a probe based upon unmasked sequence of the encoded polynucleotide having the indicated SEQ ID NO). The probe should be designed to have a Tm of approximately 80° C. (assuming 2° C. for each A or T and 4° C. for each G or C). Positive colonies can then be picked, grown in culture, and the recombinant clone isolated. Alternatively, probes designed in this manner can be used to PCR to isolate a nucleic acid molecule from the pooled clones according to methods well known in the art, e.g., by purifying the cDNA from the deposited culture pool, and using the probes in PCR reactions to produce an amplified product having the corresponding desired polynucleotide sequence.
Example 13
ATCC Deposits
The following plasmids were deposited as a bacterial culture with plasmid cDNA on Sep. 25, 1998 with the American Type Culture Collection, 1301 Parklawn Drive, Rockville, Md., USA (ATCC) as ATCC accession no. 98896:
1) Clone HX2134-4 (containing an insert corresponding to SEQ ID NO:128),
2) Clone HX2144-1 (containing an insert corresponding to SEQ ID NO: 129);
3) Clone HX2145-3 (containing an insert corresponding to SEQ ID NO: 130);
4) Clone HX2162-3 (containing an insert corresponding to SEQ ID NO: 131);
5) Clone HX2166-6 (containing an insert corresponding to SEQ ID NO: 132); and
6) Clone HX2192-1 (containing an insert corresponding to SEQ ID NO:133).
The deposit was made under the conditions specified by the Budapest Treaty on the international recognition of the deposit of microorganisms (Budapest Treaty). Constructs and polynucleotides sequences equivalent to and/or substantially equivalent to the deposited material are also considered to be within the scope of this invention. Availability of the deposited material is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.
Each of the above clones was transfected into separate bacterial cells, and were deposited as a pool of equal mixtures of all six clones in this composite deposit. Each clone can be removed from the vector in which it was deposited by EcoRI to produce the appropriately sized 0.5 kb-1.0 kb fragment for the clone. Particular clones can be obtained from the composite deposit using methods well known in the art. For example, a bacterial cell containing a particular clone can be identified by isolating single colonies on an appropriate bacterial media containing ampicillin, and identifying colonies containing the specific clone through standard colony hybridization techniques, using an oligonucleotide probe or probes designed to specifically hybridize to a sequence of one of SEQ ID NOS:128-133. The probe should be designed to have a Tm of approximately 80 EC (assuming 2 EC for each A or T and 4 EC for each G or C). Positive colonies can then be picked, grown in culture, and the recombinant clone isolated.
Example 14
A family was identified that had several members who had been diagnosed with pancreatic cancer. The family members also have a form of diabetes. The pathological features of disease in the family included progression from normal to metaplasia to dysplasia to cancer. Tissues were obtained from a member of the family diagnosed with pancreatic cancer and from a member of the family diagnosed with dysplasia of pancreatic cells, and primary cultures of ductal cells prepared according to methods well known in the art. Tissue was also obtained from an unrelated person who was diagnosed with pancreatitis, and from an unrelated person who had a normal pancreas, and primary cultures of ductal cells prepared according to methods well known in the art.
The Genomyx HIEROGLYPH™ mRNA profile kit for differential display analysis was used according to the manufacturer's instructions to identify genes that are differentially expressed in the various samples relative to one another. Briefly, mRNA was isolated from the primary ductal cell cultures, and subjected to reverse transcriptase polymerase chain reaction (PCR). The resulting cDNA was subjected to a differential display in which the cDNA from each of the samples were compared on a gel.
The cDNA fragment pattern in each sample was manually compared to the cDNA fragment pattern in every other sample on the gel. Those bands representing differentially expressed gene products (e.g., bands associated with relatively more or less cDNA in one sample relative to another) were cut from the gel, amplified, cloned, and sequenced. The following polynucleotide sequences (SEQ ID NOS:128-133) of cDNA fragments isolated from six such differentially displayed cDNA fragments were identified as being differentially regulated in pancreatic disease.
TABLE 8
|
|
Results of Differential Display
Sequence
SEQ IDCloneLength
NO.Name(bp)Results
|
128HX2134-4676Expression decreased in dysplasia only
129HX2144-1544Expression increased in cancer only
130HX2145-3432Expression decreased in dysplasia only
131HX2162-3493Expression increased in dysplasia only
132HX2166-6418Expression increased in dysplasia only
133HX2192-11063Expression decreased in dysplasia and
cancer
|
The identification of these differentially expressed polynucleotides, as well as the correlation of the relative levels of expression of the represented differentially expressed genes with the disease states of pancreatic cancer and dysplasia, indicates that the gene products of the differentially expressed polynucleotides and genes can serve as markers of these disease states, where the markers can be used either singly or in combination with one another. Examination of expression of one or more of these differentially expressed polynucleotides can thus be used in classifying the cell from which the polynucleotides are derived as, for example, cancerous, dysplastic, or normal, and can further be used in diagnosis of the subject from whom the cell sample was derived. Use of all or a subset of the differentially expressed polynucleotides as markers will increase the sensitivity and the accuracy of the diagnosis.
Example 15
Sequencing and Analysis of Differentially Expressed Polynucleotides
The sequences of the differentially expressed polynucleotides identified in Example 1 (SEQ ID NOS:128-133) were used as query sequences in the GenBank and dbEST public databases to identify possible homologous sequences. The search was performed using the BLAST program, with default settings. All six sequences were novel, i.e., no sequence present in the databases searched contained a sequence having the contiguous nucleotide sequence set forth in any of SEQ ID NOS:128-133. Moreover, each of the polynucleotides contained stretches of contiguous nucleotides for which no homologous sequence was identified. A summary of these wholly unique sequences, referred to herein as identifying sequences, is provided in Table 9 below.
TABLE 9
|
|
Identifying sequences of the differentially expressed genes of
the invention.
Identifying Sequences
SEQ ID(numbering refers to nucleotide
NO:position in Sequence Listing)
|
1281-304; 533-571
1291-62; 102-139; 183-544
1301-41; 62-182; 216-281; 319-432
1311-13; 32-137; 156-236; 255-429; 453-493
1321-101; 408-418
133327-444; 640-997; 1018-1063
|
The identifying sequences above represent exemplary minimal, contiguous nucleotides sequences of the differentially expressed polynucleotides than can be used in identification or detection of the corresponding differentially expressed genes described herein.
Example 16
Fabricating a DNA Array Using Polynucleotides Differentially Expressed in Pancreatic Cells
A DNA array is made by spotting DNA fragments onto glass microscope slides that are pretreated with poly-L-lysine. Spotting onto the array is accomplished by a robotic arrayer. The DNA is cross-linked to the glass by ultraviolet irradiation, and the free poly-L-lysine groups are blocked by treatment with 0.05% succinic anhydride, 50% 1-methyl-2-pyrrolidinone and 50% borate buffer.
The spots on the array are oligonucleotides synthesized on an ABI automated synthesizer. Each spot is one of the polynucleotides of SEQ ID NOS:128-133, each of which correspond to a gene that is differentially expressed in pancreatic cells according to varying disease states (e.g., overexpressed or underexpressed in cancerous, dysplastic, pancreatitis, and/or diabetic pancreatic cells). The polynucleotides may be present on the array in any of a variety of combinations or subsets. Some internal standards and negative control spots including non-differentially expressed sequences and/or bacterial controls are included.
mRNA from patient samples is isolated, the mRNA used to produce cDNA, amplified and subsequently labeled with fluorescent nucleotides as follows: isolated mRNA is added to a standard PCR reaction containing primers (100 pmoles each), 250 uM nucleotides, and 5 Units of Taq polymerase (Perkin Elmer). In addition, fluorescent nucleotides (Cy3-dUTP (green fluorescence) or Cy5-dUTP (red fluorescence), sold by Amersham) are added to a final concentration of 60 uM. The reaction is carried out in a Perkin Elmer thermocycler (PE9600) for 30 cycles using the following cycle profile: 92° C. for 30 seconds, 58° C. for 30 seconds, and 72° C. for 2 minutes. Unincorporated fluorescent nucleotides are removed by size exclusion chromatography (Microcon-30 concentration devices, sold by Amicon).
Buffer replacement, removal of small nucleotides and primers and sample concentration is accomplished by ultrafiltration over an Amicon microconcentrator-30 (mwco=30,000 Da) with three changes of 0.45 ml TE. The sample is reduced to 5 μl and supplemented with 1.4 μl 20×SSC and 5 μg yeast tRNA. Particles are removed from this mixture by filtration through a pre-wetted 0.45μ microspin filter (Ultrafree-MC, Millipore, Bedford, Ma.). SDS is added to a 0.28% final concentration. The fluorescently-labeled cDNA mixture is then heated to 98° C. for 2 min., quickly cooled and applied to the DNA array on a microscope slide. Hybridization proceeds under a coverslip, and the slide assembly is kept in a humidified chamber at 65° C. for 15 hours.
The slide is washed briefly in 1×SSC and 0.03% SDS, followed by a wash in 0.06% SSC. The slide is kept in a humidified chamber until fluorescence scanning was done. Fluorescence scanning and data acquisition are then accomplished using any of a variety of suitable methods well known in the art. For example, fluorescence scanning is set for 20 microns/pixel and two readings are taken per pixel. Data for channel 1 is set to collect fluorescence from Cy3 with excitation at 520 nm and emission at 550-600 nm. Channel 2 collects signals excited at 647 nm and emitted at 660-705 nm, appropriate for Cy5. No neutral density filters are applied to the signal from either channel, and the photomultiplier tube gain is set to 5. Fine adjustments are then made to the photomultiplier gain so that signals collected from the two spots are equivalent.
The data acquired from the scan of the array is then converted to any suitable form for analysis. For example, the data may be analyzed using a computer system, and the data may be displayed in a pictoral format on a computer screen, where the display shows the array as a collection of spots, each spot corresponding to a location of a different polynucleotide on the array. The spots vary in brightness according to the amount of fluorescent probe associated with the spot, which in turn is correlated with an amount of hybridized cDNA in the sample. The relative brightness of the spots on the array can be compared with one another to determine their relative intensities, either qualitatively or quantitatively.
The display of spots on the array, along with their relative brightness, provides a test sample pattern. The test sample pattern can be then compared with reference array patterns associated with positive and negative control samples on the same array, e.g., an array having polynucleotides in substantially the same locations as the array used with the test sample. The reference array patterns used in the comparison can be array patterns generated using samples from normal pancreas cells, cancerous pancreas cells, pancreatitis-associated pancreas cells, diabetic pancreas cells, and the like. A substantial or significant match between the test array pattern and a reference array pattern is indicative of a disease state of the patient from whom the test sample was obtained.
Example 17
Source of Biological Materials and Overview of Novel Polynucleotides Expressed by the Biological Materials
Candidate polynucleotides that may represent novel polynucleotides were obtained from cDNA libraries generated from selected cell lines and patient tissues. In order to obtain the candidate polynucleotides, mRNA was isolated from several selected cell lines and patient tissues, and used to construct cDNA libraries. The cells and tissues that served as sources for these cDNA libraries are summarized in Table 10 below.
Human colon cancer cell line Km12L4-A (Morikawa, et al., Cancer Research (1988) 48:6863) is derived from the KM12C cell line. The KM12C cell line (Morikawa et al. Cancer Res. (1988) 48:1943-1948), which is poorly metastatic (low metastatic) was established in culture from a Dukes' stage B2 surgical specimen (Morikawa et al. Cancer Res. (1988) 48:6863). The KM12L4-A is a highly metastatic subline derived from KM12C (Yeatman et al. Nucl. Acids. Res. (1995) 23:4007; Bao-Ling et al. Proc. Annu. Meet. Am. Assoc. Cancer. Res. (1995) 21:3269). The KM12C and KM12C-derived cell lines (e.g., KM12L4, KM12L4-A, etc.) are well-recognized in the art as a model cell line for the study of colon cancer (see, e.g., Moriakawa et al., supra; Radinsky et al. Clin. Cancer Res. (1995) 1:19; Yeatman et al., (1995) supra; Yeatman et al. Clin. Exp. Metastasis (1996) 14:246).
The MDA-MB-231 cell line (Brinkley et al. Cancer Res. (1980) 40:3118-3129) was originally isolated from pleural effusions (Cailleau, J. Natl. Cancer. Inst. (1974) 53:661), is of high metastatic potential, and forms poorly differentiated adenocarcinoma grade II in nude mice consistent with breast carcinoma. The MCF7 cell line was derived from a pleural effusion of a breast adenocarcinoma and is non-metastatic. The MV-522 cell line is derived from a human lung carcinoma and is of high metastatic potential. The UCP-3 cell line is a low metastatic human lung carcinoma cell line; the MV-522 is a high metastatic variant of UCP-3. These cell lines are well-recognized in the art as models for the study of human breast and lung cancer (see, e.g., Chandrasekaran et al., Cancer Res. (1979) 39:870 (MDA-MB-231 and MCF-7); Gastpar et al., J Med Chem (1998) 41:4965 (MDA-MB-231 and MCF-7); Ranson et al., Br J Cancer (1998) 77:1586 (MDA-MB-231 and MCF-7); Kuang et al., Nucleic Acids Res (1998) 26:1116 (MDA-MB-231 and MCF-7); Varki et al., Int J Cancer (1987) 40:46 (UCP-3); Varki et al., Tumour Biol. (1990) 11:327; (MV-522 and UCP-3); Varki et al., Anticancer Res. (1990) 10:637; (MV-522); Kelner et al., Anticancer Res (1995) 15:867 (MV-522); and Zhang et al., Anticancer Drugs (1997) 8:696 (MV522)).
The samples of libraries 15-20 are derived from two different patients (UC#2, and UC#3). The bFGF-treated HMVEC were prepared by incubation with bFGF at 10 ng/ml for 2 hrs; the VEGF-treated HMVEC were prepared by incubation with 20 ng/ml VEGF for 2 hrs. Following incubation with the respective growth factor, the cells were washed and lysis buffer added for RNA preparation.
GRRpz was derived from normal prostate epithelium. The WOca cell line is a Gleason Grade 4 cell line.
The source materials for generating the normalized prostate libraries of libraries 25 and 26 were cryopreserved prostate tumor tissue from a patient with Gleason grade 3+3 adenocarcinoma and matched normal prostate biopsies from a pool of at-risk subjects under medical surveillance. The source materials for generating the normalized prostate libraries of libraries 30 and 31 were cryopreserved prostate tumor tissue from a patient with Gleason grade 4+4 adenocarcinoma and matched normal prostate biopsies from a pool of at-risk subjects under medical surveillance.
The source materials for generating the normalized breast libraries of libraries 27, 28 and 29 were cryopreserved breast tissue from a primary breast tumor (infiltrating ductal carcinoma)(library 28), from a lymph node metastasis (library 29), or matched normal breast biopsies from a pool of at-risk subjects under medical surveillance. In each case, prostate or breast epithelia were harvested directly from frozen sections of tissue by laser capture microdissection (LCM, Arcturus Enginering Inc., Mountain View, Calif.), carried out according to methods well known in the art (see, Simone et al. Am J Pathol. 156(2):445-52 (2000)), to provide substantially homogenous cell samples.
TABLE 10
|
|
Description of cDNA Libraries
Number
Libraryof Clones in
(lib#)DescriptionLibrary
|
0Artificial library composed of deselected clones (clones with673
no associated variant or cluster)
1Human Colon Cell Line Km12 L4: High Metastatic Potential308731
(derived from Km12C)
2Human Colon Cell Line Km12C: Low Metastatic Potential284771
3Human Breast Cancer Cell Line MDA-MB-231: High326937
Metastatic Potential; micro-mets in lung
4Human Breast Cancer Cell Line MCF7: Non Metastatic318979
8Human Lung Cancer Cell Line MV-522: High Metastatic223620
Potential
9Human Lung Cancer Cell Line UCP-3: Low Metastatic312503
Potential
12Human microvascular endothelial cells (HMEC) -41938
UNTREATED (PCR (OligodT) cDNA library)
13Human microvascular endothelial cells (HMEC) - bFGF42100
TREATED (PCR (OligodT) cDNA library)
14Human microvascular endothelial cells (HMEC) - VEGF42825
TREATED (PCR (OligodT) cDNA library)
15Normal Colon - UC#2 Patient (MICRODISSECTED PCR282722
(OligodT) cDNA library)
16Colon Tumor - UC#2 Patient (MICRODISSECTED PCR298831
(OligodT) cDNA library)
17Liver Metastasis from Colon Tumor of UC#2 Patient303467
(MICRODISSECTED PCR (OligodT) cDNA library)
18Normal Colon - UC#3 Patient (MICRODISSECTED PCR36216
(OligodT) cDNA library)
19Colon Tumor - UC#3 Patient (MICRODISSECTED PCR41388
(OligodT) cDNA library)
20Liver Metastasis from Colon Tumor of UC#3 Patient30956
(MICRODISSECTED PCR (OligodT) cDNA library)
21GRRpz Cells derived from normal prostate epithelium164801
22WOca Cells derived from Gleason Grade 4 prostate cancer162088
epithelium
23Normal Lung Epithelium of Patient #1006306198
(MICRODISSECTED PCR (OligodT) cDNA library)
24Primary tumor, Large Cell Carcinoma of Patient #1006309349
(MICRODISSECTED PCR (OligodT) cDNA library)
25Normal Prostate Epithelium from Patient IF97-26811279444
26Prostate Cancer Epithelium Gleason 3 + 3 Patient IF97-26811269406
27Normal Breast Epithelium from Patient 515239494
28Primary Breast tumor from Patient 515259960
29Lymph node metastasis from Patient 515326786
30Normal Prostate Epithelium from Chiron Patient ID 884298431
31Prostate Cancer Epithelium (Gleason 4 + 4) from Chiron Patient331941
ID 884
|
Characterization of Sequences in the Libraries
After using the software program Phred (ver 0.000925.c, Green and Weing, ©11993-2000) to select those polynucleotides having the best quality sequence, the polynucleotides were compared against the public databases to identify any homologous sequences. The sequences of the isolated polynucleotides were first masked to eliminate low complexity sequences using the RepeatMasker masking program, publicly available through a web site supported by the University of Washington (See also Smit, A. F. A. and Green, P., unpublished results). Generally, masking does not influence the final search results, except to eliminate sequences of relatively little interest due to their low complexity, and to eliminate multiple “hits” based on similarity to repetitive regions common to multiple sequences, e.g., Alu repeats.
The remaining sequences were then used in a homology search of the GenBank database using the TeraBLAST program (TimeLogic, Crystal Bay, Nev.). TeraBLAST is a version of the publicly available BLAST search algorithm developed by the National Center for Biotechnology, modified to operate at an accelerated speed with increased sensitivity on a specialized computer hardware platform. The program was run with the default parameters recommended by TimeLogic to provide the best sensitivity and speed for searching DNA and protein sequences. Sequences that exhibited greater than 70% overlap, 99% identity, and a p value of less than 1×10e-40 were discarded. Sequences from this search also were discarded if the inclusive parameters were met, but the sequence was ribosomal or vector-derived.
The resulting sequences from the previous search were classified into three groups (1, 2 and 3 below) and searched in a TeraBLASTX vs. NRP (non-redundant proteins) database search: (1) unknown (no hits in the GenBank search), (2) weak similarity (greater than 45% identity and p value of less than 1×10e-5), and (3) high similarity (greater than 60% overlap, greater than 80% identity, and p value less than 1×10e-5). Sequences having greater than 70% overlap, greater than 99% identity, and p value of less than 1×10e-40 were discarded.
The remaining sequences were classified as unknown (no hits), weak similarity, and high similarity (parameters as above). Two searches were performed on these sequences. First, a TeraBLAST vs. EST database search was performed and sequences with greater than 99% overlap, greater than 99% similarity and a p value of less than 1×10e-40 were discarded. Sequences with a p value of less than 1×10e-65 when compared to a database sequence of human origin were also excluded. Second, a TeraBLASTN vs. Patent GeneSeq database was performed and sequences having greater than 99% identity, p value less than 1×10e-40, and greater than 99% overlap were discarded.
The remaining sequences were subjected to screening using other rules and redundancies in the dataset. Sequences with a p value of less than 1×10e-111 in relation to a database sequence of human origin were specifically excluded. The final result provided the sequences listed as SEQ ID NOS:134-1352 in the accompanying Sequence Listing and summarized in Table 11. Each identified polynucleotide represents sequence from at least a partial mRNA transcript.
TABLE 11
|
|
SEQ
IDCLUSTERSEQ NAMECLONE IDLIBRARY
|
|
1343573673538.O24.GZ43_504925M00084399B:E05chiron(cc187-NormBPHProstate)
1357259973538.P11.GZ43_504718M00084400A:B09chiron(cc187-NormBPHProstate)
1366459863541.A04.GZ43_504975M00084406A:B03chiron(cc187-NormBPHProstate)
1374078283541.A05.GZ43_504991M00084407A:H09chiron(cc187-NormBPHProstate)
1386491173541.A16.GZ43_505167M00084421C:B11chiron(cc187-NormBPHProstate)
1394246783541.A23.GZ43_505279M00084431C:G08chiron(cc187-NormBPHProstate)
1408542883541.B04.GZ43_504976M00084406C:A01chiron(cc187-NormBPHProstate)
1416399013541.B17.GZ43_505184M00084424A:G07chiron(cc187-NormBPHProstate)
1428422653538.G08.GZ43_504661M00084379D:A05chiron(cc187-NormBPHProstate)
1435577173538.G17.GZ43_504805M00084380C:C09chiron(cc187-NormBPHProstate)
1444599673538.G19.GZ43_504837M00084380D:B07chiron(cc187-NormBPHProstate)
1455057503538.G22.GZ43_504885M00084381C:A05chiron(cc187-NormBPHProstate)
14610535643538.H05.GZ43_504614M00084382A:D06chiron(cc187-NormBPHProstate)
1475423013538.H21.GZ43_504870M00084383B:A11chiron(cc187-NormBPHProstate)
148214463538.I08.GZ43_504663M00084385A:D02chiron(cc187-NormBPHProstate)
14911404183538.I13.GZ43_504743M00084385B:D03chiron(cc187-NormBPHProstate)
1505304533538.J22.GZ43_504888M00084388A:G03chiron(cc187-NormBPHProstate)
15112047823538.K12.GZ43_504729M00084389A:F12chiron(cc187-NormBPHProstate)
1528634753538.K23.GZ43_504905M00084390B:H04chiron(cc187-NormBPHProstate)
1534521243538.L16.GZ43_504794M00084391B:D06chiron(cc187-NormBPHProstate)
1546505203538.M02.GZ43_504571M00084392C:D03chiron(cc187-NormBPHProstate)
15511177713538.M05.GZ43_504619M00084392C:G06chiron(cc187-NormBPHProstate)
1564340743538.M08.GZ43_504667M00084393A:G07chiron(cc187-NormBPHProstate)
1578666093538.N20.GZ43_504860M00084396B:B03chiron(cc187-NormBPHProstate)
1589452473538.O07.GZ43_504653M00084397D:A09chiron(cc187-NormBPHProstate)
15910536233541.E11.GZ43_505091M00084415C:C05chiron(cc187-NormBPHProstate)
1607228783541.E14.GZ43_505139M00084419C:A09chiron(cc187-NormBPHProstate)
16112264933541.E15.GZ43_505155M00084420C:D03chiron(cc187-NormBPHProstate)
1628120313541.G17.GZ43_505189M00084423C:G11chiron(cc187-NormBPHProstate)
1637259973541.H14.GZ43_505142M00084420A:G02chiron(cc187-NormBPHProstate)
16411915473541.I15.GZ43_505159M00084420D:C07chiron(cc187-NormBPHProstate)
165213703541.I17.GZ43_505191M00084423D:B05chiron(cc187-NormBPHProstate)
1664183203541.I18.GZ43_505207M00084424D:G07chiron(cc187-NormBPHProstate)
167245803541.J19.GZ43_505224M00084427B:D01chiron(cc187-NormBPHProstate)
1686475873541.K09.GZ43_505065M00084413C:A11chiron(cc187-NormBPHProstate)
16912259893541.L19.GZ43_505226M00084427C:D04chiron(cc187-NormBPHProstate)
17010798633541.M02.GZ43_504955M00084403D:D04chiron(cc187-NormBPHProstate)
17111368033541.M07.GZ43_505035M00084410C:F10chiron(cc187-NormBPHProstate)
1725282813541.M18.GZ43_505211M00084425A:A01chiron(cc187-NormBPHProstate)
1736609073541.O04.GZ43_504989M00084406B:C03chiron(cc187-NormBPHProstate)
1744025883541.O13.GZ43_505133M00084418D:A04chiron(cc187-NormBPHProstate)
1759471683541.O23.GZ43_505293M00084432B:C05chiron(cc187-NormBPHProstate)
17612239483541.P05.GZ43_505006M00084408D:E06chiron(cc187-NormBPHProstate)
1774261383541.P22.GZ43_505278M00084431C:B02chiron(cc187-NormBPHProstate)
17810378873544.A09.GZ43_505439M00084447D:F03chiron(cc187-NormBPHProstate)
1794683343544.A13.GZ43_505503M00084454A:G08cbiron(cc187-NormBPHProstate)
18011404093544.A14.GZ43_505519M00084456A:H04chiron(cc187-NormBPHProstate)
1815557263544.A17.GZ43_505567M00084463A:B07chiron(cc187-NormBPHProstate)
1827269223544.B02.GZ43_505328M00084437C:G05chiron(cc187-NormBPHProstate)
1834025163544.B09.GZ43_505440M00084448B:D11chiron(cc187-NormBPHProstate)
1848120313544.B18.GZ43_505584M00084467A:D06chiron(cc187-NormBPHProstate)
1854481773544.E05.GZ43_505379M00084441D:E09chiron(cc187-NormBPHProstate)
1865057503544.E18.GZ43_505587M00084466B:E01chiron(cc187-NormBPHProstate)
1875083223544.F06.GZ43_505396M00084443C:H06chiron(cc187-NormBPHProstate)
18812240723544.F16.GZ43_505556M00084461C:D06chiron(cc187-NormBPHProstate)
1898013544.G06.GZ43_505397M00084443A:E10chiron(cc187-NormBPHProstate)
1907481013544.G10.GZ43_505461M00084449A:D09chiron(cc187-NormBPHProstate)
19112241073544.G11.GZ43_505477M00084450C:A09chiron(cc187-NormBPHProstate)
19212268453544.G12.GZ43_505493M00084452B:F07chiron(cc187-NormBPHProstate)
19310737673544.H03.GZ43_505350M00084439B:A08chiron(cc187-NormBPHProstate)
19412247523544.H15.GZ43_505542M00084459A:F10chiron(cc187-NormBPHProstate)
19510524803544.H24.GZ43_505686M00084434B:E06chiron(cc187-NormBPHProstate)
1962450313544.I07.GZ43_505415M00084444D:F09chiron(cc187-NormBPHProstate)
1974944993544.I15.GZ43_505543M00084458A:G06chiron(cc187-NormBPHProstate)
19811385933544.I20.GZ43_505623M00084469A:C09chiron(cc187-NormBPHProstate)
19911396913544.J04.GZ43_505368M00084441B:E05chiron(cc187-NormBPHProstate)
2007906933544.J11.GZ43_505480M00084451D:A03chiron(cc187-NormBPHProstate)
20111170033544.J13.GZ43_505512M00084455D:B03chiron(cc187-NormBPHProstate)
2028447403544.J23.GZ43_505672M00084475B:D03chiron(cc187-NormBPHProstate)
2034525643544.K16.GZ43_505561M00084460D:B04chiron(cc187-NormBPHProstate)
2048628233544.L11.GZ43_505482M00084451D:F06chiron(cc187-NormBPHProstate)
205193673544.L13.GZ43_505514M00084455D:G03chiron(cc187-NormBPHProstate)
20611946563544.M06.GZ43_505403M00084443B:C02chiron(cc187-NormBPHProstate)
20712248793544.M10.GZ43_505467M00084449B:C09chiron(cc187-NormBPHProstate)
2084549043544.N07.GZ43_505420M00084446A:A05chiron(cc187-NormBPHProstate)
2096766653544.N12.GZ43_505500M00084453D:B12chiron(cc187-NormBPHProstate)
2105428253544.N19.GZ43_505612M00084468C:E07chiron(cc187-NormBPHProstate)
2114119603544.O03.GZ43_505357M00084438D:H04chiron(cc187-NormBPHProstate)
2129367953544.O10.GZ43_505469M00084449C:C01chiron(cc187-NormBPHProstate)
21312834373544.O15.GZ43_505549M00084458B:G05chiron(cc187-NormBPHProstate)
2144021503544.O20.GZ43_505629M00084469B:F08chiron(cc187-NormBPHProstate)
2154547333544.P18.GZ43_505598M00084468A:A09chiron(cc187-NormBPHProstate)
21612110323547.A04.GZ43_505743M00084483A:C06chiron(cc187-NormBPHProstate)
2174527633547.A11.GZ43_505855M00084493A:E03chiron(cc187-NormBPHProstate)
2185282813547.A24.GZ43_506063M00084475C:G11chiron(cc187-NormBPHProstate)
21911368033547.C05.GZ43_505761M00084484C:B11chiron(cc187-NormBPHProstate)
2204548263547.C17.GZ43_505953M00084500D:B11chiron(cc187-NormBPHProstate)
22110548073547.C23.GZ43_506049M00084510D:D05chiron(cc187-NormBPHProstate)
2227263863547.D19.GZ43_505986M00084504C:F05chiron(cc187-NormBPHProstate)
22312237053547.D23.GZ43_506050M00084511D:A02chiron(cc187-NormBPHProstate)
2243984393547.E04.GZ43_505747M00084483A:E05chiron(cc187-NormBPHProstate)
2258331743547.F02.GZ43_505716M00084480B:A05chiron(cc187-NormBPHProstate)
2265009193547.F10.GZ43_505844M00084492B:F03chiron(cc187-NormBPHProstate)
22712260643547.F20.GZ43_506004M00084506A:E08chiron(cc187-NormBPHProstate)
2285555093547.G02.GZ43_505717M00084479B:E04chiron(cc187-NormBPHProstate)
2296538173547.G09.GZ43_505829M00084490A:C12chiron(cc187-NormBPHProstate)
2304782123547.G22.GZ43_506037M00084509D:C02chiron(cc187-NormBPHProstate)
23110540743547.H12.GZ43_505878M00084495B:C11chiron(cc187-NormBPHProstate)
23210600213547.H14.GZ43_505910M00084497D:D03chiron(cc187-NormBPHProstate)
23312273523547.I07.GZ43_505799M00084487C:H06chiron(cc187-NormBPHProstate)
23482933547.I16.GZ43_505943M00084499C:C11chiron(cc187-NormBPHProstate)
2354771103547.I17.GZ43_505959M00084501A:D06chiron(cc187-NormBPHProstate)
23612240393547.I20.GZ43_506007M00084505C:H08chiron(cc187-NormBPHProstate)
2375423013547.J05.GZ43_505768M00084485C:B04chiron(cc187-NormBPHProstate)
2384552113547.J10.GZ43_505848M00084492C:B05chiron(cc187-NormBPHProstate)
23910563693547.J20.GZ43_506008M00084506C:A05chiron(cc187-NormBPHProstate)
2405498143547.J22.GZ43_506040M00084510C:F02chiron(cc187-NormBPHProstate)
2415096733547.K01.GZ43_505705M00084477C:C07chiron(cc187-NormBPHProstate)
2427362563547.L09.GZ43_505834M00084491A:E08chiron(cc187-NormBPHProstate)
24311398493547.L11.GZ43_505866M00084494C:C01chiron(cc187-NormBPHProstate)
24412239383547.L16.GZ43_505946M00084500C:D01chiron(cc187-NormBPHProstate)
24510594453547.L22.GZ43_506042M00084510C:F05chiron(cc187-NormBPHProstate)
2464782123547.M02.GZ43_505723M00084479D:E10chiron(cc187-NormBPHProstate)
24711404183547.M07.GZ43_505803M00084487D:F04chiron(cc187-NormBPHProstate)
2485349433547.M08.GZ43_505819M00084489A:D12chiron(cc187-NormBPHProstate)
2497080253547.M16.GZ43_505947M00084499D:A10chiron(cc187-NormBPHProstate)
25011384193547.N06.GZ43_505788M00084487B:A06chiron(cc187-NormBPHProstate)
25112265883547.O03.GZ43_505741M00084481D:C06chiron(cc187-NormBPHProstate)
2524616693547.O07.GZ43_505805M00084487D:F07chiron(cc187-NormBPHProstate)
25310600213547.O14.GZ43_505917M00084497B:C12chiron(cc187-NormBPHProstate)
2543079853547.P18.GZ43_505982M00084503D:G10chiron(cc187-NormBPHProstate)
2558408523547.P21.GZ43_506030M00084509A:E10chiron(cc187-NormBPHProstate)
2564022863547.P22.GZ43_506046M00084510C:H01chiron(cc187-NormBPHProstate)
2574519943550.A12.GZ43_506255M00084513C:C10chiron(cc187-NormBPHProstate)
2584516793550.A16.GZ43_506319M00084514A:A03chiron(cc187-NormBPHProstate)
2594506073550.B06.GZ43_506160M00084515D:G03chiron(cc187-NormBPHProstate)
2608875603550.C01.GZ43_506081M00084517C:D06chiron(cc187-NormBPHProstate)
2617273963550.C22.GZ43_506417M00084519B:D01chiron(cc187-NormBPHProstate)
26212243793550.D16.GZ43_506322M00084520B:A12chiron(cc187-NormBPHProstate)
26311370963550.D23.GZ43_506434M00084521A:E11chiron(cc187-NormBPHProstate)
26410524663550.E02.GZ43_506099M00084521B:E11chiron(cc187-NormBPHProstate)
26510649753550.E06.GZ43_506163M00084521C:H11chiron(cc187-NormBPHProstate)
2661800923550.F06.GZ43_506164M00084523C:A05chiron(cc187-NormBPHProstate)
26712242693550.F08.GZ43_506196M00084523C:C10chiron(cc187-NormBPHProstate)
26810535643550.F20.GZ43_506388M00084524D:D02chiron(cc187-NormBPHProstate)
2696778583550.F22.GZ43_506420M00084525A:E08chiron(cc187-NormBPHProstate)
27012255003550.G02.GZ43_506101M00084525D:H01chiron(cc187-NormBPHProstate)
27110540383550.G08.GZ43_506197M00084526C:G09chiron(cc187-NormBPHProstate)
27210378873550.G10.GZ43_506229M00084526D:E09chiron(cc187-NormBPHProstate)
2739640803550.G15.GZ43_506309M00084527C:H07chiron(cc187-NormBPHProstate)
2745538973550.G23.GZ43_506437M00084528C:F06chiron(cc187-NormBPHProstate)
2757553913550.H10.GZ43_506230M00084530D:G07chiron(cc187-NormBPHProstate)
2766441743550.H21.GZ43_506406M00084533A:C04chiron(cc187-NormBPHProstate)
2778939813550.H23.GZ43_506438M00084533B:B10chiron(cc187-NormBPHProstate)
2788215363550.I03.GZ43_506119M00084534B:E12chiron(cc187-NormBPHProstate)
27912273363550.I19.GZ43_506375M00084535D:C12chiron(cc187-NormBPHProstate)
2809913663550.I21.GZ43_506407M00084536B:A03chiron(cc187-NormBPHProstate)
2815498143550.J05.GZ43_506152M00084536D:F07chiron(cc187-NormBPHProstate)
2824025883550.J11.GZ43_506248M00084537B:C05chiron(cc187-NormBPHProstate)
2837101943550.K05.GZ43_506153M00084539D:D11chiron(cc187-NormBPHProstate)
28412227093550.K09.GZ43_506217M00084540B:B08chiron(cc187-NormBPHProstate)
28510537643550.K14.GZ43_506297M00084540D:B12chiron(cc187-NormBPHProstate)
28610625373550.L16.GZ43_506330M00084545C:C05chiron(cc187-NormBPHProstate)
2879645933550.L19.GZ43_506378M00084546C:C06chiron(cc187-NormBPHProstate)
28810540383550.L23.GZ43_506442M00084547B:B10chiron(cc187-NormBPHProstate)
28912234773550.M21.GZ43_506411M00084553B:F04chiron(cc187-NormBPHProstate)
2904025163550.N01.GZ43_506092M00084553D:G05chiron(cc187-NormBPHProstate)
2915170143550.N07.GZ43_506188M00084554C:D05chiron(cc187-NormBPHProstate)
29212235053550.O03.GZ43_506125M00084558D:A04chiron(cc187-NormBPHProstate)
2938727873550.O04.GZ43_506141M00084558D:G08chiron(cc187-NormBPHProstate)
2944053663550.O08.GZ43_506205M00084559B:F10chiron(cc187-NormBPHProstate)
295483433550.O15.GZ43_506317M00084560A:G08chiron(cc187-NormBPHProstate)
29610741603550.O17.GZ43_506349M00084560B:F12chiron(cc187-NormBPHProstate)
29712257193550.O18.GZ43_506365M00084560C:G05chiron(cc187-NormBPHProstate)
2988567033550.O21.GZ43_506413M00084561C:D07chiron(cc187-NormBPHProstate)
2999701653550.P18.GZ43_506366M00084565A:D10chiron(cc187-NormBPHProstate)
3004948903550.P23.GZ43_506446M00084565D:F08chiron(cc187-NormBPHProstate)
30112850393553.A09.GZ43_506591M00084587C:A07chiron(cc187-NormBPHProstate)
30212004533553.B07.GZ43_506560M00084584B:G07chiron(cc187-NormBPHProstate)
30312196173553.B16.GZ43_506704M00084602D:B09chiron(cc187-NormBPHProstate)
30410429183553.B22.GZ43_506800M00084612C:B01chiron(cc187-NormBPHProstate)
30512260863553.D04.GZ43_506514M00084576A:E12chiron(cc187-NormBPHProstate)
3068610253553.D07.GZ43_506562M00084584B:H12chiron(cc187-NormBPHProstate)
30712245053553.D14.GZ43_506674M00084598D:H05chiron(cc187-NormBPHProstate)
3086797243553.D19.GZ43_506754M00084605D:G09chiron(cc187-NormBPHProstate)
3092346673553.E08.GZ43_506579M00084585D:H12chiron(cc187-NormBPHProstate)
3104485763553.E09.GZ43_506595M00084587C:G07chiron(cc187-NormBPHProstate)
3114506073553.F12.GZ43_506644M00084595C:C07chiron(cc187-NormBPHProstate)
3124523223553.F13.GZ43_506660M00084597A:F06chiron(cc187-NormBPHProstate)
31312253843553.F19.GZ43_506756M00084607A:B03chiron(cc187-NormBPHProstate)
3149742233553.G05.GZ43_506533M00084578B:E12chiron(cc187-NormBPHProstate)
3158457153553.G06.GZ43_506549M00084581B:E06chiron(cc187-NormBPHProstate)
31611389973553.G07.GZ43_506565M00084583D:H12chiron(cc187-NormBPHProstate)
3174782123553.G21.GZ43_506789M00084609C:F10chiron(cc187-NormBPHProstate)
3188671483553.H06.GZ43_506550M00084582C:H03chiron(cc187-NormBPHProstate)
31912142023553.H09.GZ43_506598M00084588B:D02chiron(cc187-NormBPHProstate)
3205858993553.H21.GZ43_506790M00084610D:H04chiron(cc187-NormBPHProstate)
3218634753553.I13.GZ43_506663M00084596D:E10chiron(cc187-NormBPHProstate)
3227259823553.I16.GZ43_506711M00084602C:E04chiron(cc187-NormBPHProstate)
323170753553.J12.GZ43_506648M00084595D:D08chiron(cc187-NormBPHProstate)
32410548133553.J14.GZ43_506680M00084599D:C02chiron(cc187-NormBPHProstate)
32510649753553.J16.GZ43_506712M00084603A:B07chiron(cc187-NormBPHProstate)
32610550893553.J17.GZ43_506728M00084604A:D02chiron(cc187-NormBPHProstate)
3275518483553.J22.GZ43_506808M00084613A:A01chiron(cc187-NormBPHProstate)
3285858993553.J24.GZ43_506840M00084568D:A02chiron(cc187-NormBPHProstate)
32912118993553.K01.GZ43_506473M00084569D:B04chiron(cc187-NormBPHProstate)
3304604993553.K02.GZ43_506489M00084571C:D05chiron(cc187-NormBPHProstate)
331391153553.K03.GZ43_506505M00084573D:G11chiron(cc187-NormBPHProstate)
3322429013553.K05.GZ43_506537M00084578C:G09chiron(cc187-NormBPHProstate)
3337198923553.K07.GZ43_506569M00084584B:A02chiron(cc187-NormBPHProstate)
33410856383553.K15.GZ43_506697M00084600D:B10chiron(cc187-NormBPHProstate)
3354494653538.A11.GZ43_504703M00084363A:C02chiron(cc187-NormBPHProstate)
3365428253538.A24.GZ43_504911M00084364C:B06chiron(cc187-NormBPHProstate)
33710655313538.B01.GZ43_504544M00084364D:F08chiron(cc187-NormBPHProstate)
3389858593538.B20.GZ43_504848M00084367D:E06chiron(cc187-NormBPHProstate)
3394091823538.C01.GZ43_504545M00084368D:C02chiron(cc187-NormBPHProstate)
34012232713538.C02.GZ43_504561M00084368D:D03chiron(cc187-NormBPHProstate)
3414457423538.D06.GZ43_504626M00084372D:H11chiron(cc187-NormBPHProstate)
3424516793538.D09.GZ43_504674M00084373A:F08chiron(cc187-NormBPHProstate)
34311413713538.D21.GZ43_504866M00084374A:A10chiron(cc187-NormBPHProstate)
34410147343538.E15.GZ43_504771M00084376A:E06chiron(cc187-NormBPHProstate)
34512269323538.F02.GZ43_504564M00084377B:E11chiron(cc187-NormBPHProstate)
34612273033538.F08.GZ43_504660M00084377D:E08chiron(cc187-NormBPHProstate)
3475613903553.K23.GZ43_506825M00084614C:G05chiron(cc187-NormBPHProstate)
3485297093553.K24.GZ43_506841M00084567B:F03chiron(cc187-NormBPHProstate)
34912277813553.L02.GZ43_506490M00084572D:F07chiron(cc187-NormBPHProstate)
35011385723553.L04.GZ43_506522M00084577B:C08chiron(cc187-NormBPHProstate)
35111170033553.L21.GZ43_506794M00084611A:A06chiron(cc187-NormBPHProstate)
35212280333553.M12.GZ43_506651M00084595B:C08chiron(cc187-NormBPHProstate)
3539611193553.M23.GZ43_506827M00084614D:A08chiron(cc187-NormBPHProstate)
3546115923553.N01.GZ43_506476M00084571A:C02chiron(cc187-NormBPHProstate)
3555551153553.N02.GZ43_506492M00084573A:A10chiron(cc187-NormBPHProstate)
3568567033553.N04.GZ43_506524M00084577B:D04chiron(cc187-NormBPHProstate)
3578460563553.N07.GZ43_506572M00084585B:D06chiron(cc187-NormBPHProstate)
3586402773553.N08.GZ43_506588M00084587B:H07chiron(cc187-NormBPHProstate)
3592141923553.O07.GZ43_506573M00084584B:F09chiron(cc187-NormBPHProstate)
36012261603553.O18.GZ43_506749M00084604D:D08chiron(cc187-NormBPHProstate)
36112279683553.O23.GZ43_506829M00084614D:B07chiron(cc187-NormBPHProstate)
36212242693553.P03.GZ43_506510M00084575A:A11chiron(cc187-NormBPHProstate)
3637745203553.P05.GZ43_506542M00084580B:B05chiron(cc187-NormBPHProstate)
36412274573553.P12.GZ43_506654M00084596A:G03chiron(cc187-NormBPHProstate)
36511101433553.P18.GZ43_506750M00084605B:H04chiron(cc187-NormBPHProstate)
3665541893553.P21.GZ43_506798M00084611B:A11chiron(cc187-NormBPHProstate)
36747453556.A03.GZ43_506879M00084620D:E05chiron(cc187-NormBPHProstate)
36811947313556.A06.GZ43_506927M00084633B:A06chiron(cc187-NormBPHProstate)
3699701653556.B06.GZ43_506928M00084634A:D01chiron(cc187-NormBPHProstate)
37012244223556.B09.GZ43_506976M00084640D:A08chiron(cc187-NormBPHProstate)
3716134113556.B10.GZ43_506992M00084642C:F10chiron(cc187-NormBPHProstate)
37210563693556.B14.GZ43_507056M00084647C:E12chiron(cc187-NormBPHProstate)
373843473556.C13.GZ43_507041M00084645D:G02chiron(cc187-NormBPHProstate)
37411385933556.C15.GZ43_507073M00084648A:F08chiron(cc187-NormBPHProstate)
3758457153556.C18.GZ43_507121M00084654A:E04chiron(cc187-NormBPHProstate)
3769712263556.C24.GZ43_507217M00084615D:H12chiron(cc187-NormBPHProstate)
37711385933556.D15.GZ43_507074M00084648D:F05chiron(cc187-NormBPHProstate)
3784135053556.D20.GZ43_507154M00084657C:E01chiron(cc187-NormBPHProstate)
37912241073556.D23.GZ43_507202M00084666A:C04chiron(cc187-NormBPHProstate)
3807745203556.E13.GZ43_507043M00084646A:D02chiron(cc187-NormBPHProstate)
3815731693556.E24.GZ43_507219M00084616A:G03chiron(cc187-NormBPHProstate)
38210534173556.F10.GZ43_506996M00084642D:E08chiron(cc187-NormBPHProstate)
3837101943556.G15.GZ43_507077M00084648B:F06chiron(cc187-NormBPHProstate)
3845584843556.H01.GZ43_506854M00084618C:A03chiron(cc187-NormBPHProstate)
38512118993556.H02.GZ43_506870M00084620A:E08chiron(cc187-NormBPHProstate)
3868232713556.H12.GZ43_507030M00084645C:F07chiron(cc187-NormBPHProstate)
3873769003556.H20.GZ43_507158M00084657D:B10chiron(cc187-NormBPHProstate)
3887265843556.I02.GZ43_506871M00084619A:E04chiron(cc187-NormBPHProstate)
3897259823556.I14.GZ43_507063M00084647C:A05chiron(cc187-NormBPHProstate)
39012118993556.J05.GZ43_506920M00084633A:B12chiron(cc187-NormBPHProstate)
3918570313556.J07.GZ43_506952M00084636C:A06chiron(cc187-NormBPHProstate)
39210548133556.J14.GZ43_507064M00084647D:C05chiron(cc187-NormBPHProstate)
39312268623556.J16.GZ43_507096M00084651B:G10chiron(cc187-NormBPHProstate)
39412244223556.K04.GZ43_506905M00084630D:F09chiron(cc187-NormBPHProstate)
3955297093556.K12.GZ43_507033M00084645B:A06chiron(cc187-NormBPHProstate)
3964508823556.K13.GZ43_507049M00084646B:B03chiron(cc187-NormBPHProstate)
39712240393556.K17.GZ43_507113M00084652D:G11chiron(cc187-NormBPHProstate)
39812245983556.L08.GZ43_506970M00084638D:A05chiron(cc187-NormBPHProstate)
3993982113556.L09.GZ43_506986M00084641B:F08chiron(cc187-NormBPHProstate)
40012451883556.L16.GZ43_507098M00084651C:H01chiron(cc187-NormBPHProstate)
4015580813556.L23.GZ43_507210M00084666C:A06chiron(cc187-NormBPHProstate)
40212243793556.M02.GZ43_506875M00084619A:G10chiron(cc187-NormBPHProstate)
4037335383556.M11.GZ43_507019M00084644A:H05chiron(cc187-NormBPHProstate)
4047273963556.M23.GZ43_507211M00084664D:E05chiron(cc187-NormBPHProstate)
405168723556.N02.GZ43_506876M00084620B:F05chiron(cc187-NormBPHProstate)
40611398493556.N04.GZ43_506908M00084631D:G01chiron(cc187-NormBPHProstate)
40711225283556.N05.GZ43_506924M00084633A:H05chiron(cc187-NormBPHProstate)
40810773193556.N06.GZ43_506940M00084634C:H02chiron(cc187-NormBPHProstate)
40911160873556.N21.GZ43_507180M00084659C:G05chiron(cc187-NormBPHProstate)
41011985633556.O08.GZ43_506973M00084638A:E10chiron(cc187-NormBPHProstate)
4115850993556.O13.GZ43_507053M00084646B:D07chiron(cc187-NormBPHProstate)
4126505203556.P07.GZ43_506958M00084637B:E01chiron(cc187-NormBPHProstate)
4138449573559.A04.GZ43_507279M00084673B:H11chiron(cc187-NormBPHProstate)
41410770333559.A20.GZ43_507535M00084702A:B08chiron(cc187-NormBPHProstate)
41511871743559.A24.GZ43_507599M00084667C:A03chiron(cc187-NormBPHProstate)
4164027893559.B04.GZ43_507280M00084675A:E02chiron(cc187-NormBPHProstate)
4178637683559.B06.GZ43_507312M00084681B:G11chiron(cc187-NormBPHProstate)
4186502633559.B08.GZ43_507344M00084684C:D02chiron(cc187-NormBPHProstate)
41910540383559.B10.GZ43_507376M00084687A:A03chiron(cc187-NormBPHProstate)
420383559.B18.GZ43_507504M00084700A:C10chiron(cc187-NormBPHProstate)
42112273243559.C06.GZ43_507313M00084679D:G12chiron(cc187-NormBPHProstate)
42212503733559.D21.GZ43_507554M00084704A:C12chiron(cc187-NormBPHProstate)
42312279123559.E06.GZ43_507315M00084680A:F08chiron(cc187-NormBPHProstate)
42410666543559.E09.GZ43_507363M00084685C:B12chiron(cc187-NormBPHProstate)
4257032043559.E20.GZ43_507539M00084702B:C12chiron(cc187-NormBPHProstate)
42612279123559.F07.GZ43_507332M00084683B:A01chiron(cc187-NormBPHProstate)
4271418703559.F17.GZ43_507492M00084699A:G05chiron(cc187-NormBPHProstate)
428155773559.H09.GZ43_507366M00084686B:B04chiron(cc187-NormBPHProstate)
4291297863559.H22.GZ43_507574M00084705C:D01chiron(cc187-NormBPHProstate)
4304548263559.H24.GZ43_507606M00084668D:D08chiron(cc187-NormBPHProstate)
4316475873559.I05.GZ43_507303M00084676B:E02chiron(cc187-NormBPHProstate)
4329150123559.J04.GZ43_507288M00084675B:A04chiron(cc187-NormBPHProstate)
43331553559.J20.GZ43_507544M00084703B:D09chiron(cc187-NormBPHProstate)
43412109533559.K16.GZ43_507481M00084696D:H04chiron(cc187-NormBPHProstate)
4355760403559.K17.GZ43_507497M00084698B:D02chiron(cc187-NormBPHProstate)
4369452473559.L01.GZ43_507242M00084670B:A09chiron(cc187-NormBPHProstate)
43711974443559.L14.GZ43_507450M00084694D:F04chiron(cc187-NormBPHProstate)
4385731693559.L19.GZ43_507530M00084701C:E08chiron(cc187-NormBPHProstate)
4393872563559.M02.GZ43_507259M00084671A:C12chiron(cc187-NormBPHProstate)
44012279933559.M09.GZ43_507371M00084685D:B11chiron(cc187-NormBPHProstate)
44111173923559.N05.GZ43_507308M00084678C:C11chiron(cc187-NormBPHProstate)
4427265843559.N18.GZ43_507516M00084700D:E09chiron(cc187-NormBPHProstate)
4434969623559.N21.GZ43_507564M00084704C:B09chiron(cc187-NormBPHProstate)
4444023783559.O01.GZ43_507245M00084669C:A10chiron(cc187-NormBPHProstate)
4459913663559.O05.GZ43_507309M00084677C:F03chiron(cc187-NormBPHProstate)
4468605533559.O07.GZ43_507341M00084683A:B12chiron(cc187-NormBPHProstate)
4476446873559.O20.GZ43_507549M00084703A:E04chiron(cc187-NormBPHProstate)
4481297863559.P10.GZ43_507390M00084687C:F12chiron(cc187-NormBPHProstate)
44910625373559.P15.GZ43_507470M00084696C:A07chiron(cc187-NormBPHProstate)
45011972593559.P18.GZ43_507518M00084700D:H04chiron(cc187-NormBPHProstate)
4511646183559.P24.GZ43_507614M00084669A:A05chiron(cc187-NormBPHProstate)
45212252643562.A01.GZ43_507615M00084707D:H03chiron(cc187-NormBPHProstate)
45312207083562.A15.GZ43_507839M00084727A:A02chiron(cc187-NormBPHProstate)
4547271363562.B22.GZ43_507952M00084737A:C09chiron(cc187-NormBPHProstate)
45512255953562.C23.GZ43_507969M00084738B:A09chiron(cc187-NormBPHProstate)
4567265223562.D10.GZ43_507762M00084720A:A01chiron(cc187-NormBPHProstate)
4578469203562.E01.GZ43_507619M00084708A:A11chiron(cc187-NormBPHProstate)
4584483563562.E03.GZ43_507651M00084710B:G07chiron(cc187-NormBPHProstate)
45912547333562.E12.GZ43_507795M00084722A:H12chiron(cc187-NormBPHProstate)
4604539013562.F19.GZ43_507908M00084732B:A04chiron(cc187-NormBPHProstate)
4615304533562.F20.GZ43_507924M00084734A:H01chiron(cc187-NormBPHProstate)
46212536703562.G13.GZ43_507813M00084723D:G09chiron(cc187-NormBPHProstate)
4639714653562.G19.GZ43_507909M00084731C:G07chiron(cc187-NormBPHProstate)
4642703562.H11.GZ43_507782M00084721C:F09chiron(cc187-NormBPHProstate)
4659701653562.H12.GZ43_507798M00084722D:A03chiron(cc187-NormBPHProstate)
4667265223562.I01.GZ43_507623M00084708B:A06chiron(cc187-NormBPHProstate)
46710537993562.I02.GZ43_507639M00084709C:B02chiron(cc187-NormBPHProstate)
4688487013562.I13.GZ43_507815M00084724A:C02chiron(cc187-NormBPHProstate)
46912608463562.I15.GZ43_507847M00084727A:G09chiron(cc187-NormBPHProstate)
47012570963562.J09.GZ43_507752M00084718D:C04chiron(cc187-NormBPHProstate)
47112530873562.J13.GZ43_507816M00084724D:F04chiron(cc187-NormBPHProstate)
4724479503562.K04.GZ43_507673M00084711B:A05chiron(cc187-NormBPHProstate)
4733939483562.K08.GZ43_507737M00084716D:H03chiron(cc187-NormBPHProstate)
4748939813562.L12.GZ43_507802M00084722D:G04chiron(cc187-NormBPHProstate)
47512248813562.N24.GZ43_507996M00084707D:B08chiron(cc187-NormBPHProstate)
47611412833562.O11.GZ43_507789M00084721B:C11chiron(cc187-NormBPHProstate)
4777421013562.O18.GZ43_507901M00084730B:A09chiron(cc187-NormBPHProstate)
478340283562.O20.GZ43_507933M00084734A:E04chiron(cc187-NormBPHProstate)
47912546743562.P21.GZ43_507950M00084736B:H03chiron(cc187-NormBPHProstate)
48012264133562.P23.GZ43_507982M00084740C:B08chiron(cc187-NormBPHProstate)
48153753565.A23.GZ43_508351M00084742A:F07chiron(cc187-NormBPHProstate)
4826285703565.B05.GZ43_508064M00084743A:E03chiron(cc187-NormBPHProstate)
48310550183565.B13.GZ43_508192M00084743D:G01chiron(cc187-NormBPHProstate)
48411390883565.B14.GZ43_508208M00084743D:H04chiron(cc187-NormBPHProstate)
4854535223565.C04.GZ43_508049M00084745A:A08chiron(cc187-NormBPHProstate)
4865518913565.C06.GZ43_508081M00084745A:H04chiron(cc187-NormBPHProstate)
4877003543565.C17.GZ43_508257M00084746B:B04chiron(cc187-NormBPHProstate)
48811160873565.D14.GZ43_508210M00084747D:G02chiron(cc187-NormBPHProstate)
4896404623565.D17.GZ43_508258M00084748A:D09chiron(cc187-NormBPHProstate)
4904771103565.D19.GZ43_508290M00084748A:H02chiron(cc187-NormBPHProstate)
491342013565.E16.GZ43_508243M00084750C:B08chiron(cc187-NormBPHProstate)
49212252643565.G07.GZ43_508101M00084755A:D02chiron(cc187-NormBPHProstate)
493168723565.G09.GZ43_508133M00084755B:A04chiron(cc187-NormBPHProstate)
49411715183565.G22.GZ43_508341M00084755D:E06chiron(cc187-NormBPHProstate)
49512615803565.H06.GZ43_508086M00084756B:H01chiron(cc187-NormBPHProstate)
49612618923565.H10.GZ43_508150M00084756C:H01chiron(cc187-NormBPHProstate)
4974546123565.H11.GZ43_508166M00084756D:C04chiron(cc187-NormBPHProstate)
49812583983565.H15.GZ43_508230M00084757A:D01chiron(cc187-NormBPHProstate)
49912593943565.H23.GZ43_508358M00084757B:F11chiron(cc187-NormBPHProstate)
50012251063565.H24.GZ43_508374M00084757B:F05chiron(cc187-NormBPHProstate)
5019367953565.K15.GZ43_508233M00084760D:D09chiron(cc187-NormBPHProstate)
5025524323565.L22.GZ43_508346M00084763D:A04chiron(cc187-NormBPHProstate)
5034776923565.M15.GZ43_508235M00084764D:G08chiron(cc187-NormBPHProstate)
50410550633565.M20.GZ43_508315M00084765B:A10chiron(cc187-NormBPHProstate)
50512584563565.N12.GZ43_508188M00084766B:E03chiron(cc187-NormBPHProstate)
50612593193565.N13.GZ43_508204M00084766B:F02chiron(cc187-NormBPHProstate)
50710523993565.N19.GZ43_508300M00084766D:F12chiron(cc187-NormBPHProstate)
50810660413565.O02.GZ43_508029M00084767B:D10chiron(cc187-NormBPHProstate)
50912598723565.O03.GZ43_508045M00084767B:F06chiron(cc187-NormBPHProstate)
51010550293565.O07.GZ43_508109M00084767D:B04chiron(cc187-NormBPHProstate)
51112187933565.O15.GZ43_508237M00084768B:E09chiron(cc187-NormBPHProstate)
5121418703565.P03.GZ43_508046M00084769C:H03chiron(cc187-NormBPHProstate)
51324243565.P09.GZ43_508142M00084770B:G12chiron(cc187-NormBPHProstate)
5144777083565.P22.GZ43_508350M00084771D:A01chiron(cc187-NormBPHProstate)
51512266433565.P24.GZ43_508382M00084771D:G03chiron(cc187-NormBPHProstate)
51612242263568.A10.GZ43_508545M00084810D:B10chiron(cc187-NormBPHProstate)
51711719853568.B02.GZ43_508418M00084812A:C02chiron(cc187-NormBPHProstate)
51811932053568.B05.GZ43_508466M00084812A:E05chiron(cc187-NormBPHProstate)
5197101943568.C22.GZ43_508739M00084817A:H11chiron(cc187-NormBPHProstate)
52012253353568.D23.GZ43_508756M00084820D:A03chiron(cc187-NormBPHProstate)
5214027943568.E17.GZ43_508661M00084822B:G11chiron(cc187-NormBPHProstate)
5223806343568.E20.GZ43_508709M00084822C:D06chiron(cc187-NormBPHProstate)
5233899953568.F06.GZ43_508486M00084823A:H01chiron(cc187-NormBPHProstate)
5244513903568.F07.GZ43_508502M00084823A:H06chiron(cc187-NormBPHProstate)
52511372593568.F11.GZ43_508566M00084823D:E05chiron(cc187-NormBPHProstate)
52612519503568.F12.GZ43_508582M00084823D:E06chiron(cc187-NormBPHProstate)
52712509953568.F22.GZ43_508742M00084824C:C10chiron(cc187-NormBPHProstate)
52810524663568.G10.GZ43_508551M00084826B:D12chiron(cc187-NormBPHProstate)
5296217023568.G12.GZ43_508583M00084826B:E11chiron(cc187-NormBPHProstate)
5306172923568.G24.GZ43_508775M00084827D:D04chiron(cc187-NormBPHProstate)
53180453568.H20.GZ43_508712M00084829B:F06chiron(cc187-NormBPHProstate)
53212247523568.J10.GZ43_508554M00084833A:G07chiron(cc187-NormBPHProstate)
53312109533568.J22.GZ43_508746M00084833D:B04chiron(cc187-NormBPHProstate)
5347463893568.K01.GZ43_508411M00084834A:A03chiron(cc187-NormBPHProstate)
5358459313568.K04.GZ43_508459M00084834B:G02chiron(cc187-NormBPHProstate)
5368475163568.L04.GZ43_508460M00084835D:H03chiron(cc187-NormBPHProstate)
53711101433568.M03.GZ43_508445M00084837B:E06chiron(cc187-NormBPHProstate)
53812004533568.M13.GZ43_508605M00084838A:F12chiron(cc187-NormBPHProstate)
53912566023568.N11.GZ43_508574M00084839C:B09chiron(cc187-NormBPHProstate)
54011939703568.O17.GZ43_508671M00084841B:H09chiron(cc187-NormBPHProstate)
54112565643568.P04.GZ43_508464M00084842C:B07chiron(cc187-NormBPHProstate)
54212577323568.P18.GZ43_508688M00084843A:D06chiron(cc187-NormBPHProstate)
54378563568.P19.GZ43_508704M00084843A:G01chiron(cc187-NormBPHProstate)
5444496973571.A04.GZ43_508833M00084843D:C06chiron(cc187-NormBPHProstate)
54511369723571.A07.GZ43_508881M00084843D:F05chiron(cc187-NormBPHProstate)
54611408653571.A08.GZ43_508897M00084844A:E10chiron(cc187-NormBPHProstate)
54710798633571.A11.GZ43_508945M00084844B:H08chiron(cc187-NormBPHProstate)
54812255003571.A14.GZ43_508993M00084844C:F04chiron(cc187-NormBPHProstate)
54911890273571.A22.GZ43_509121M00084845A:E02chiron(cc187-NormBPHProstate)
55010696323571.B13.GZ43_508978M00084845C:H05chiron(cc187-NormBPHProstate)
55110696323571.B22.GZ43_509122M00084846B:H07chiron(cc187-NormBPHProstate)
5523876103571.C08.GZ43_508899M00084847B:G05chiron(cc187-NormBPHProstate)
55312611343571.D04.GZ43_508836M00084849A:H08chiron(cc187-NormBPHProstate)
5545998203571.D07.GZ43_508884M00084849B:F11chiron(cc187-NormBPHProstate)
5554183203571.E02.GZ43_508805M00084850C:A11chiron(cc187-NormBPHProstate)
55612245933571.E10.GZ43_508933M00084850D:H02chiron(cc187-NormBPHProstate)
55711160873571.E16.GZ43_509029M00084851C:F10chiron(cc187-NormBPHProstate)
5589858593571.F06.GZ43_508870M00084853A:F08chiron(cc187-NormBPHProstate)
55911932363571.F16.GZ43_509030M00084853D:A12chiron(cc187-NormBPHProstate)
5605049043571.F23.GZ43_509142M00084853D:G03chiron(cc187-NormBPHProstate)
5612429013571.G22.GZ43_509127M00084855D:H05chiron(cc187-NormBPHProstate)
56212268453571.G24.GZ43_509159M00084856B:A12chiron(cc187-NormBPHProstate)
5631403143571.H01.GZ43_508792M00084856B:D03chiron(cc187-NormBPHProstate)
56412609053571.H10.GZ43_508936M00084857A:G05chiron(cc187-NormBPHProstate)
5655567723571.H12.GZ43_508968M00084857B:A09chiron(cc187-NormBPHProstate)
56611761823571.H16.GZ43_509032M00084857C:E11chiron(cc187-NormBPHProstate)
56711980723571.H18.GZ43_509064M00084857D:A11chiron(cc187-NormBPHProstate)
5686774343571.I11.GZ43_508953M00084858C:B01chiron(cc187-NormBPHProstate)
56911932363571.J07.GZ43_508890M00084859C:D09chiron(cc187-NormBPHProstate)
57010550893571.J08.GZ43_508906M00084859C:H05chiron(cc187-NormBPHProstate)
57110548843571.J09.GZ43_508922M00084859D:B03chiron(cc187-NormBPHProstate)
5724944993571.J14.GZ43_509002M00084860B:A01chiron(cc187-NormBPHProstate)
5736864183571.L01.GZ43_508796M00084862B:B01chiron(cc187-NormBPHProstate)
57411404093571.M17.GZ43_509053M00084865D:B04chiron(cc187-NormBPHProstate)
57512604803571.M19.GZ43_509085M00084865D:G02chiron(cc187-NormBPHProstate)
5764025163571.M24.GZ43_509165M00084866B:A03chiron(cc187-NormBPHProstate)
57710523743571.N09.GZ43_508926M00084866C:H04chiron(cc187-NormBPHProstate)
57811416203571.N14.GZ43_509006M00084867A:C11chiron(cc187-NormBPHProstate)
57912553303571.N17.GZ43_509054M00084867B:A03chiron(cc187-NormBPHProstate)
58011936993571.N22.GZ43_509134M00084867C:G02chiron(cc187-NormBPHProstate)
58110548843571.O08.GZ43_508911M00084868B:D01chiron(cc187-NormBPHProstate)
5828666093574.A20.GZ43_509473M00084902B:A10chiron(cc187-NormBPHProstate)
58310593323574.B01.GZ43_509170M00084876D:A06chiron(cc187-NormBPHProstate)
5845677003574.B04.GZ43_509218M00084880B:D03chiron(cc187-NormBPHProstate)
58512552683574.B10.GZ43_509314M00084888D:A11chiron(cc187-NormBPHProstate)
5864016823574.B14.GZ43_509378M00084894A:G09chiron(cc187-NormBPHProstate)
5874771103574.B24.GZ43_509538M00084874D:E03chiron(cc187-NormBPHProstate)
5885540933574.C09.GZ43_509299M00084887C:C07chiron(cc187-NormBPHProstate)
58912250443574.C10.GZ43_509315M00084888C:D12chiron(cc187-NormBPHProstate)
5902026713574.C12.GZ43_509347M00084890B:E02chiron(cc187-NormBPHProstate)
591898333574.C14.GZ43_509379M00084893C:A12chiron(cc187-NormBPHProstate)
59212258043574.C16.GZ43_509411M00084896B:G10chiron(cc187-NormBPHProstate)
59312250443574.C23.GZ43_509523M00084907C:C01chiron(cc187-NormBPHProstate)
5945567723574.D02.GZ43_509188M00084878B:B12chiron(cc187-NormBPHProstate)
59511932363574.D12.GZ43_509348M00084890D:F09chiron(cc187-NormBPHProstate)
5966766653574.E02.GZ43_509189M00084877D:G07chiron(cc187-NormBPHProstate)
5975550593574.E03.GZ43_509205M00084879A:A04chiron(cc187-NormBPHProstate)
5984508823574.E14.GZ43_509381M00084893C:B01chiron(cc187-NormBPHProstate)
59910537993574.F10.GZ43_509318M00084889A:B07chiron(cc187-NormBPHProstate)
6004022863574.F18.GZ43_509446M00084900C:A04chiron(cc187-NormBPHProstate)
60110523643574.F23.GZ43_509526M00084908C:F07chiron(cc187-NormBPHProstate)
6024962333574.G07.GZ43_509271M00084884D:D03chiron(cc187-NormBPHProstate)
60311934733574.G11.GZ43_509335M00084889C:A04chiron(cc187-NormBPHProstate)
60412232713574.H07.GZ43_509272M00084885D:A12chiron(cc187-NormBPHProstate)
6054948903574.I02.GZ43_509193M00084877D:H09chiron(cc187-NormBPHProstate)
6064471513574.I07.GZ43_509273M00084885A:C01chiron(cc187-NormBPHProstate)
60712240093574.J11.GZ43_509338M00084889D:G06chiron(cc187-NormBPHProstate)
60812242263574.J14.GZ43_509386M00084894B:F11chiron(cc187-NormBPHProstate)
60910549793574.J23.GZ43_509530M00084908D:B11chiron(cc187-NormBPHProstate)
61012255223574.K12.GZ43_509355M00084890C:A06chiron(cc187-NormBPHProstate)
61112264133574.K20.GZ43_509483M00084902C:F05chiron(cc187-NormBPHProstate)
61212237053574.L07.GZ43_509276M00084886A:C06chiron(cc187-NormBPHProstate)
61312586463574.M03.GZ43_509213M00084879B:E01chiron(cc187-NormBPHProstate)
6148424593574.M23.GZ43_509533M00084908A:F03chiron(cc187-NormBPHProstate)
61512557453574.N04.GZ43_509230M00084880D:A10chiron(cc187-NormBPHProstate)
6166526513574.N10.GZ43_509326M00084889B:C02chiron(cc187-NormBPHProstate)
6176507153574.N12.GZ43_509358M00084891D:A02chiron(cc187-NormBPHProstate)
6186285703574.N20.GZ43_509486M00084904A:D03chiron(cc187-NormBPHProstate)
61910531213574.P07.GZ43_509280M00084886B:D06chiron(cc187-NormBPHProstate)
62011420063574.P17.GZ43_509440M00084899D:B01chiron(cc187-NormBPHProstate)
62112268623577.A06.GZ43_509633M00084909C:G02chiron(cc187-NormBPHProstate)
6229452473577.A18.GZ43_509825M00084910D:E07chiron(cc187-NormBPHProstate)
6239150123577.B12.GZ43_509730M00084912D:A09chiron(cc187-NormBPHProstate)
6244467473577.B15.GZ43_509778M00084912D:G06chiron(cc187-NormBPHProstate)
6258199173577.B19.GZ43_509842M00084913B:F05chiron(cc187-NormBPHProstate)
62612245473577.E19.GZ43_509845M00084919C:B04chiron(cc187-NormBPHProstate)
62712495473577.F02.GZ43_509574M00084919D:B08chiron(cc187-NormBPHProstate)
6283573673577.G07.GZ43_509655M00084921C:E04chiron(cc187-NormBPHProstate)
6297254383577.G13.GZ43_509751M00084922A:C08chiron(cc187-NormBPHProstate)
63011938193577.H06.GZ43_509640M00084923D:B05chiron(cc187-NormBPHProstate)
63112243783577.H08.GZ43_509672M00084923D:F11chiron(cc187-NormBPHProstate)
632930733577.H18.GZ43_509832M00084925A:B08chiron(cc187-NormBPHProstate)
6337378823577.I01.GZ43_509561M00084925C:G01chiron(cc187-NormBPHProstate)
6344539583577.I17.GZ43_509817M00084926B:C05chiron(cc187-NormBPHProstate)
63510769303577.J04.GZ43_509610M00084927A:C01chiron(cc187-NormBPHProstate)
63612599553577.K06.GZ43_509643M00084928D:F06chiron(cc187-NormBPHProstate)
63711954033577.K14.GZ43_509771M00084929C:B02chiron(cc187-NormBPHProstate)
63812249783577.K23.GZ43_509915M00084930B:E12chiron(cc187-NormBPHProstate)
63912497313577.L10.GZ43_509708M00084930D:B08chiron(cc187-NormBPHProstate)
640529393577.N10.GZ43_509710M00084935B:E10chiron(cc187-NormBPHProstate)
6417269223577.N14.GZ43_509774M00084935C:E07chiron(cc187-NormBPHProstate)
6425521423577.O17.GZ43_509823M00084937D:B04chiron(cc187-NormBPHProstate)
64311408393577.O22.GZ43_509903M00084938B:A11chiron(cc187-NormBPHProstate)
64412227093577.P02.GZ43_509584M00084938B:F12chiron(cc187-NormBPHProstate)
6456402773577.P07.GZ43_509664M00084938C:G06chiron(cc187-NormBPHProstate)
6466548483577.P23.GZ43_509920M00084940B:F06chiron(cc187-NormBPHProstate)
64712254573580.A04.GZ43_509985M00084941B:E07chiron(cc187-NormBPHProstate)
6485257593580.A09.GZ43_510065M00084941C:H04chiron(cc187-NormBPHProstate)
6491601123580.A13.GZ43_510129M00084941D:C10chiron(cc187-NormBPHProstate)
65011395223580.A14.GZ43_510145M00084941D:H02chiron(cc187-NormBPHProstate)
65110368453580.B01.GZ43_509938M00084942C:B10chiron(cc187-NormBPHProstate)
65212239073580.C01.GZ43_509939M00084944D:E05chiron(cc187-NormBPHProstate)
6536401573580.C03.GZ43_509971M00084945A:D10chiron(cc187-NormBPHProstate)
65411324133580.C05.GZ43_510003M00084945A:H10chiron(cc187-NormBPHProstate)
6556217023580.D07.GZ43_510036M00084946D:H05chiron(cc187-NormBPHProstate)
65612278623580.D22.GZ43_510276M00084948B:F04chiron(cc187-NormBPHProstate)
6579913663580.E02.GZ43_509957M00084948D:B08chiron(cc187-NormBPHProstate)
6584392973580.E08.GZ43_510053M00084949B:B12chiron(cc187-NormBPHProstate)
6594166873580.E10.GZ43_510085M00084949B:H11chiron(cc187-NormBPHProstate)
66010749093580.E19.GZ43_510229M00084950D:A06chiron(cc187-NormBPHProstate)
66112278623580.E21.GZ43_510261M00084950D:F05chiron(cc187-NormBPHProstate)
6625665483580.E23.GZ43_510293M00084951A:D04chiron(cc187-NormBPHProstate)
6634041213580.G03.GZ43_509975M00084953D:D03chiron(cc187-NormBPHProstate)
6648286953580.G13.GZ43_510135M00084954C:A03chiron(cc187-NormBPHProstate)
6656176293580.G14.GZ43_510151M00084954C:B12chiron(cc187-NormBPHProstate)
6664536573580.G18.GZ43_510215M00084954D:A05chiron(cc187-NormBPHProstate)
6675482173580.G19.GZ43_510231M00084954D:D12chiron(cc187-NormBPHProstate)
6684535823580.G20.GZ43_510247M00084954D:E01chiron(cc187-NormBPHProstate)
66911398833580.G24.GZ43_510311M00084955A:E08chiron(cc187-NormBPHProstate)
67011225283580.H12.GZ43_510120M00084956B:B05chiron(cc187-NormBPHProstate)
6711682253580.H16.GZ43_510184M00084956C:G09chiron(cc187-NormBPHProstate)
67210741603580.H22.GZ43_510280M00084957B:H07chiron(cc187-NormBPHProstate)
67311067303580.I06.GZ43_510025M00084958B:E10chiron(cc187-NormBPHProstate)
67410680363580.I08.GZ43_510057M00084958C:B03chiron(cc187-NormBPHProstate)
67511417533580.I18.GZ43_510217M00084959B:C07chiron(cc187-NormBPHProstate)
6766491173580.J10.GZ43_510090M00084960D:D02chiron(cc187-NormBPHProstate)
67712279133580.J12.GZ43_510122M00084961A:C07chiron(cc187-NormBPHProstate)
6783997383580.J18.GZ43_510218M00084961C:A06chiron(cc187-NormBPHProstate)
6795015563580.J20.GZ43_510250M00084961C:F01chiron(cc187-NormBPHProstate)
680170753580.J21.GZ43_510266M00084961D:H03chiron(cc187-NormBPHProstate)
6815320623580.K03.GZ43_509979M00084962C:F10chiron(cc187-NormBPHProstate)
6824087113580.K05.GZ43_510011M00084963D:D07chiron(cc187-NormBPHProstate)
6836406963580.K21.GZ43_510267M00084966A:A08chiron(cc187-NormBPHProstate)
68411369723580.L09.GZ43_510076M00084967B:B10chiron(cc187-NormBPHProstate)
6858493333580.L10.GZ43_510092M00084967B:D09chiron(cc187-NormBPHProstate)
68612227093580.L12.GZ43_510124M00084967C:D10chiron(cc187-NormBPHProstate)
6877385253580.L13.GZ43_510140M00084967C:D12chiron(cc187-NormBPHProstate)
68810531213580.L17.GZ43_510204M00084968A:D01chiron(cc187-NormBPHProstate)
68912263233580.M01.GZ43_509949M00084968C:D10chiron(cc187-NormBPHProstate)
6907075903580.M16.GZ43_510189M00084969C:F03chiron(cc187-NormBPHProstate)
6913766593580.M17.GZ43_510205M00084969C:H11chiron(cc187-NormBPHProstate)
6926379153580.M18.GZ43_510221M00084969D:C11chiron(cc187-NormBPHProstate)
69310594453580.M23.GZ43_510301M00084970A:C11chiron(cc187-NormBPHProstate)
69411384193580.N10.GZ43_510094M00084970C:G03chiron(cc187-NormBPHProstate)
69512264943580.N11.GZ43_510110M00084970C:H09chiron(cc187-NormBPHProstate)
6964279043580.N14.GZ43_510158M00084970D:B01chiron(cc187-NormBPHProstate)
6974508823580.N15.GZ43_510174M00084970D:E08chiron(cc187-NormBPHProstate)
6989617573580.N23.GZ43_510302M00084971C:G07chiron(cc187-NormBPHProstate)
69910600213580.O02.GZ43_509967M00084972B:H03chiron(cc187-NormBPHProstate)
7003786293580.O06.GZ43_510031M00084973A:A01chiron(cc187-NormBPHProstate)
701124203580.O07.GZ43_510047M00084973A:B06chiron(cc187-NormBPHProstate)
70211389973580.O08.GZ43_510063M00084973A:C01chiron(cc187-NormBPHProstate)
7038438313580.P04.GZ43_510000M00084974D:F11chiron(cc187-NormBPHProstate)
7048628233580.P05.GZ43_510016M00084975A:G05chiron(cc187-NormBPHProstate)
7058368793580.P14.GZ43_510160M00084976B:A08chiron(cc187-NormBPHProstate)
70612262673580.P19.GZ43_510240M00084976C:C12chiron(cc187-NormBPHProstate)
7077254383583.B06.GZ43_510402M00084980C:B07chiron(cc187-NormBPHProstate)
7086134113583.B07.GZ43_510418M00084980C:E06chiron(cc187-NormBPHProstate)
7098610253583.B10.GZ43_510466M00084980D:D02chiron(cc187-NormBPHProstate)
7105577173583.B11.GZ43_510482M00084980D:H08chiron(cc187-NormBPHProstate)
7114004073583.D15.GZ43_510548M00084987A:D09chiron(cc187-NormBPHProstate)
71211934543583.D22.GZ43_510660M00084987B:H12chiron(cc187-NormBPHProstate)
71310649753583.E11.GZ43_510485M00084988B:B08chiron(cc187-NormBPHProstate)
7147776703583.E13.GZ43_510517M00084988C:A04chiron(cc187-NormBPHProstate)
71510856453583.E15.GZ43_510549M00084988C:B01chiron(cc187-NormBPHProstate)
7165096733583.E17.GZ43_510581M00084988C:G03chiron(cc187-NormBPHProstate)
71711186513583.F24.GZ43_510694M00084992D:B02chiron(cc187-NormBPHProstate)
71811191393583.G09.GZ43_510455M00084994A:H04chiron(cc187-NormBPHProstate)
7197080253583.G16.GZ43_510567M00084994D:F11chiron(cc187-NormBPHProstate)
7206983073583.G17.GZ43_510583M00084994D:H04chiron(cc187-NormBPHProstate)
7215371183583.G21.GZ43_510647M00084995B:B08chiron(cc187-NormBPHProstate)
7224477313583.H03.GZ43_510360M00084996B:D08chiron(cc187-NormBPHProstate)
72312264843583.H12.GZ43_510504M00084997D:H09chiron(cc187-NormBPHProstate)
72410599683583.H13.GZ43_510520M00084998A:C12chiron(cc187-NormBPHProstate)
7254944993583.H15.GZ43_510552M00084998B:A04chiron(cc187-NormBPHProstate)
7264969623583.J02.GZ43_510346M00085003C:D03chiron(cc187-NormBPHProstate)
7274924893583.K08.GZ43_510443M00085006C:C07chiron(cc187-NormBPHProstate)
72811165033583.K10.GZ43_510475M00085006D:C10chiron(cc187-NormBPHProstate)
7295520363583.K11.GZ43_510491M00085006D:C04chiron(cc187-NormBPHProstate)
7305015563583.K14.GZ43_510539M00085007A:B03chiron(cc187-NormBPHProstate)
7311646183583.K17.GZ43_510587M00085007B:C07chiron(cc187-NormBPHProstate)
73212747593583.K23.GZ43_510683M00085008B:H11chiron(cc187-NormBPHProstate)
7334043083583.L05.GZ43_510396M00085009B:F10chiron(cc187-NormBPHProstate)
7342345863583.L08.GZ43_510444M00085009C:C01chiron(cc187-NormBPHProstate)
7356491173583.L09.GZ43_510460M00085009D:A02chiron(cc187-NormBPHProstate)
7363535283583.L17.GZ43_510588M00085010C:H01chiron(cc187-NormBPHProstate)
737944133583.L21.GZ43_510652M00085011B:A01chiron(cc187-NormBPHProstate)
7384014243583.M08.GZ43_510445M00085012C:A08chiron(cc187-NormBPHProstate)
7394164703583.M10.GZ43_510477M00085012C:D06chiron(cc187-NormBPHProstate)
7403802053583.M13.GZ43_510525M00085013A:E06chiron(cc187-NormBPHProstate)
74110540693583.N09.GZ43_510462M00085015A:C09chiron(cc187-NormBPHProstate)
7428368793583.O03.GZ43_510367M00085017C:A11chiron(cc187-NormBPHProstate)
7434515053583.O11.GZ43_510495M00085018C:B09chiron(cc187-NormBPHProstate)
7447478053583.O17.GZ43_510591M00085019C:D05chiron(cc187-NormBPHProstate)
74511101433583.P09.GZ43_510464M00085021C:F06chiron(cc187-NormBPHProstate)
7468215363583.P19.GZ43_510624M00085022B:B03chiron(cc187-NormBPHProstate)
7472346673583.P22.GZ43_510672M00085022B:F05chiron(cc187-NormBPHProstate)
74812699423590.A12.GZ43_512274M00085023D:E11chiron(cc187-NormBPHProstate)
74912245933590.B01.GZ43_512099M00085025A:D11chiron(cc187-NormBPHProstate)
7503866123590.B16.GZ43_512339M00085026D:A01chiron(cc187-NormBPHProstate)
75112505813590.B21.GZ43_512419M00085027A:C02chiron(cc187-NormBPHProstate)
7525570633590.C20.GZ43_512404M00085029A:C02chiron(cc187-NormBPHProstate)
75311369773590.D03.GZ43_512133M00085029D:E12chiron(cc187-NormBPHProstate)
754182253590.D19.GZ43_512389M00085031B:E03chiron(cc187-NormBPHProstate)
7557277303590.D23.GZ43_512453M00085031C:D05chiron(cc187-NormBPHProstate)
75612247043590.E08.GZ43_512214M00085032C:F04chiron(cc187-NormBPHProstate)
75712506603590.E10.GZ43_512246M00085032D:C03chiron(cc187-NormBPHProstate)
7588469203590.F01.GZ43_512103M00085034B:E11chiron(cc187-NormBPHProstate)
7599658143590.F16.GZ43_512343M00085035B:C12chiron(cc187-NormBPHProstate)
7604392973590.G01.GZ43_512104M00085035D:D09chiron(cc187-NormBPHProstate)
7617463893590.G02.GZ43_512120M00085035D:E04chiron(cc187-NormBPHProstate)
7626774343590.H04.GZ43_512153M00085038A:B10chiron(cc187-NormBPHProstate)
76310550893590.H06.GZ43_512185M00085038A:C06chiron(cc187-NormBPHProstate)
7647729853590.H09.GZ43_512233M00085038D:D10chiron(cc187-NormBPHProstate)
7656249713590.H12.GZ43_512281M00085039B:E09chiron(cc187-NormBPHProstate)
7666896393590.H16.GZ43_512345M00085039D:F09chiron(cc187-NormBPHProstate)
76712264883590.I16.GZ43_512346M00085047A:H02chiron(cc187-NormBPHProstate)
7684034883590.J01.GZ43_512107M00085047D:F03chiron(cc187-NormBPHProstate)
7699150123590.J02.GZ43_512123M00085047D:F08chiron(cc187-NormBPHProstate)
77010147343590.J18.GZ43_512379M00085049B:E03chiron(cc187-NormBPHProstate)
77117983590.J21.GZ43_512427M00085050A:B06chiron(cc187-NormBPHProstate)
7726115923590.J22.GZ43_512443M00085050A:E11chiron(cc187-NormBPHProstate)
7733748533590.K06.GZ43_512188M00085051A:G03chiron(cc187-NormBPHProstate)
7745941163590.K10.GZ43_512252M00085051C:A01chiron(cc187-NormBPHProstate)
7755518913590.K19.GZ43_512396M00085052B:E04chiron(cc187-NormBPHProstate)
7763899953590.L08.GZ43_512221M00085053C:D07chiron(cc187-NormBPHProstate)
7774839183590.L10.GZ43_512253M00085053D:D04chiron(cc187-NormBPHProstate)
7784119603590.M03.GZ43_512142M00085056A:G12chiron(cc187-NormBPHProstate)
7795320623590.M04.GZ43_512158M00085056B:B06chiron(cc187-NormBPHProstate)
7809422003590.M09.GZ43_512238M00085056D:B12chiron(cc187-NormBPHProstate)
7814706983590.N04.GZ43_512159M00085058A:H02chiron(cc187-NormBPHProstate)
78210884023590.N19.GZ43_512399M00085059B:H11chiron(cc187-NormBPHProstate)
78312263043590.N21.GZ43_512431M00085059B:H07chiron(cc187-NormBPHProstate)
7848694533590.O08.GZ43_512224M00085060B:C05chiron(cc187-NormBPHProstate)
7857375023596.C02.GZ43_512500M00085121A:D10chiron(cc187-NormBPHProstate)
7864044183596.C20.GZ43_512788M00085123A:E07chiron(cc187-NormBPHProstate)
7874044183596.C22.GZ43_512820M00085123B:C04chiron(cc187-NormBPHProstate)
7885559673596.D01.GZ43_512485M00085123C:G11chiron(cc187-NormBPHProstate)
78911841343596.D07.GZ43_512581M00085124A:G04chiron(cc187-NormBPHProstate)
790898333596.D09.GZ43_512613M00085124B:G05chiron(cc187-NormBPHProstate)
7914948903596.D17.GZ43_512741M00085125C:H06chiron(cc187-NormBPHProstate)
7924011663596.E08.GZ43_512598M00085127C:C03chiron(cc187-NormBPHProstate)
7936459863596.E22.GZ43_512822M00085129B:C02chiron(cc187-NormBPHProstate)
79412258703596.F10.GZ43_512631M00085131D:A06chiron(cc187-NormBPHProstate)
7952698293596.G13.GZ43_512680M00085141C:G06chiron(cc187-NormBPHProstate)
7967228783596.H04.GZ43_512537M00085142D:F04chiron(cc187-NormBPHProstate)
7974546123596.H10.GZ43_512633M00085143C:D05chiron(cc187-NormBPHProstate)
7986428693596.H17.GZ43_512745M00085144B:C12chiron(cc187-NormBPHProstate)
7997708343596.H22.GZ43_512825M00085144D:G03chiron(cc187-NormBPHProstate)
80011890273596.I06.GZ43_512570M00085145C:D02chiron(cc187-NormBPHProstate)
80111890273596.I16.GZ43_512730M00085146B:C01chiron(cc187-NormBPHProstate)
80211896183596.J04.GZ43_512539M00085147C:A04chiron(cc187-NormBPHProstate)
8037776703596.J13.GZ43_512683M00085148B:H01chiron(cc187-NormBPHProstate)
80412262433596.K14.GZ43_512700M00085151A:B04chiron(cc187-NormBPHProstate)
80512265103596.K15.GZ43_512716M00085151A:H09chiron(cc187-NormBPHProstate)
8065170073596.L01.GZ43_512493M00085152B:A06chiron(cc187-NormBPHProstate)
80710708983596.L08.GZ43_512605M00085155B:F10chiron(cc187-NormBPHProstate)
80811173923596.L13.GZ43_512685M00085156A:G04chiron(cc187-NormBPHProstate)
80912273563596.N02.GZ43_512511M00085164C:G05chiron(cc187-NormBPHProstate)
81010429183596.N12.GZ43_512671M00085166C:A08chiron(cc187-NormBPHProstate)
8115524323596.N15.GZ43_512719M00085166D:C10chiron(cc187-NormBPHProstate)
8125850993596.N16.GZ43_512735M00085167A:G02chiron(cc187-NormBPHProstate)
8132222163596.N21.GZ43_512815M00085167C:D06chiron(cc187-NormBPHProstate)
8146505203596.O10.GZ43_512640M00085168D:D04chiron(cc187-NormBPHProstate)
8155577173596.O12.GZ43_512672M00085169A:H12chiron(cc187-NormBPHProstate)
816903596.P03.GZ43_512529M00085171D:F05chiron(cc187-NormBPHProstate)
8177478053596.P04.GZ43_512545M00085172A:G05chiron(cc187-NormBPHProstate)
8184599673596.P07.GZ43_512593M00085172C:F06chiron(cc187-NormBPHProstate)
8194546923596.P08.GZ43_512609M00085173A:B07chiron(cc187-NormBPHProstate)
820124203596.P10.GZ43_512641M00085173B:A08chiron(cc187-NormBPHProstate)
82111299283596.P21.GZ43_512817M00085175B:A03chiron(cc187-NormBPHProstate)
82279443599.A04.GZ43_512914M00085176C:B11chiron(cc187-NormBPHProstate)
8236373673599.A23.GZ43_513218M00085178D:F01chiron(cc187-NormBPHProstate)
82412272533599.B15.GZ43_513091M00085182B:E04chiron(cc187-NormBPHProstate)
82510538613599.B16.GZ43_513107M00085182B:H10chiron(cc187-NormBPHProstate)
8264013543599.C03.GZ43_512900M00085184D:B08chiron(cc187-NormBPHProstate)
82711173923599.C17.GZ43_513124M00085187B:C11chiron(cc187-NormBPHProstate)
82811169923599.D03.GZ43_512901M00085190B:C09chiron(cc187-NormBPHProstate)
8298321483599.D05.GZ43_512933M00085190B:H04chiron(cc187-NormBPHProstate)
83010815483599.D07.GZ43_512965M00085190C:D10chiron(cc187-NormBPHProstate)
83111385783599.D10.GZ43_513013M00085191A:B03chiron(cc187-NormBPHProstate)
8328473953599.E01.GZ43_512870M00085194C:B12chiron(cc187-NormBPHProstate)
833116833599.E05.GZ43_512934M00085194D:F04chiron(cc187-NormBPHProstate)
83412276233599.F17.GZ43_513127M00085201C:C12chiron(cc187-NormBPHProstate)
8355559673599.F24.GZ43_513239M00085203A:E06chiron(cc187-NormBPHProstate)
83612268143599.H05.GZ43_512937M00085209C:F11chiron(cc187-NormBPHProstate)
83712278463599.H23.GZ43_513225M00085214D:G01chiron(cc187-NormBPHProstate)
83812267513599.J11.GZ43_513035M00085220D:E06chiron(cc187-NormBPHProstate)
839903599.K02.GZ43_512892M00085222D:D07chiron(cc187-NormBPHProstate)
840898333599.K04.GZ43_512924M00085223A:G01chiron(cc187-NormBPHProstate)
8418545733599.K23.GZ43_513228M00085226C:F08chiron(cc187-NormBPHProstate)
84210660413599.L04.GZ43_512925M00085228B:C10chiron(cc187-NormBPHProstate)
843422853599.L15.GZ43_513101M00085229B:C10chiron(cc187-NormBPHProstate)
8446618023599.M04.GZ43_512926M00085230B:G08chiron(cc187-NormBPHProstate)
8455524283599.M22.GZ43_513214M00085242A:C06chiron(cc187-NormBPHProstate)
8469968883599.M24.GZ43_513246M00085243A:D07chiron(cc187-NormBPHProstate)
8475555093599.N09.GZ43_513007M00085244C:D03chiron(cc187-NormBPHProstate)
84810806343599.N16.GZ43_513119M00085245C:D07chiron(cc187-NormBPHProstate)
8491800923599.N20.GZ43_513183M00085245D:G07chiron(cc187-NormBPHProstate)
8506577483599.N24.GZ43_513247M00085246B:G12chiron(cc187-NormBPHProstate)
8515557263599.O06.GZ43_512960M00085247A:F05chiron(cc187-NormBPHProstate)
85210550293599.O17.GZ43_513136M00085248B:G12chiron(cc187-NormBPHProstate)
8534271823599.P05.GZ43_512945M00085249C:C11chiron(cc187-NormBPHProstate)
85412282773602.A09.GZ43_513378M00085252B:H07chiron(cc187-NormBPHProstate)
85511426323602.B18.GZ43_513523M00085255B:F11chiron(cc187-NormBPHProstate)
85611083323602.B21.GZ43_513571M00085255D:B09chiron(cc187-NormBPHProstate)
8577345683602.B22.GZ43_513587M00085255D:E12chiron(cc187-NormBPHProstate)
85810856383602.C24.GZ43_513620M00085259A:H05chiron(cc187-NormBPHProstate)
85911367213602.D06.GZ43_513333M00085259D:B06chiron(cc187-NormBPHProstate)
86012846833602.D11.GZ43_513413M00085260C:A10chiron(cc187-NormBPHProstate)
8619354603602.E04.GZ43_513302M00085262A:A02chiron(cc187-NormBPHProstate)
8624258243602.E06.GZ43_513334M00085262C:E04chiron(cc187-NormBPHProstate)
8634302013602.E13.GZ43_513446M00085263C:F12chiron(cc187-NormBPHProstate)
8646469353602.E21.GZ43_513574M00085264C:F04chiron(cc187-NormBPHProstate)
86510649633602.F12.GZ43_513431M00083691C:E12chiron(cc187-
ProstateCancer4 + 4)
86611299283602.G03.GZ43_513288M00083692C:D09chiron(cc187-
ProstateCancer4 + 4)
8675850993602.G17.GZ43_513512M00083694C:F09chiron(cc187-
ProstateCancer4 + 4)
86811940853602.I07.GZ43_513354M00083698B:H01chiron(cc187-
ProstateCancer4 + 4)
8699933602.I11.GZ43_513418M00083698D:E01chiron(cc187-
ProstateCancer4 + 4)
87031233602.I15.GZ43_513482M00083699B:C12chiron(cc187-
ProstateCancer4 + 4)
87111936893602.J13.GZ43_513451M00083701D:G09chiron(cc187-
ProstateCancer4 + 4)
872197773602.K03.GZ43_513292M00083704C:C04chiron(cc187-
ProstateCancer4 + 4)
8736373673602.K06.GZ43_513340M00083706A:D02chiron(cc187-
ProstateCancer4 + 4)
874137093602.L20.GZ43_513565M00083710D:B09chiron(cc187-
ProstateCancer4 + 4)
87511314093602.N03.GZ43_513295M00083714C:F04chiron(cc187-
ProstateCancer4 + 4)
8764547333602.N06.GZ43_513343M00083715A:B11chiron(cc187-
ProstateCancer4 + 4)
8774543493605.A15.gz43_513858M00083722B:A07chiron(cc187-
ProstateCancer4 + 4)
87812509953605.C16.gz43_513876M00083726B:E07chiron(cc187-
ProstateCancer4 + 4)
87911324133605.E19.gz43_513926M00083729C:F10chiron(cc187-
ProstateCancer4 + 4)
8803973133605.G13.gz43_513832M00083733B:D08chiron(cc187-
ProstateCancer4 + 4)
88112922813605.H10.gz43_513785M00083735C:H12chiron(cc187-
ProstateCancer4 + 4)
88212488613605.H21.gz43_513961M00083736A:D10chiron(cc187-
ProstateCancer4 + 4)
88312922623605.I19.gz43_513930M00083738C:D05chiron(cc187-
ProstateCancer4 + 4)
8844962333605.J16.gz43_513883M00083740C:G01chiron(cc187-
ProstateCancer4 + 4)
88510537933605.K19.gz43_513932M00083745A:A10chiron(cc187-
ProstateCancer4 + 4)
8867345683605.M17.gz43_513902M00083749D:B09chiron(cc187-
ProstateCancer4 + 4)
8873963203605.N04.gz43_513695M00083750D:G12chiron(cc187-
ProstateCancer4 + 4)
8884535223605.N09.gz43_513775M00085266B:C06chiron(cc187-
ProstateCancer4 + 4)
8894183203605.N12.gz43_513823M00085266D:C09chiron(cc187-
ProstateCancer4 + 4)
89010537473605.N16.gz43_513887M00085267B:D06chiron(cc187-
ProstateCancer4 + 4)
8916446873608.B06.gz43_514099M00085282C:F05chiron(cc187-
ProstateCancer4 + 4)
8928453543608.B12.gz43_514195M00085293B:B05chiron(cc187-
ProstateCancer4 + 4)
8938503353608.B24.gz43_514387M00085273A:A09chiron(cc187-
ProstateCancer4 + 4)
8948424593608.C18.gz43_514292M00085301C:H11chiron(cc187-
ProstateCancer4 + 4)
8956538173608.E17.gz43_514278M00085299B:G01chiron(cc187-
ProstateCancer4 + 4)
8962575473608.E20.gz43_514326M00085304B:D11chiron(cc187-
ProstateCancer4 + 4)
89712274543608.F13.gz43_514215M00085294D:G03chiron(cc187-
ProstateCancer4 + 4)
89810538543608.G09.gz43_514152M00085287C:G08chiron(cc187-
ProstateCancer4 + 4)
89912532343608.H05.gz43_514089M00085280D:F06chiron(cc187-
ProstateCancer4 + 4)
90012553303608.H14.gz43_514233M00085296D:H10chiron(cc187-
ProstateCancer4 + 4)
9019746353608.H18.gz43_514297M00085302C:B06chiron(cc187-
ProstateCancer4 + 4)
9028726463608.J17.gz43_514283M00085301B:C10chiron(cc187-
ProstateCancer4 + 4)
9039394453608.J24.gz43_514395M00085273B:F08chiron(cc187-
ProstateCancer4 + 4)
9047758203608.K03.gz43_514060M00085277D:C11chiron(cc187-
ProstateCancer4 + 4)
9054524773608.K14.gz43_514236M00085296C:C09chiron(cc187-
ProstateCancer4 + 4)
9066548483608.L07.gz43_514125M00085284D:A09chiron(cc187-
ProstateCancer4 + 4)
90712257343608.L14.gz43_514237M00085297A:A11chiron(cc187-
ProstateCancer4 + 4)
9084041973608.N09.gz43_514159M00085288C:C09chiron(cc187-
ProstateCancer4 + 4)
9098662373608.N19.gz43_514319M00085304A:B11chiron(cc187-
ProstateCancer4 + 4)
9106502633608.N20.gz43_514335M00085305B:F10chiron(cc187-
ProstateCancer4 + 4)
91111413713608.O04.gz43_514080M00085278D:F03chiron(cc187-
ProstateCancer4 + 4)
91212575833608.P22.gz43_514369M00085309A:D11chiron(cc187-
ProstateCancer4 + 4)
91312524753611.A17.gz43_514658M00085312C:B09chiron(cc187-
ProstateCancer4 + 4)
9143862553611.B11.gz43_514563M00085314C:F01chiron(cc187-
ProstateCancer4 + 4)
91510791963611.B16.gz43_514643M00085315C:B03chiron(cc187-
ProstateCancer4 + 4)
91612584563611.C09.gz43_514532M00085317B:G09chiron(cc187-
ProstateCancer4 + 4)
9174034883611.E07.gz43_514502M00085322D:G05chiron(cc187-
ProstateCancer4 + 4)
9188570313611.E12.gz43_514582M00085323C:H07chiron(cc187-
ProstateCancer4 + 4)
91912926003611.E20.gz43_514710M00085324B:F10chiron(cc187-
ProstateCancer4 + 4)
92012268623611.F15.gz43_514631M00085326B:D08chiron(cc187-
ProstateCancer4 + 4)
9218570313611.H10.gz43_514553M00085331C:C07chiron(cc187-
ProstateCancer4 + 4)
92212922983611.H22.gz43_514745M00085332D:B03chiron(cc187-
ProstateCancer4 + 4)
92311830793611.I04.gz43_514458M00085333B:B10chiron(cc187-
ProstateCancer4 + 4)
92412580113611.I13.gz43_514602M00085334A:D10chiron(cc187-
ProstateCancer4 + 4)
9256778583611.J04.gz43_514459M00085335B:D09chiron(cc187-
ProstateCancer4 + 4)
92610537473611.J15.gz43_514635M00085336C:G09chiron(cc187-
ProstateCancer4 + 4)
9274045893611.J17.gz43_514667M00085336D:B10chiron(cc187-
ProstateCancer4 + 4)
9287277303611.J22.gz43_514747M00085337B:F08chiron(cc187-
ProstateCancer4 + 4)
9294518193611.K01.gz43_514412M00085337C:E09chiron(cc187-
ProstateCancer4 + 4)
9305516913611.K12.gz43_514588M00085339C:C04chiron(cc187-
ProstateCancer4 + 4)
93110798633611.L22.gz43_514749M00085341C:H08chiron(cc187-
ProstateCancer4 + 4)
93210537473611.M18.gz43_514686M00085344A:G08chiron(cc187-
ProstateCancer4 + 4)
93312557453611.M24.gz43_514782M00085344D:B07chiron(cc187-
ProstateCancer4 + 4)
93412544183611.N01.gz43_514415M00085344D:F01chiron(cc187-
ProstateCancer4 + 4)
9355040383611.N09.gz43_514543M00085345B:C09chiron(cc187-
ProstateCancer4 + 4)
9369742233611.O16.gz43_514656M00085349A:C08chiron(cc187-
ProstateCancer4 + 4)
93712239363611.P08.gz43_514529M00085350D:G05chiron(cc187-
ProstateCancer4 + 4)
93812922893614.C18.gz43_515060M00085358B:A04chiron(cc187-
ProstateCancer4 + 4)
93912924233614.D14.gz43_514997M00085360B:G03chiron(cc187-
ProstateCancer4 + 4)
94012557453614.D21.gz43_515109M00085361A:A09chiron(cc187-
ProstateCancer4 + 4)
94112587143614.E06.gz43_514870M00085361D:F06chiron(cc187-
ProstateCancer4 + 4)
94212584913614.F22.gz43_515127M00085365C:C09chiron(cc187-
ProstateCancer4 + 4)
94310791963614.G20.gz43_515096M00085367B:C02chiron(cc187-
ProstateCancer4 + 4)
94412575313614.H09.gz43_514921M00085368B:A02chiron(cc187-
ProstateCancer4 + 4)
94510552563614.H22.gz43_515129M00085369D:G04chiron(cc187-
ProstateCancer4 + 4)
94612924393614.J07.gz43_514891M00085373C:B06chiron(cc187-
ProstateCancer4 + 4)
9477614603614.K22.gz43_515132M00085380B:E10chiron(cc187-
ProstateCancer4 + 4)
9488323473614.L13.gz43_514989M00085381C:A05chiron(cc187-
ProstateCancer4 + 4)
9498554283614.M08.gz43_514910M00085383C:C05chiron(cc187-
ProstateCancer4 + 4)
9507661343614.O02.gz43_514816M00085389B:B05chiron(cc187-
ProstateCancer4 + 4)
9518759783614.O07.gz43_514896M00085389C:H04chiron(cc187-
ProstateCancer4 + 4)
95212265353614.O16.gz43_515040M00085390D:A03chiron(cc187-
ProstateCancer4 + 4)
95312047823614.P11.gz43_514961M00085393A:D06chiron(cc187-
ProstateCancer4 + 4)
95412939723614.P16.gz43_515041M00085393D:F12chiron(cc187-
ProstateCancer4 + 4)
9558493333617.B16.gz43_515411M00085434C:G06chiron(cc187-
ProstateCancer4 + 4)
9567214893617.C21.gz43_515492M00085444C:C02chiron(cc187-
ProstateCancer4 + 4)
9573973133617.F10.gz43_515319M00085419A:G09chiron(cc187-
ProstateCancer4 + 4)
95812924363617.H16.gz43_515417M00085435A:B11chiron(cc187-
ProstateCancer4 + 4)
9599638803617.I01.gz43_515178M00085396B:G04chiron(cc187-
ProstateCancer4 + 4)
96012279723617.L16.gz43_515421M00085435B:C05chiron(cc187-
ProstateCancer4 + 4)
9618759783617.L21.gz43_515501M00085446A:D04chiron(cc187-
ProstateCancer4 + 4)
96210649633617.M08.gz43_515294M00085415A:D12chiron(cc187-
ProstateCancer4 + 4)
96312245053617.M13.gz43_515374M00085428B:G02chiron(cc187-
ProstateCancer4 + 4)
9644539013617.N05.gz43_515247M00085406A:F03chiron(cc187-
ProstateCancer4 + 4)
96512924233617.N10.gz43_515327M00085419C:H05chiron(cc187-
ProstateCancer4 + 4)
96612738443617.N14.gz43_515391M00085432A:H08chiron(cc187-
ProstateCancer4 + 4)
96712276233617.N19.gz43_515471M00085441D:H10chiron(cc187-
ProstateCancer4 + 4)
96812922623617.P11.gz43_515345M00085422D:D07chiron(cc187-
ProstateCancer4 + 4)
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ProstateCancer4 + 4)
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ProstateCancer4 + 4)
11631893553664.D06.gz43_520606M00086176C:A06chiron(cc187-
ProstateCancer4 + 4)
11649659473664.D12.gz43_520702M00086178A:D07chiron(cc187-
ProstateCancer4 + 4)
116512610353664.D17.gz43_520782M00086178D:H12chiron(cc187-
ProstateCancer4 + 4)
11667614603664.E18.gz43_520799M00086183A:H04chiron(cc187-
ProstateCancer4 + 4)
11675049043664.E23.gz43_520879M00086184C:C10chiron(cc187-
ProstateCancer4 + 4)
116812279683664.E24.gz43_520895M00086184C:D04chiron(cc187-
ProstateCancer4 + 4)
11696060403664.G12.gz43_520705M00086191A:G09chiron(cc187-
ProstateCancer4 + 4)
117012929323664.G20.gz43_520833M00086193A:F04chiron(cc187-
ProstateCancer4 + 4)
117110695783664.H15.gz43_520754M00086196A:F07chiron(cc187-
ProstateCancer4 + 4)
117212924393664.H22.gz43_520866M00086197B:A03chiron(cc187-
ProstateCancer4 + 4)
11736526513664.J12.gz43_520708M00086202C:A07chiron(cc187-
ProstateCancer4 + 4)
11744492763664.J23.gz43_520884M00086203C:H04chiron(cc187-
ProstateCancer4 + 4)
11755482173664.K16.gz43_520773M00086206A:E10chiron(cc187-
ProstateCancer4 + 4)
117610553263664.K19.gz43_520821M00086206C:C01chiron(cc187-
ProstateCancer4 + 4)
117712939463664.L21.gz43_520854M00086209B:H12chiron(cc187-
ProstateCancer4 + 4)
117812565643664.O22.gz43_520873M00086225B:E01chiron(cc187-
ProstateCancer4 + 4)
11799638803664.P12.gz43_520714M00086227B:E06chiron(cc187-
ProstateCancer4 + 4)
118080453664.P18.gz43_520810M00086228A:F11chiron(cc187-
ProstateCancer4 + 4)
11816099143665.A23.gz43_521259M00086233C:F01chiron(cc187-
ProstateCancer4 + 4)
11828536963665.B01.gz43_520908M00086233D:A03chiron(cc187-
ProstateCancer4 + 4)
11834184393665.B12.gz43_521084M00086235A:F05chiron(cc187-
ProstateCancer4 + 4)
11848408523665.E11.gz43_521071M00086247A:G11chiron(cc187-
ProstateCancer4 + 4)
11855551153665.E20.gz43_521215M00086248A:H09chiron(cc187-
ProstateCancer4 + 4)
118610612063665.H20.gz43_521218M00086259A:F11chiron(cc187-
ProstateCancer4 + 4)
11877338403665.K01.gz43_520917M00086266B:E10chiron(cc187-
ProstateCancer4 + 4)
11887321833665.M01.gz43_520919M00086270C:D08chiron(cc187-
ProstateCancer4 + 4)
118912922813665.M21.gz43_521239M00086272D:E04chiron(cc187-
ProstateCancer4 + 4)
119012273523665.M23.gz43_521271M00086272D:H11chiron(cc187-
ProstateCancer4 + 4)
11919588443665.N24.gz43_521288M00086276D:F10chiron(cc187-
ProstateCancer4 + 4)
1192463665.O06.gz43_521001M00086277B:E06chiron(cc187-
ProstateCancer4 + 4)
119311386273665.O14.gz43_521129M00086279A:B07chiron(cc187-
ProstateCancer4 + 4)
119412243893665.O15.gz43_521145M00086279C:A08chiron(cc187-
ProstateCancer4 + 4)
11956721923665.O19.gz43_521209M00086279D:C07chiron(cc187-
ProstateCancer4 + 4)
11967248973665.O21.gz43_521241M00086280A:E02chiron(cc187-
ProstateCancer4 + 4)
119712923893665.O23.gz43_521273M00086280B:G09chiron(cc187-
ProstateCancer4 + 4)
119812747363665.P13.gz43_521114M00086282A:B10chiron(cc187-
ProstateCancer4 + 4)
119912925823666.A07.gz43_521387M00086285B:C10chiron(cc187-
ProstateCancer4 + 4)
1200997743666.A19.gz43_521579M00086286B:F08chiron(cc187-
ProstateCancer4 + 4)
12017421013666.A24.gz43_521659M00086286C:H02chiron(cc187-
ProstateCancer4 + 4)
12027661343666.B11.gz43_521452M00086288A:B05chiron(cc187-
ProstateCancer4 + 4)
120312503733666.C18.gz43_521565M00086291A:E10chiron(cc187-
ProstateCancer4 + 4)
120410888473666.D02.gz43_521310M00086291D:B08chiron(cc187-
ProstateCancer4 + 4)
120512939463666.D11.gz43_521454M00086294B:E11chiron(cc187-
ProstateCancer4 + 4)
120634623666.D15.gz43_521518M00086294C:G05chiron(cc187-
ProstateCancer4 + 4)
12077277643666.D16.gz43_521534M00086294D:F08chiron(cc187-
ProstateCancer4 + 4)
12083967243666.F22.gz43_521632M00086301A:D04chiron(cc187-
ProstateCancer4 + 4)
120911420193666.G12.gz43_521473M00086302A:E06chiron(cc187-
ProstateCancer4 + 4)
12107655003666.I12.gz43_521475M00086310A:F04chiron(cc187-
ProstateCancer4 + 4)
121111972593666.L01.gz43_521302M00086322A:E02chiron(cc187-
ProstateCancer4 + 4)
121211384723666.L06.gz43_521382M00086322D:D05chiron(cc187-
ProstateCancer4 + 4)
12138694533666.L11.gz43_521462M00086323B:C04chiron(cc187-
ProstateCancer4 + 4)
121410882513666.L23.gz43_521654M00086324D:D01chiron(cc187-
ProstateCancer4 + 4)
121512924133666.M16.gz43_521543M00086327B:D10chiron(cc187-
ProstateCancer4 + 4)
121612747363666.N06.gz43_521384M00086328B:G12chiron(cc187-
ProstateCancer4 + 4)
121712599553667.A15.gz43_524557M00086336B:A08chiron(cc187-
ProstateCancer4 + 4)
121880453754.A08.gz43_532949M00083799D:F10chiron(cc187-NormBPHProstate)
121912279993754.A13.gz43_533029M00083800C:E07chiron(cc187-NormBPHProstate)
12206379153754.A16.gz43_533077M00083801B:H03chiron(cc187-NormBPHProstate)
12217649783754.B04.gz43_532886M00083803B:F11chiron(cc187-NormBPHProstate)
12225009193754.805.gz43_532902M00083803B:F12chiron(cc187-NormBPHProstate)
122312241333754.B07.gz43_532934M00083803C:F03chiron(cc187-NormBPHProstate)
12248316383754.B08.gz43_532950M00083804A:H12chiron(cc187-NormBPHProstate)
122512297923754.B10.gz43_532982M00083804B:C03chiron(cc187-NormBPHProstate)
122612236183754.C22.gz43_533175M00083809B:E08chiron(cc187-NormBPHProstate)
12274537673754.D19.gz43_533128M00083812C:G02chiron(cc187-NormBPHProstate)
12287335383754.E12.gz43_533017M00083814D:A10chiron(cc187-NormBPHProstate)
12294014243754.E20.gz43_533145M00083815C:H08chiron(cc187-NormBPHProstate)
123010810493754.F01.gz43_532842M00083816B:D08chiron(cc187-NormBPHProstate)
123111931243754.F08.gz43_532954M00083817B:A11chiron(cc187-NormBPHProstate)
123210856383754.F11.gz43_533002M00083817B:G09chiron(cc187-NormBPHProstate)
12334270933754.F15.gz43_533066M00083817D:A08chiron(cc187-NormBPHProstate)
12345140903754.F20.gz43_533146M00083818A:E09chiron(cc187-NormBPHProstate)
123510804013754.G03.gz43_532875M00083818C:A02chiron(cc187-NormBPHProstate)
12365677003754.G08.gz43_532955M00083819B:E10chiron(cc187-NormBPHProstate)
12378286953754.G18.gz43_533115M00083820B:C03chiron(cc187-NormBPHProstate)
12387175833754.H08.gz43_532956M00083831D:H11chiron(cc187-NormBPHProstate)
12394542143754.I01.gz43_532845M00083834B:F09chiron(cc187-NormBPHProstate)
12401697623754.I03.gz43_532877M00083834C:E02chiron(cc187-NormBPHProstate)
124112279123754.J01.gz43_532846M00083838A:E05chiron(cc187-NormBPHProstate)
124212187933754.J05.gz43_532910M00083838C:F07chiron(cc187-NormBPHProstate)
12438550953754.J10.gz43_532990M00083839A:H03chiron(cc187-NormBPHProstate)
124411769923754.J12.gz43_533022M00083839B:G09chiron(cc187-NormBPHProstate)
124510737673754.J24.gz43_533214M00083841A:G01chiron(cc187-NormBPHProstate)
12464520153754.K14.gz43_533055M00083844A:E12chiron(cc187-NormBPHProstate)
124712244543754.K17.gz43_533103M00083844B:C04chiron(cc187-NormBPHProstate)
12489617573754.K20.gz43_533151M00083844C:C04chiron(cc187-NormBPHProstate)
12494037383754.M08.gz43_532961M00083849C:F11chiron(cc187-NormBPHProstate)
12504553863754.N16.gz43_533090M00084246A:D03chiron(cc187-NormBPHProstate)
12515563393754.N19.gz43_533138M00084246B:D10chiron(cc187-NormBPHProstate)
12526609073754.N22.gz43_533186M00084246B:H03chiron(cc187-NormBPHProstate)
12534043083754.O18.gz43_533123M00084248B:C06chiron(cc187-NormBPHProstate)
125412025703754.O23.gz43_533203M00084248D:H09chiron(cc187-NormBPHProstate)
125512269323754.P13.gz43_533044M00084251D:C05chiron(cc187-NormBPHProstate)
125610548073754.P17.gz43_533108M00084252B:H01chiron(cc187-NormBPHProstate)
125710530613756.A02.gz43_533237M00085806B:A10chiron(cc187-
ProstateCancer4 + 4)
125810797653756.A11.gz43_533381M00085825C:D12chiron(cc187-
ProstateCancer4 + 4)
125910923913756.A13.gz43_533413M00085830B:E09chiron(cc187-
ProstateCancer4 + 4)
126012263883756.B03.gz43_533254M00085809A:E04chiron(cc187-
ProstateCancer4 + 4)
12612563756.B04.gz43_533270M00085811B:D12chiron(cc187-
ProstateCancer4 + 4)
12627302283756.B15.gz43_533446M00085835D:F06chiron(cc187-
ProstateCancer4 + 4)
126312550373756.B21.gz43_533542M00085859B:A11chiron(cc187-
ProstateCancer4 + 4)
126410807163756.B22.gz43_533558M00085861C:A03chiron(cc187-
ProstateCancer4 + 4)
12657774853756.C06.gz43_533303M00085814B:G08chiron(cc187-
ProstateCancer4 + 4)
12664496873756.C16.gz43_533463M00085837C:H08chiron(cc187-
ProstateCancer4 + 4)
126712599063756.D08.gz43_533336M00085819D:C02chiron(cc187-
ProstateCancer4 + 4)
126824543756.D18.gz43_533496M00085846A:H10chiron(cc187-
ProstateCancer4 + 4)
126911065143756.D24.gz43_533592M00085804B:F09chiron(cc187-
ProstateCancer4 + 4)
127010530783756.E01.gz43_533225M00085805A:G07chiron(cc187-
ProstateCancer4 + 4)
127110530143756.E06.gz43_533305M00085814C:C12chiron(cc187-
ProstateCancer4 + 4)
12725589763756.E12.gz43_533401M00085827C:F03chiron(cc187-
ProstateCancer4 + 4)
12734921113756.E22.gz43_533561M00085860D:H02chiron(cc187-
ProstateCancer4 + 4)
127492493756.F11.gz43_533386M00085826D:B03chiron(cc187-
ProstateCancer4 + 4)
127511387993756.F16.gz43_533466M00085839B:B12chiron(cc187-
ProstateCancer4 + 4)
127610522163756.G07.gz43_533323M00085817B:B08chiron(cc187-
ProstateCancer4 + 4)
12777148403756.G12.gz43_533403M00085827D:D01chiron(cc187-
ProstateCancer4 + 4)
12783725413756.G14.gz43_533435M00085832D:G02chiron(cc187-
ProstateCancer4 + 4)
12793740983756.I03.gz43_533261M00085808C:E12chiron(cc187-
ProstateCancer4 + 4)
128087563756.J05.gz43_533294M00085814A:C02chiron(cc187-
ProstateCancer4 + 4)
12814869613756.K03.gz43_533263M00085808D:E01chiron(cc187-
ProstateCancer4 + 4)
12826177143756.K07.gz43_533327M00085817C:C10chiron(cc187-
ProstateCancer4 + 4)
128329463756.K15.gz43_533455M00085835B:E11chiron(cc187-
ProstateCancer4 + 4)
128412279633756.K18.gz43_533503M00085844C:H11chiron(cc187-
ProstateCancer4 + 4)
128512061613756.K20.gz43_533535M00085854C:E06chiron(cc187-
ProstateCancer4 + 4)
12866085603756.L02.gz43_533248M00085807D:G11chiron(cc187-
ProstateCancer4 + 4)
1287161553756.L03.gz43_533264M00085810A:A10chiron(cc187-
ProstateCancer4 + 4)
128811944313756.L19.gz43_533520M00085854A:D09chiron(cc187-
ProstateCancer4 + 4)
12891747303756.M06.gz43_533313M00085815C:E11chiron(cc187-
ProstateCancer4 + 4)
12903727413756.M07.gz43_533329M00085817C:G05chiron(cc187-
ProstateCancer4 + 4)
12913800703756.M20.gz43_533537M00085854C:F04chiron(cc187-
ProstateCancer4 + 4)
12929746353756.N18.gz43_533506M00085849D:G06chiron(cc187-
ProstateCancer4 + 4)
129312527473756.N21.gz43_533554M00085860B:D10chiron(cc187-
ProstateCancer4 + 4)
12945257593756.O03.gz43_533267M00085809A:C05chiron(cc187-
ProstateCancer4 + 4)
12953861093756.O07.gz43_533331M00085817D:C04chiron(cc187-
ProstateCancer4 + 4)
12964013423756.O08.gz43_533347M00085819C:F06chiron(cc187-
ProstateCancer4 + 4)
129712601773756.P08.gz43_533348M00085820D:D02chiron(cc187-
ProstateCancer4 + 4)
12987204543759.C01.gz43_533607M00085068C:B03chiron(cc187-NormBPHProstate)
12998758433759.D15.gz43_533832M00085092D:D09chiron(cc187-NormBPHProstate)
13004028513759.H08.gz43_533724M00085082C:B04chiron(cc187-NormBPHProstate)
1301213703759.H15.gz43_533836M00085100A:A12chiron(cc187-NormBPHProstate)
130211962683759.H17.gz43_533868M00085105C:D01chiron(cc187-NormBPHProstate)
13031854323759.H23.gz43_533964M00085066B:D12chiron(cc187-NormBPHProstate)
130412681613759.I05.gz43_533677M00085076C:A07chiron(cc187-NormBPHProstate)
1305447373759.I19.gz43_533901M00085107D:H08chiron(cc187-NormBPHProstate)
13064007343759.K05.gz43_533679M00085076C:H01chiron(cc187-NormBPHProstate)
13074135733759.K17.gz43_533871M00085104C:A10chiron(cc187-NormBPHProstate)
130810666543759.L02.gz43_533632M00085071B:D07chiron(cc187-NormBPHProstate)
130912263653759.L09.gz43_533744M00085084A:E12chiron(cc187-NormBPHProstate)
1310157593759.L10.gz43_533760M00085085C:D10chiron(cc187-NormBPHProstate)
13111403723759.L15.gz43_533840M00085100A:H07chiron(cc187-NormBPHProstate)
131211385723759.L24.gz43_533984M00085068B:A07chiron(cc187-NormBPHProstate)
131367903759.M19.gz43_533905M00085108A:C12chiron(cc187-NormBPHProstate)
131412281473759.N08.gz43_533730M00085083A:E04chiron(cc187-NormBPHProstate)
1315436783759.N16.gz43_533858M00085103D:H12chiron(cc187-NormBPHProstate)
131611169923759.N23.gz43_533970M00085066D:A05chiron(cc187-NormBPHProstate)
13173932613759.O16.gz43_533859M00085101C:H03chiron(cc187-NormBPHProstate)
13183784593759.P03.gz43_533652M00085074B:A07chiron(cc187-NormBPHProstate)
13192427073759.P13.gz43_533812M00085090C:C09chiron(cc187-NormBPHProstate)
1320693759.P15.gz43_533844M00085100B:C12chiron(cc187-NormBPHProstate)
1321126133759.P17.gz43_533876M00085105D:H02chiron(cc187-NormBPHProstate)
132210521083762.A09.gz43_534117M00084772C:G12chiron(cc187-NormBPHProstate)
132311915473762.A16.gz43_534229M00084773C:H08chiron(cc187-NormBPHProstate)
13242049853762.A19.gz43_534277M00084773C:F08chiron(cc187-NormBPHProstate)
132510548133762.A20.gz43_534293M00084773C:D04chiron(cc187-NormBPHProstate)
13266872233762.B05.gz43_534054M00084774B:C10chiron(cc187-NormBPHProstate)
132712257193762.B15.gz43_534214M00084775C:E05chiron(cc187-NormBPHProstate)
132811395223762.C20.gz43_534295M00084779D:H10chiron(cc187-NormBPHProstate)
13294087113762.C23.gz43_534343M00084779B:D03chiron(cc187-NormBPHProstate)
13307708343762.D03.gz43_534024M00084780C:F08chiron(cc187-NormBPHProstate)
13319617903762.D04.gz43_534040M00084780D:D07chiron(cc187-NormBPHProstate)
133211184703762.D18.gz43_534264M00084781A:H09chiron(cc187-NormBPHProstate)
13331403143762.D19.gz43_534280M00084781A:E05chiron(cc187-NormBPHProstate)
13346846383762.D22.gz43_534328M00084781A:A05chiron(cc187-NormBPHProstate)
133510546483762.E01.gz43_533993M00084782D:H08chiron(cc187-NormBPHProstate)
13364542143762.E10.gz43_534137M00084782D:D10chiron(cc187-NormBPHProstate)
1337778563762.E15.gz43_534217M00084783C:A09chiron(cc187-NormBPHProstate)
133847453762.E23.gz43_534345M00084783C:G08chiron(cc187-NormBPHProstate)
13395570633762.F08.gz43_534106M00084784B:A02chiron(cc187-NormBPHProstate)
134010873183762.F22.gz43_534330M00084787B:D12chiron(cc187-NormBPHProstate)
134110563693762.G18.gz43_534267M00084789D:B10chiron(cc187-NormBPHProstate)
13429968883762.H12.gz43_534172M00084791D:D01chiron(cc187-NormBPHProstate)
13439686473762.I07.gz43_534093M00084794B:C01chiron(cc187-NormBPHProstate)
13444537673762.J03.gz43_534030M00084796D:B01chiron(cc187-NormBPHProstate)
134510147343762.J18.gz43_534270M00084799C:E08chiron(cc187-NormBPHProstate)
1346866123762.K02.gz43_534015M00084800B:H09chiron(cc187-NormBPHProstate)
13475516913762.K20.gz43_534303M00084802A:H09chiron(cc187-NormBPHProstate)
13486915123762.L18.gz43_534272M00084804C:H10chiron(cc187-NormBPHProstate)
134912590693762.L20.gz43_534304M00084804B:E01chiron(cc187-NormBPHProstate)
13506836503762.M04.gz43_534049M00084805D:E02chiron(cc187-NormBPHProstate)
13514471513762.M17.gz43_534257M00084807D:F07chiron(cc187-NormBPHProstate)
135210520583762.M23.gz43_534353M00084808D:A07chiron(cc187-NormBPHProstate)
|
Summary of Polynucleotides of the Invention
Table 11 (inserted prior to claims) provides a summary of polynucleotides isolated as described. Specifically, Table 11 provides: 1) the SEQ ID NO (“SEQ ID”) assigned to each sequence for use in the present specification; 2) the Cluster Identification No. (“CLUSTER”); 3) the Sequence Name assigned to each sequence; 3) the sequence name (“SEQ NAME”) used as an internal identifier of the sequence; 4) the name assigned to the clone from which the sequence was isolated (“CLONE ID”); and 5) the name of the library from which the sequence was isolated (“LIBRARY”). Because at least some of the provided polynucleotides represent partial mRNA transcripts, two or more polynucleotides may represent different regions of the same mRNA transcript and the same gene and/or may be contained within the same clone. Thus, for example, if two or more SEQ ID NOS: are identified as belonging to the same clone, then either sequence can be used to obtain the full-length mRNA or gene. Clones which comprise the sequences described herein were deposited as set out in the tables indicated below (see Example entitled “Deposit Information”).
Example 18
Contig Assembly
The sequences of the polynucleotides provided in the present invention can be used to extend the sequence information of the gene to which the polynucleotides correspond (e.g., a gene, or mRNA encoded by the gene, having a sequence of the polynucleotide described herein). This expanded sequence information can in turn be used to further characterize the corresponding gene, which in turn provides additional information about the nature of the gene product (e.g., the normal function of the gene product). The additional information can serve to provide additional evidence of the gene product's use as a therapeutic target, and provide further guidance as to the types of agents that can modulate its activity.
For example, a contig was assembled using the sequence of a polynucleotide described herein. A “contig” is a contiguous sequence of nucleotides that is assembled from nucleic acid sequences having overlapping (e.g., shared or substantially similar) sequence information. The sequences of publicly-available ESTs (Expressed Sequence Tags) and the sequences of various of the above-described polynucleotides were used in the contig assembly. The contig was assembled using the software program Sequencher, version 4.05, according to the manufacturer's instructions. The sequence information obtained in the contig assembly was then used to obtain a consensus sequence derived from the contig using the Sequencher program. The resulting consensus sequence was used to search both the public databases as well as databases internal to the applicants to match the consensus polynucleotide with homology data and/or differential gene expressed data.
The final result provided the sequences listed as SEQ ID NOS: 1353-1561 in the accompanying Sequence Listing and summarized in Tables 12 and 13 (inserted prior to claims). Table 12 provides a summary of the consensus sequences assembled as described. Specifically, Table 3 provides: 1) the SEQ ID NO (“SEQ ID”) assigned to each consensus sequence for use in the present specification; 2) the Cluster Identification No. (“CLUSTER”); and 3) the consensus sequence name (“CONSENSUS SEQ NAME”) used as an internal identifier of the sequence.
TABLE 12
|
|
SEQ IDCLUSTERCONSENSUS SEQ NAME
|
|
135346Clu46.con_5
13548293Clu8293.con_1
135534201Clu34201.con_1
135648343Clu48343.con_1
135789833Clu89833.con_2
1358140314Clu140314.con_2
1359141870Clu141870.con_1
1360180092Clu180092.con_1
1361189355Clu189355.con_1
1362234667Clu234667.con_1
1363242901Clu242901.con_1
1364307985Clu307985.con_1
1365387610Clu387610.con_1
1366389995Clu389995.con_2
1367393948Clu393948.con_1
1368397313Clu397313.con_1
1369398439Clu398439.con_1
1370402150Clu402150.con_1
1371402789Clu402789.con_1
1372403488Clu403488.con_1
1373413505Clu413505.con_1
1374418320Clu418320.con_1
1375424678Clu424678.con_1
1376426138Clu426138.con_1
1377427904Clu427904.con_1
1378447151Clu447151.con_1
1379452564Clu452564.con_1
1380454826Clu454826.con_1
1381460499Clu460499.con_1
1382477110Clu477110.con_1
1383477708Clu477708.con_1
1384478212Clu478212.con_1
1385484459Clu484459.con_1
1386494499Clu494499.con_1
1387494890Clu494890.con_1
1388500919Clu500919.con_1
1389504038Clu504038.con_1
1390504904Clu504904.con_2
1391505750Clu505750.con_1
1392517444Clu517444.con_1
1393528281Clu528281.con_1
1394529709Clu529709.con_1
1395532062Clu532062.con_1
1396542301Clu542301.con_1
1397542825Clu542825.con_1
1398548217Clu548217.con_1
1399548277Clu548277.con_1
1400554189Clu554189.con_2
1401555115Clu555115.con_2
1402555967Clu555967.con_1
1403557717Clu557717.con_1
1404566548Clu566548.con_1
1405567700Clu567700.con_2
1406573169Clu573169.con_1
1407585099Clu585099.con_1
1408585899Clu585899.con_1
1409609914Clu609914.con_1
1410613411Clu613411.con_1
1411621702Clu621702.con_1
1412637915Clu637915.con_1
1413640157Clu640157.con_1
1414640277Clu640277.con_1
1415645986Clu645986.con_1
1416647587Clu647587.con_2
1417652651Clu652651.con_1
1418653817Clu653817.con_1
1419660907Clu660907.con_1
1420661802Clu661802.con_1
1421676665Clu676665.con_1
1422684638Clu684638.con_1
1423691512Clu691512.con_1
1424700354Clu700354.con_1
1425708025Clu708025.con_1
1426710194Clu710194.con_1
1427733840Clu733840.con_1
1428734568Clu734568.con_1
1429742101Clu742101.con_1
1430747805Clu747805.con_1
1431761460Clu761460.con_1
1432765500Clu765500.con_1
1433770834Clu770834.con_1
1434774520Clu774520.con_1
1435777670Clu777670.con_1
1436812031Clu812031.con_1
1437821536Clu821536.con_1
1438823271Clu823271.con_1
1439845354Clu845354.con_1
1440846056Clu846056.con_1
1441854573Clu854573.con_1
1442863768Clu863768.con_1
1443869453Clu869453.con_1
1444875978Clu875978.con_2
1445893981Clu893981.con_1
1446945247Clu945247.con_1
1447947168Clu947168.con_1
1448961757Clu961757.con_1
1449968647Clu968647.con_1
1450970165Clu970165.con_1
1451991366Clu991366.con_1
14521014734Clu1014734.con_1
14531037887Clu1037887.con_1
14541052399Clu1052399.con_1
14551052466Clu1052466.con_1
14561053121Clu1053121.con_1
14571053514Clu1053514.con_1
14581053747Clu1053747.con_1
14591053799Clu1053799.con_1
14601053854Clu1053854.con_1
14611054038Clu1054038.con_1
14621054069Clu1054069.con_1
14631054074Clu1054074.con_1
14641054807Clu1054807.con_1
14651054813Clu1054813.con_1
14661054884Clu1054884.con_1
14671055018Clu1055018.con_1
14681055063Clu1055063.con_1
14691055089Clu1055089.con_1
14701055256Clu1055256.con_1
14711055326Clu1055326.con_1
14721056369Clu1056369.con_1
14731059332Clu1059332.con_1
14741059445Clu1059445.con_1
14751060021Clu1060021.con_1
14761061206Clu1061206.con_1
14771062537Clu1062537.con_1
14781064975Clu1064975.con_1
14791065531Clu1065531.con_1
14801066041Clu1066041.con_1
14811069578Clu1069578.con_1
14821069632Clu1069632.con_1
14831073767Clu1073767.con_1
14841074160Clu1074160.con_1
14851076930Clu1076930.con_1
14861077033Clu1077033.con_1
14871079196Clu1079196.con_1
14881079863Clu1079863.con_1
14891085638Clu1085638.con_1
14901085645Clu1085645.con_1
14911088847Clu1088847.con_1
14921088930Clu1088930.con_1
14931108332Clu1108332.con_2
14941110143Clu1110143.con_1
14951116087Clu1116087.con_1
14961131409Clu1131409.con_1
14971132413Clu1132413.con_1
14981136803Clu1136803.con_1
14991138419Clu1138419.con_1
15001138593Clu1138593.con_1
15011139522Clu1139522.con_2
15021139691Clu1139691.con_1
15031142019Clu1142019.con_1
15041171518Clu1171518.con_2
15051176182Clu1176182.con_1
15061182447Clu1182447.con_1
15071183079Clu1183079.con_1
15081184134Clu1184134.con_1
15091189027Clu1189027.con_1
15101191547Clu1191547.con_1
15111193236Clu1193236.con_1
15121210953Clu1210953.con_1
15131211899Clu1211899.con_1
15141218793Clu1218793.con_1
15151223271Clu1223271.con_1
15161223477Clu1223477.con_1
15171223907Clu1223907.con_1
15181223938Clu1223938.con_1
15191223948Clu1223948.con_1
15201224039Clu1224039.con_1
15211224226Clu1224226.con_1
15221224379Clu1224379.con_1
15231224422Clu1224422.con_1
15241224547Clu1224547.con_1
15251224704Clu1224704.con_1
15261224752Clu1224752.con_1
15271224881Clu1224881.con_2
15281225500Clu1225500.con_1
15291225595Clu1225595.con_1
15301225719Clu1225719.con_2
15311225734Clu1225734.con_1
15321226064Clu1226064.con_1
15331226304Clu1226304.con_1
15341226413Clu1226413.con_1
15351226932Clu1226932.con_1
15361227623Clu1227623.con_1
15371227781Clu1227781.con_1
15381227862Clu1227862.con_2
15391227912Clu1227912.con_1
15401227968Clu1227968.con_1
15411228277Clu1228277.con_1
15421230257Clu1230257.con_1
15431245188Clu1245188.con_1
15441250373Clu1250373.con_1
15451256564Clu1256564.con_2
15461259069Clu1259069.con_2
15471274736Clu1274736.con_1
15481283437Clu1283437.con_2
15491292262Clu1292262.con_1
15501292281Clu1292281.con_1
15511292289Clu1292289.con_1
15521292413Clu1292413.con_1
15531292423Clu1292423.con_1
15541292436Clu1292436.con_1
15551292439Clu1292439.con_1
15561292582Clu1292582.con_1
15571292600Clu1292600.con_1
15581292932Clu1292932.con_1
15591292996Clu1292996.con_1
15601293946Clu1293946.con_1
15611293972Clu1293972.con_1
|
A correlation between the polynucleotide used in consensus sequence assembly as described above and the corresponding consensus sequence is contained in Table 13. Specifically Table 13 provides: 1) the SEQ ID NO of the consensus sequence (“CONSENSUS SEQ ID”); 2) the consensus sequence name (“CONSENSUS SEQ NAME”) used as an internal identifier of the sequence; 3) the SEQ ID NO of the polynucleotide (“POLYNTD SEQ ID”) of SEQ ID NOS: 134-1352 used in assembly of the consensus sequence; and 4) the sequence name (“POLYNTD SEQ NAME”) of the polynucleotide of SEQ ID NOS: 134-1352 used in assembly of the consensus sequence.
TABLE 13
|
|
CON-
SEN-
SUSCONSENSUS SEQPOLYNTD
SEQ IDNAMESEQ IDPOLYNTD SEQ NAME
|
|
1353Clu46.con_511923665.O06.gz43_521001
1354Clu8293.con_12343547.I16.GZ43_505943
1355Clu34201.con_14913565.E16.GZ43_508243
1356Clu48343.con_12953550.O15.GZ43_506317
1357Clu89833.con_25913574.C14.GZ43_509379
1357Clu89833.con_28403599.K04.GZ43_512924
1358Clu140314.con_25633571.H01.GZ43_508792
1358Clu140314.con_213333762.D19.gz43_534280
1359Clu141870.con_14273559.F17.GZ43_507492
1359Clu141870.con_15123565.P03.GZ43_508046
1360Clu180092.con_18493599.N20.GZ43_513183
1360Clu180092.con_12663550.F06.GZ43_506164
1361Clu189355.con_111633664.D06.gz43_520606
1361Clu189355.con_111093661.E23.gz43_519727
1361Clu189355.con_111573663.N12.gz43_520328
1362Clu234667.con_13093553.E08.GZ43_506579
1362Clu234667.con_17473583.P22.GZ43_510672
1363Clu242901.con_13323553.K05.GZ43_506537
1363Clu242901.con_15613571.G22.GZ43_509127
1364Clu307985.con_12543547.P18.GZ43_505982
1365Clu387610.con_15523571.C08.GZ43_508899
1366Clu389995.con_25233568.F06.GZ43_508486
1366Clu389995.con_27763590.L08.GZ43_512221
1367Clu393948.con_14733562.K08.GZ43_507737
1368Clu397313.con_18803605.G13.gz43_513832
1368Clu397313.con_19573617.F10.gz43_515319
1369Clu398439.con_12243547.E04.GZ43_505747
1370Clu402150.con_12143544.O20.GZ43_505629
1371Clu402789.con_14163559.B04.GZ43_507280
1372Clu403488.con_17683590.J01.GZ43_512107
1372Clu403488.con_19173611.E07.gz43_514502
1373Clu413505.con_13783556.D20.GZ43_507154
1374Clu418320.con_11663541.I18.GZ43_505207
1374Clu418320.con_15553571.E02.GZ43_508805
1374Clu418320.con_18893605.N12.gz43_513823
1375Clu424678.con_11393541.A23.GZ43_505279
1376Clu426138.con_11773541.P22.GZ43_505278
1377Clu427904.con_16963580.N14.GZ43_510158
1378Clu447151.con_113513762.M17.gz43_534257
1378Clu447151.con_16063574.I07.GZ43_509273
1379Clu452564.con_12033544.K16.GZ43_505561
1379Clu452564.con_110343632.M13.gz43_517517
1380Clu454826.con_12203547.C17.GZ43_505953
1380Clu454826.con_14303559.H24.GZ43_507606
1381Clu460499.con_13303553.K02.GZ43_506489
1382Clu477110.con_12353547.I17.GZ43_505959
1382Clu477110.con_14903565.D19.GZ43_508290
1382Clu477110.con_15873574.B24.GZ43_509538
1383Clu477708.con_15143565.P22.GZ43_508350
1384Clu478212.con_13173553.G21.GZ43_506789
1384Clu478212.con_12463547.M02.GZ43_505723
1384Clu478212.con_12303547.G22.GZ43_506037
1385Clu484459.con_110523635.M18.gz43_517981
1385Clu484459.con_111253662.C15.gz43_519981
1386Clu494499.con_11973544.I15.GZ43_505543
1386Clu494499.con_15723571.J14.GZ43_509002
1386Clu494499.con_17253583.H15.GZ43_510552
1387Clu494890.con_13003550.P23.GZ43_506446
1387Clu494890.con_16053574.I02.GZ43_509193
1387Clu494890.con_17913596.D17.GZ43_512741
1388Clu500919.con_112223754.B05.gz43_532902
1388Clu500919.con_12263547.F10.GZ43_505844
1389Clu504038.con_19353611.N09.gz43_514543
1389Clu504038.con_111123661.G20.gz43_519681
1390Clu504904.con_211673664.E23.gz43_520879
1391Clu505750.con_11453538.G22.GZ43_504885
1391Clu505750.con_11863544.E18.GZ43_505587
1392Clu517444.con_110213629.H10.gz43_517080
1392Clu517444.con_111343662.K03.gz43_519797
1393Clu528281.con_11723541.M18.GZ43_505211
1393Clu528281.con_12183547.A24.GZ43_506063
1394Clu529709.con_13483553.K24.GZ43_506841
1394Clu529709.con_13953556.K12.GZ43_507033
1395Clu532062.con_16813580.K03.GZ43_509979
1395Clu532062.con_17793590.M04.GZ43_512158
1396Clu542301.con_12373547.J05.GZ43_505768
1396Clu542301.con_11473538.H21.GZ43_504870
1397Clu542825.con_12103544.N19.GZ43_505612
1397Clu542825.con_13363538.A24.GZ43_504911
1398Clu548217.con_16673580.G19.GZ43_510231
1398Clu548217.con_111753664.K16.gz43_520773
1399Clu548277.con_110063626.I20.gz43_516857
1400Clu554189.con_23663553.P21.GZ43_506798
1401Clu555115.con_211853665.E20.gz43_521215
1402Clu555967.con_18353599.F24.GZ43_513239
1402Clu555967.con_17883596.D01.GZ43_512485
1403Clu557717.con_18153596.O12.GZ43_512672
1403Clu557717.con_11433538.G17.GZ43_504805
1403Clu557717.con_17103583.B11.GZ43_510482
1404Clu566548.con_16623580.E23.GZ43_510293
1405Clu567700.con_25843574.B04.GZ43_509218
1406Clu573169.con_13813556.E24.GZ43_507219
1406Clu573169.con_14383559.L19.GZ43_507530
1407Clu585099.con_14113556.O13.GZ43_507053
1407Clu585099.con_18123596.N16.GZ43_512735
1407Clu585099.con_18673602.G17.GZ43_513512
1408Clu585899.con_13283553.J24.GZ43_506840
1408Clu585899.con_13203553.H21.GZ43_506790
1409Clu609914.con_111813665.A23.gz43_521259
1409Clu609914.con_111143661.H24.gz43_519746
1410Clu613411.con_13713556.B10.GZ43_506992
1410Clu613411.con_17083583.B07.GZ43_510418
1411Clu621702.con_15293568.G12.GZ43_508583
1411Clu621702.con_16553580.D07.GZ43_510036
1412Clu637915.con_16923580.M18.GZ43_510221
1412Clu637915.con_112203754.A16.gz43_533077
1413Clu640157.con_16533580.C03.GZ43_509971
1414Clu640277.con_13583553.N08.GZ43_506588
1414Clu640277.con_16453577.P07.GZ43_509664
1415Clu645986.con_11363541.A04.GZ43_504975
1415Clu645986.con_17933596.E22.GZ43_512822
1416Clu647587.con_24313559.I05.GZ43_507303
1417Clu652651.con_16163574.N10.GZ43_509326
1417Clu652651.con_110433635.D07.gz43_517796
1417Clu652651.con_111733664.J12.gz43_520708
1418Clu653817.con_12293547.G09.GZ43_505829
1418Clu653817.con_18953608.E17.gz43_514278
1419Clu660907.con_11733541.O04.GZ43_504989
1419Clu660907.con_112523754.N22.gz43_533186
1420Clu661802.con_18443599.M04.GZ43_512926
1421Clu676665.con_15963574.E02.GZ43_509189
1421Clu676665.con_12093544.N12.GZ43_505500
1422Clu684638.con_113343762.D22.gz43_534328
1422Clu684638.con_110673643.F07.gz43_518566
1423Clu691512.con_19773620.E23.gz43_516133
1423Clu691512.con_111223662.A13.gz43_519947
1423Clu691512.con_113483762.L18.gz43_534272
1424Clu700354.con_14873565.C17.GZ43_508257
1425Clu708025.con_12493547.M16.GZ43_505947
1425Clu708025.con_17193583.G16.GZ43_510567
1426Clu710194.con_12833550.K05.GZ43_506153
1426Clu710194.con_13833556.G15.GZ43_507077
1426Clu710194.con_15193568.C22.GZ43_508739
1427Clu733840.con_110563635.P18.gz43_517984
1427Clu733840.con_111313662.J08.gz43_519876
1427Clu733840.con_111873665.K01.gz43_520917
1428Clu734568.con_18573602.B22.GZ43_513587
1428Clu734568.con_18863605.M17.gz43_513902
1429Clu742101.con_14773562.O18.GZ43_507901
1429Clu742101.con_112013666.A24.gz43_521659
1430Clu747805.con_17443583.O17.GZ43_510591
1430Clu747805.con_18173596.P04.GZ43_512545
1431Clu761460.con_19473614.K22.gz43_515132
1431Clu761460.con_110453635.F06.gz43_517782
1431Clu761460.con_111663664.E18.gz43_520799
1432Clu765500.con_112103666.I12.gz43_521475
1433Clu770834.con_17993596.H22.GZ43_512825
1433Clu770834.con_113303762.D03.gz43_534024
1434Clu774520.con_13633553.P05.GZ43_506542
1434Clu774520.con_13803556.E13.GZ43_507043
1435Clu777670.con_17143583.E13.GZ43_510517
1435Clu777670.con_18033596.J13.GZ43_512683
1436Clu812031.con_11623541.G17.GZ43_505189
1436Clu812031.con_11843544.B18.GZ43_505584
1437Clu821536.con_12783550.I03.GZ43_506119
1437Clu821536.con_17463583.P19.GZ43_510624
1438Clu823271.con_13863556.H12.GZ43_507030
1439Clu845354.con_18923608.B12.gz43_514195
1440Clu846056.con_13573553.N07.GZ43_506572
1440Clu846056.con_110603638.H07.gz43_518184
1441Clu854573.con_110353632.M19.gz43_517613
1441Clu854573.con_18413599.K23.GZ43_513228
1442Clu863768.con_14173559.B06.GZ43_507312
1443Clu869453.con_17843590.O08.GZ43_512224
1444Clu875978.con_29513614.O07.gz43_514896
1444Clu875978.con_29613617.L21.gz43_515501
1445Clu893981.con_12773550.H23.GZ43_506438
1445Clu893981.con_14743562.L12.GZ43_507802
1446Clu945247.con_16223577.A18.GZ43_509825
1446Clu945247.con_11583538.O07.GZ43_504653
1446Clu945247.con_14363559.L01.GZ43_507242
1447Clu947168.con_11753541.O23.GZ43_505293
1448Clu961757.con_16983580.N23.GZ43_510302
1448Clu961757.con_112483754.K20.gz43_533151
1449Clu968647.con_111043646.P17.gz43_519120
1449Clu968647.con_113433762.I07.gz43_534093
1450Clu970165.con_12993550.P18.GZ43_506366
1450Clu970165.con_13693556.B06.GZ43_506928
1450Clu970165.con_14653562.H12.GZ43_507798
1451Clu991366.con_12803550.I21.GZ43_506407
1451Clu991366.con_14453559.O05.GZ43_507309
1451Clu991366.con_16573580.E02.GZ43_509957
1452Clu1014734.con_13443538.E15.GZ43_504771
1452Clu1014734.con_17703590.J18.GZ43_512379
1452Clu1014734.con_113453762.J18.gz43_534270
1453Clu1037887.con_11783544.A09.GZ43_505439
1453Clu1037887.con_12723550.G10.GZ43_506229
1454Clu1052399.con_15073565.N19.GZ43_508300
1455Clu1052466.con_12643550.E02.GZ43_506099
1455Clu1052466.con_15283568.G10.GZ43_508551
1456Clu1053121.con_16193574.P07.GZ43_509280
1456Clu1053121.con_16883580.L17.GZ43_510204
1457Clu1053514.con_110533635.O01.gz43_517711
1457Clu1053514.con_111553663.N09.gz43_520280
1458Clu1053747.con_18903605.N16.gz43_513887
1458Clu1053747.con_19323611.M18.gz43_514686
1459Clu1053799.con_14673562.I02.GZ43_507639
1460Clu1053854.con_18983608.G09.gz43_514152
1460Clu1053854.con_110393632.P07.gz43_517424
1461Clu1054038.con_12713550.G08.GZ43_506197
1461Clu1054038.con_12883550.L23.GZ43_506442
1461Clu1054038.con_14193559.B10.GZ43_507376
1462Clu1054069.con_17413583.N09.GZ43_510462
1463Clu1054074.con_12313547.H12.GZ43_505878
1464Clu1054807.con_12213547.C23.GZ43_506049
1464Clu1054807.con_112563754.P17.gz43_533108
1465Clu1054813.con_13923556.J14.GZ43_507064
1465Clu1054813.con_113253762.A20.gz43_534293
1465Clu1054813.con_13243553.J14.GZ43_506680
1466Clu1054884.con_15813571.O08.GZ43_508911
1466Clu1054884.con_15713571.J09.GZ43_508922
1467Clu1055018.con_14833565.B13.GZ43_508192
1468Clu1055063.con_15043565.M20.GZ43_508315
1469Clu1055089.con_13263553.J17.GZ43_506728
1469Clu1055089.con_15703571.J08.GZ43_508906
1469Clu1055089.con_17633590.H06.GZ43_512185
1470Clu1055256.con_19453614.H22.gz43_515129
1470Clu1055256.con_110373632.N21.gz43_517646
1470Clu1055256.con_111563663.N10.gz43_520296
1471Clu1055326.con_110303632.G01.gz43_517319
1471Clu1055326.con_111763664.K19.gz43_520821
1472Clu1056369.con_12393547.J20.GZ43_506008
1472Clu1056369.con_13723556.B14.GZ43_507056
1472Clu1056369.con_113413762.G18.gz43_534267
1473Clu1059332.con_15833574.B01.GZ43_509170
1474Clu1059445.con_12453547.L22.GZ43_506042
1475Clu1060021.con_12323547.H14.GZ43_505910
1475Clu1060021.con_12533547.O14.GZ43_505917
1476Clu1061206.con_19733620.E12.gz43_515957
1476Clu1061206.con_110883646.C06.gz43_518931
1476Clu1061206.con_111863665.H20.gz43_521218
1477Clu1062537.con_12863550.L16.GZ43_506330
1477Clu1062537.con_14493559.P15.GZ43_507470
1478Clu1064975.con_13253553.J16.GZ43_506712
1478Clu1064975.con_17133583.E11.GZ43_510485
1478Clu1064975.con_12653550.E06.GZ43_506163
1479Clu1065531.con_13373538.B01.GZ43_504544
1480Clu1066041.con_15083565.O02.GZ43_508029
1480Clu1066041.con_18423599.L04.GZ43_512925
1481Clu1069578.con_111713664.H15.gz43_520754
1482Clu1069632.con_15503571.B13.GZ43_508978
1482Clu1069632.con_15513571.B22.GZ43_509122
1483Clu1073767.con_11933544.H03.GZ43_505350
1483Clu1073767.con_112453754.J24.gz43_533214
1484Clu1074160.con_12963550.O17.GZ43_506349
1484Clu1074160.con_16723580.H22.GZ43_510280
1485Clu1076930.con_16353577.J04.GZ43_509610
1485Clu1076930.con_19833620.K24.gz43_516155
1486Clu1077033.con_14143559.A20.GZ43_507535
1487Clu1079196.con_19153611.B16.gz43_514643
1487Clu1079196.con_19433614.G20.gz43_515096
1488Clu1079863.con_11703541.M02.GZ43_504955
1488Clu1079863.con_15473571.A11.GZ43_508945
1488Clu1079863.con_19313611.L22.gz43_514749
1489Clu1085638.con_112323754.F11.gz43_533002
1489Clu1085638.con_13343553.K15.GZ43_506697
1489Clu1085638.con_18583602.C24.GZ43_513620
1490Clu1085645.con_17153583.E15.GZ43_510549
1491Clu1088847.con_111163661.J15.gz43_519604
1491Clu1088847.con_112043666.D02.gz43_521310
1491Clu1088847.con_110593638.F15.gz43_518310
1492Clu1088930.con_19993623.N23.gz43_516526
1493Clu1108332.con_28563602.B21.GZ43_513571
1494Clu1110143.con_13653553.P18.GZ43_506750
1494Clu1110143.con_15373568.M03.GZ43_508445
1495Clu1116087.con_14093556.N21.GZ43_507180
1495Clu1116087.con_14883565.D14.GZ43_508210
1495Clu1116087.con_15573571.E16.GZ43_509029
1496Clu1131409.con_18753602.N03.GZ43_513295
1496Clu1131409.con_111353662.L05.gz43_519830
1497Clu1132413.con_16543580.C05.GZ43_510003
1497Clu1132413.con_18793605.E19.gz43_513926
1498Clu1136803.con_11713541.M07.GZ43_505035
1498Clu1136803.con_12193547.C05.GZ43_505761
1499Clu1138419.con_12503547.N06.GZ43_505788
1500Clu1138593.con_11983544.I20.GZ43_505623
1500Clu1138593.con_13743556.C15.GZ43_507073
1500Clu1138593.con_13773556.D15.GZ43_507074
1501Clu1139522.con_26503580.A14.GZ43_510145
1501Clu1139522.con_213283762.C20.gz43_534295
1502Clu1139691.con_11993544.J04.GZ43_505368
1503Clu1142019.con_112093666.G12.gz43_521473
1504Clu1171518.con_24943565.G22.GZ43_508341
1505Clu1176182.con_15663571.H16.GZ43_509032
1506Clu1182447.con_110943646.H16.gz43_519096
1507Clu1183079.con_19233611.I04.gz43_514458
1508Clu1184134.con_17893596.D07.GZ43_512581
1509Clu1189027.con_15493571.A22.GZ43_509121
1509Clu1189027.con_18003596.I06.GZ43_512570
1509Clu1189027.con_18013596.I16.GZ43_512730
1510Clu1191547.con_11643541.I15.GZ43_505159
1510Clu1191547.con_113233762.A16.gz43_534229
1511Clu1193236.con_15953574.D12.GZ43_509348
1511Clu1193236.con_15693571.J07.GZ43_508890
1511Clu1193236.con_15593571.F16.GZ43_509030
1512Clu1210953.con_15333568.J22.GZ43_508746
1512Clu1210953.con_14343559.K16.GZ43_507481
1513Clu1211899.con_13293553.K01.GZ43_506473
1513Clu1211899.con_13853556.H02.GZ43_506870
1513Clu1211899.con_13903556.J05.GZ43_506920
1514Clu1218793.con_15113565.O15.GZ43_508237
1514Clu1218793.con_112423754.J05.gz43_532910
1515Clu1223271.con_13403538.C02.GZ43_504561
1515Clu1223271.con_16043574.H07.GZ43_509272
1516Clu1223477.con_12893550.M21.GZ43_506411
1517Clu1223907.con_16523580.C01.GZ43_509939
1518Clu1223938.con_12443547.L16.GZ43_505946
1519Clu1223948.con_11763541.P05.GZ43_505006
1520Clu1224039.con_12363547.I20.GZ43_506007
1520Clu1224039.con_13973556.K17.GZ43_507113
1521Clu1224226.con_15163568.A10.GZ43_508545
1521Clu1224226.con_16083574.J14.GZ43_509386
1522Clu1224379.con_12623550.D16.GZ43_506322
1522Clu1224379.con_14023556.M02.GZ43_506875
1523Clu1224422.con_13703556.B09.GZ43_506976
1523Clu1224422.con_13943556.K04.GZ43_506905
1524Clu1224547.con_16263577.E19.GZ43_509845
1525Clu1224704.con_17563590.E08.GZ43_512214
1526Clu1224752.con_15323568.J10.GZ43_508554
1526Clu1224752.con_11943544.H15.GZ43_505542
1527Clu1224881.con_24753562.N24.GZ43_507996
1527Clu1224881.con_211113661.G16.gz43_519617
1528Clu1225500.con_12703550.G02.GZ43_506101
1528Clu1225500.con_15483571.A14.GZ43_508993
1528Clu1225500.con_110663643.E24.gz43_518837
1529Clu1225595.con_14553562.C23.GZ43_507969
1530Clu1225719.con_22973550.O18.GZ43_506365
1530Clu1225719.con_213273762.B15.gz43_534214
1531Clu1225734.con_19073608.L14.gz43_514237
1532Clu1226064.con_12273547.F20.GZ43_506004
1533Clu1226304.con_17833590.N21.GZ43_512431
1534Clu1226413.con_16113574.K20.GZ43_509483
1534Clu1226413.con_14803562.P23.GZ43_507982
1535Clu1226932.con_13453538.F02.GZ43_504564
1535Clu1226932.con_112553754.P13.gz43_533044
1536Clu1227623.con_18343599.F17.GZ43_513127
1536Clu1227623.con_19673617.N19.gz43_515471
1537Clu1227781.con_13493553.L02.GZ43_506490
1537Clu1227781.con_110143626.P14.gz43_516768
1538Clu1227862.con_26563580.D22.GZ43_510276
1538Clu1227862.con_26613580.E21.GZ43_510261
1539Clu1227912.con_112413754.J01.gz43_532846
1539Clu1227912.con_14263559.F07.GZ43_507332
1539Clu1227912.con_14233559.E06.GZ43_507315
1540Clu1227968.con_111683664.E24.gz43_520895
1540Clu1227968.con_13613553.O23.GZ43_506829
1541Clu1228277.con_18543602.A09.GZ43_513378
1542Clu1230257.con_110683643.G20.gz43_518775
1542Clu1230257.con_110863646.A13.gz43_519041
1543Clu1245188.con_14003556.L16.GZ43_507098
1543Clu1245188.con_111423663.E04.gz43_520191
1544Clu1250373.con_14223559.D21.GZ43_507554
1544Clu1250373.con_111413663.C19.gz43_520429
1544Clu1250373.con_112033666.C18.gz43_521565
1545Clu1256564.con_25413568.P04.GZ43_508464
1545Clu1256564.con_211783664.O22.gz43_520873
1546Clu1259069.con_213493762.L20.gz43_534304
1547Clu1274736.con_111983665.P13.gz43_521114
1547Clu1274736.con_112163666.N06.gz43_521384
1548Clu1283437.con_22133544.O15.GZ43_505549
1548Clu1283437.con_29813620.J18.gz43_516058
1549Clu1292262.con_18833605.I19.gz43_513930
1549Clu1292262.con_19683617.P11.gz43_515345
1549Clu1292262.con_111173661.K22.gz43_519717
1550Clu1292281.con_18813605.H10.gz43_513785
1550Clu1292281.con_111893665.M21.gz43_521239
1551Clu1292289.con_19383614.C18.gz43_515060
1551Clu1292289.con_110983646.K14.gz43_519067
1552Clu1292413.con_110163629.B14.gz43_517138
1552Clu1292413.con_112153666.M16.gz43_521543
1552Clu1292413.con_19763620.E19.gz43_516069
1553Clu1292423.con_19393614.D14.gz43_514997
1553Clu1292423.con_19653617.N10.gz43_515327
1554Clu1292436.con_19583617.H16.gz43_515417
1554Clu1292436.con_110743643.I24.gz43_518841
1555Clu1292439.con_19463614.J07.gz43_514891
1555Clu1292439.con_111723664.H22.gz43_520866
1556Clu1292582.con_111993666.A07.gz43_521387
1556Clu1292582.con_110403635.A06.gz43_517777
1557Clu1292600.con_111153661.I22.gz43_519715
1557Clu1292600.con_19193611.E20.gz43_514710
1557Clu1292600.con_110643638.N05.gz43_518158
1558Clu1292932.con_111543663.M24.gz43_520519
1558Clu1292932.con_111703664.G20.gz43_520833
1559Clu1292996.con_110183629.E01.gz43_516933
1559Clu1292996.con_110363632.N13.gz43_517518
1559Clu1292996.con_111613664.A11.gz43_520683
1560Clu1293946.con_111773664.L21.gz43_520854
1560Clu1293946.con_112053666.D11.gz43_521454
1561Clu1293972.con_110993646.L17.gz43_519116
1561Clu1293972.con_19543614.P16.gz43_515041
1561Clu1293972.con_110103626.N07.gz43_516654
|
Example 19
Additional Gene Characterization
Sequences of the polynucleotides of SEQ ID NOS: 134-1352 were used as a query sequence in a TeraBLASTN search of the DoubleTwist Human Genome Sequence Database (DoubleTwist, Inc., Oakland, Calif.), which contains all the human genomic sequences that have been assembled into a contiguous model of the human genome. Predicted cDNA and protein sequences were obtained where a polynucleotide of the invention was homologous to a predicted full-length gene sequence. Alternatively, a sequence of a contig or consensus sequence described herein could be used directly as a query sequence in a TeraBLASTN search of the DoubleTwist Human Genome Sequence Database.
The final results of the search provided the predicted cDNA sequences listed as SEQ ID NOS: 1562-1618 in the accompanying Sequence Listing and summarized in Table 14 (inserted prior to claims), and the predicted protein sequences listed as SEQ ID SEQ ID NOS:1619-1675 in the accompanying Sequence Listing and summarized in Table 15 (inserted prior to claims). Specifically, Table 14 provides: 1) the SEQ ID NO (“SEQ ID”) assigned to each cDNA sequence for use in the present specification; 2) the cDNA sequence name (“cDNA SEQ NAME”) used as an internal identifier of the sequence; 3) the chromosome (“CHROM”) containing the gene corresponding to the cDNA sequence; and 4) the exon (“EXON”) of the gene corresponding to the cDNA sequence to which the polynucleotide of SEQ ID NOS: 134-1352 maps. Table 15 provides: 1) the SEQ ID NO (“SEQ ID”) assigned to each protein sequence for use in the present specification; 2) the protein sequence name (“PROTEIN SEQ NAME”) used as an internal identifier of the sequence; 3) the chromosome (“CHROM”) containing the gene corresponding to the cDNA sequence; and 4) the exon (“EXON”) of the gene corresponding to the cDNA and protein sequence to which the polynucleotide of SEQ ID NOS: 134-1352 maps.
TABLE 14
|
|
SEQ IDcDNA SEQ NAMECHROMEXON
|
1562NTN_004511S11.3_4Chr(1)Exons(14)
1563NTN_004754S1.3_4Chr(1)Exons(5)
1564NTN_005530S2.3_3Chr(3)Exons(5)
1565NTN_005530S2.3_4Chr(3)Exons(6)
1566NTN_005564S1.3_3Chr(3)Exons(2)
1567NTN_005635S2.3_1Chr(3)Exons(10)
1568NTN_005962S3.3_4Chr(3)Exons(9)
1569NTN_006051S4.3_2Chr(4)Exons(17)
1570NTN_006051S5.3_5Chr(4)Exons(6)
1571NTN_007011S7.3_9Chr(5)Exons(16)
1572NTN_007122S8.3_8Chr(6)Exons(12)
1573NTN_007592S2.3_10Chr(6)Exons(3)
1574NTN_007592S3.3_5Chr(6)Exons(1)
1575NTN_007844S7.3_3Chr(7)Exons(3)
1576NTN_007867S7.3_3Chr(7)Exons(15)
1577NTN_007867S8.3_1Chr(7)Exons(14)
1578NTN_008059S2.3_3Chr(8)Exons(8)
1579NTN_008338S4.3_6Chr(9)Exons(4)
1580NTN_008470S7.3_1Chr(9)Exons(13)
1581NTN_008627S7.3_5Chr(10)Exons(13)
1582NTN_008769S8.3_1Chr(10)Exons(7)
1583NTN_008858S2.3_2Chr(10)Exons(5)
1584NTN_009296S1.3_1Chr(11)Exons(6)
1585NTN_009296S3.3_2Chr(11)Exons(3)
1586NTN_009526S2.3_3Chr(12)Exons(11)
1587NTN_009526S2.3_5Chr(12)Exons(11)
1588NTN_009848S8.3_5Chr(13)Exons(3)
1589NTN_009866S24.3_5Chr(13)Exons(5)
1590NTN_010018S2.3_5Chr(14)Exons(12)
1591NTN_010164S3.3_8Chr(14)Exons(3)
1592NTN_010289S6.3_5Chr(15)Exons(2)
1593NTN_010663S1.3_1Chr(17)Exons(4)
1594NTN_010757S4.3_2Chr(17)Exons(25)
1595NTN_011059S6.3_4Chr(18)Exons(2)
1596NTN_011130S2.3_3Chr(19)Exons(6)
1597NTN_011266S2.2_3Chr(19)Exons(1)
1598NTN_011361S6.3_7Chr(20)Exons(6)
1599NTN_011430S6.3_6Chr(20)Exons(22)
1600NTN_011512S51.3_3Chr(21)Exons(11)
1601NTN_017582S2.3_6Chr(9)Exons(2)
1602NTN_019508S1.3_6Chr(10)Exons(8)
1603NTN_019721S6.3_1Chr(Y)Exons(5)
1604NTN_022448S1.3_2Chr(3)Exons(11)
1605NTN_022456S1.3_2Chr(3)Exons(6)
1606NTN_022526S1.3_2Chr(3)Exons(10)
1607NTN_022807S2.3_1Chr(4)Exons(2)
1608NTN_022948S1.3_1Chr(4)Exons(4)
1609NTN_022948S1.3_5Chr(4)Exons(4)
1610NTN_023142S2.3_4Chr(5)Exons(2)
1611NTN_023860S1.3_1Chr(8)Exons(3)
1612NTN_024001S1.3_4Chr(9)Exons(6)
1613NTN_024871S1.3_7Chr(17)Exons(4)
1614NTN_024882S1.3_3Chr(17)Exons(8)
1615NTN_025842S13.2_1Chr(11)Exons(10)
1616NTN_025864S1.1_1Chr(12)Exons(3)
1617NTN_025907S4.2_3Chr(17)Exons(5)
1618NTN_026331S1.1_1Chr(7)Exons(14)
|
TABLE 15
|
|
|
SEQ ID
PROTEIN SEQ NAME
CHROM
EXON
|
|
1619
NTP_004511S11.3_4
Chr(1)
Exons(14)
|
1620
NTP_004754S1.3_4
Chr(1)
Exons(5)
|
1621
NTP_005530S2.3_3
Chr(3)
Exons(5)
|
1622
NTP_005530S2.3_4
Chr(3)
Exons(6)
|
1623
NTP_005564S1.3_3
Chr(3)
Exons(2)
|
1624
NTP_005635S2.3_1
Chr(3)
Exons(10)
|
1625
NTP_005962S3.3_4
Chr(3)
Exons(9)
|
1626
NTP_006051S4.3_2
Chr(4)
Exons(17)
|
1627
NTP_006051S5.3_5
Chr(4)
Exons(6)
|
1628
NTP_007011S7.3_9
Chr(5)
Exons(16)
|
1629
NTP_007122S8.3_8
Chr(6)
Exons(12)
|
1630
NTP_007592S2.3_10
Chr(6)
Exons(3)
|
1631
NTP_007592S3.3_5
Chr(6)
Exons(1)
|
1632
NTP_007844S7.3_3
Chr(7)
Exons(3)
|
1633
NTP_007867S7.3_3
Chr(7)
Exons(15)
|
1634
NTP_007867S8.3_1
Chr(7)
Exons(14)
|
1635
NTP_008059S2.3_3
Chr(8)
Exons(8)
|
1636
NTP_008338S4.3_6
Chr(9)
Exons(4)
|
1637
NTP_008470S7.3_1
Chr(9)
Exons(13)
|
1638
NTP_008627S7.3_5
Chr(10)
Exons(13)
|
1639
NTP_008769S8.3_1
Chr(10)
Exons(7)
|
1640
NTP_008858S2.3_2
Chr(10)
Exons(5)
|
1641
NTP_009296S1.3_1
Chr(11)
Exons(6)
|
1642
NTP_009296S3.3_2
Chr(11)
Exons(3)
|
1643
NTP_009526S2.3_3
Chr(12)
Exons(11)
|
1644
NTP_009526S2.3_5
Chr(12)
Exons(11)
|
1645
NTP_009848S8.3_5
Chr(13)
Exons(3)
|
1646
NTP_009866S24.3_5
Chr(13)
Exons(5)
|
1647
NTP_010018S2.3_5
Chr(14)
Exons(12)
|
1648
NTP_010164S3.3_8
Chr(14)
Exons(3)
|
1649
NTP_010289S6.3_5
Chr(15)
Exons(2)
|
1650
NTP_010663S1.3_1
Chr(17)
Exons(4)
|
1651
NTP_010757S4.3_2
Chr(17)
Exons(25)
|
1652
NTP_011059S6.3_4
Chr(18)
Exons(2)
|
1653
NTP_011130S2.3_3
Chr(19)
Exons(6)
|
1654
NTP_011266S2.2_3
Chr(19)
Exons(1)
|
1655
NTP_011361S6.3_7
Chr(20)
Exons(6)
|
1656
NTP_011430S6.3_6
Chr(20)
Exons(22)
|
1657
NTP_011512S51.3_3
Chr(21)
Exons(11)
|
1658
NTP_017582S2.3_6
Chr(9)
Exons(2)
|
1659
NTP_019508S1.3_6
Chr(10)
Exons(8)
|
1660
NTP_019721S6.3_1
Chr(Y)
Exons(5)
|
1661
NTP_022448S1.3_2
Chr(3)
Exons(11)
|
1662
NTP_022456S1.3_2
Chr(3)
Exons(6)
|
1663
NTP_022526S1.3_2
Chr(3)
Exons(10)
|
1664
NTP_022807S2.3_1
Chr(4)
Exons(2)
|
1665
NTP_022948S1.3_1
Chr(4)
Exons(4)
|
1666
NTP_022948S1.3_5
Chr(4)
Exons(4)
|
1667
NTP_023142S2.3_4
Chr(5)
Exons(2)
|
1668
NTP_023860S1.3_1
Chr(8)
Exons(3)
|
1669
NTP_024001S1.3_4
Chr(9)
Exons(6)
|
1670
NTP_024871S1.3_7
Chr(17)
Exons(4)
|
1671
NTP_024882S1.3_3
Chr(17)
Exons(8)
|
1672
NTP_025842S13.2_1
Chr(11)
Exons(10)
|
1673
NTP_025864S1.1_1
Chr(12)
Exons(3)
|
1674
NTP_025907S4.2_3
Chr(17)
Exons(5)
|
1675
NTP_026331S1.1_1
Chr(7)
Exons(14)
|
|
A correlation between the polynucleotide used as a query sequence as described above and the corresponding predicted cDNA and protein sequences is contained in Table 16. Specifically Table 16 provides: 1) the SEQ ID NO of the cDNA (“cDNA SEQ ID”); 2) the cDNA sequence name (“cDNA SEQ NAME”) used as an internal identifier of sequence; 3) the SEQ ID NO of the protein (“PROTEIN SEQ ID”) encoded by the cDNA sequence 4) the sequence name of the protein (“PROTEIN SEQ NAME”) encoded by the cDNA sequence; 5) the SEQ ID NO of the polynucleotide (“POLYNTD SEQ ID”) of SEQ ID NOS: 134-1352 that maps to the cDNA and protein; and 6) the sequence name (“POLYNTD SEQ NAME”) of the polynucleotide of SEQ ID NOS: 134-1352 that maps to the DNA and protein.
TABLE 16
|
|
cDNA
SEQPROTEINPROTEIN SEQPOLYNTDPOLYNTD SEQ
IDcDNA SEQ NAMESEQ IDNAMESEQ IDNAME
|
|
1562NTN_004511S11.3_41619NTP_004511S11.3_41823544.B02.GZ43_505328
1563NTN_004754S1.3_41620NTP_004754S1.3_48963608.E20.gz43_514326
1564NTN_005530S2.3_31621NTP_005530S2.3_36623580.E23.GZ43_510293
1565NTN_005530S2.3_41622NTP_005530S2.3_46623580.E23.GZ43_510293
1566NTN_005564S1.3_31623NTP_005564S1.3_33833556.G15.GZ43_507077
1566NTN_005564S1.3_31623NTP_005564S1.3_32833550.K05.GZ43_506153
1566NTN_005564S1.3_31623NTP_005564S1.3_35193568.C22.GZ43_508739
1567NTN_005635S2.3_11624NTP_005635S2.3_15513571.B22.GZ43_509122
1567NTN_005635S2.3_11624NTP_005635S2.3_15503571.B13.GZ43_508978
1568NTN_005962S3.3_41625NTP_005962S3.3_43863556.H12.GZ43_507030
1569NTN_006051S4.3_21626NTP_006051S4.3_24483559.P10.GZ43_507390
1570NTN_006051S5.3_51627NTP_006051S5.3_54483559.P10.GZ43_507390
1571NTN_007011S7.3_91628NTP_007011S7.3_99833620.K24.gz43_516155
1571NTN_007011S7.3_91628NTP_007011S7.3_96353577.J04.GZ43_509610
1572NTN_007122S8.3_81629NTP_007122S8.3_813373762.E15.gz43_534217
1572NTN_007122S8.3_81629NTP_007122S8.3_813373762.E15.gz43_534217
1573NTN_007592S2.3_101630NTP_007592S2.3_1010053626.G01.gz43_516551
1574NTN_007592S3.3_51631NTP_007592S3.3_510573638.A02.gz43_518097
1575NTN_007844S7.3_31632NTP_007844S7.3_310383632.O06.gz43_517407
1576NTN_007867S7.3_31633NTP_007867S7.3_35663571.H16.GZ43_509032
1577NTN_007867S8.3_11634NTP_007867S8.3_15663571.H16.GZ43_509032
1578NTN_008059S2.3_31635NTP_008059S2.3_37663590.H16.GZ43_512345
1579NTN_008338S4.3_61636NTP_008338S4.3_610143626.P14.gz43_516768
1579NTN_008338S4.3_61636NTP_008338S4.3_63493553.L02.GZ43_506490
1580NTN_008470S7.3_11637NTP_008470S7.3_112333754.F15.gz43_533066
1581NTN_008627S7.3_51638NTP_008627S7.3_59563617.C21.gz43_515492
1582NTN_008769S8.3_11639NTP_008769S8.3_12073544.M10.GZ43_505467
1582NTN_008769S8.3_11639NTP_008769S8.3_12073544.M10.GZ43_505467
1583NTN_008858S2.3_21640NTP_008858S2.3_28443599.M04.GZ43_512926
1584NTN_009296S1.3_11641NTP_009296S1.3_19553617.B16.gz43_515411
1585NTN_009296S3.3_21642NTP_009296S3.3_28833605.I19.gz43_513930
1585NTN_009296S3.3_21642NTP_009296S3.3_211173661.K22.gz43_519717
1585NTN_009296S3.3_21642NTP_009296S3.3_29683617.P11.gz43_515345
1586NTN_009526S2.3_31643NTP_009526S2.3_31663541.I18.GZ43_505207
1586NTN_009526S2.3_31643NTP_009526S2.3_35553571.E02.GZ43_508805
1586NTN_009526S2.3_31643NTP_009526S2.3_38893605.N12.gz43_513823
1587NTN_009526S2.3_51644NTP_009526S2.3_55553571.E02.GZ43_508805
1587NTN_009526S2.3_51644NTP_009526S2.3_51663541.I18.GZ43_505207
1587NTN_009526S2.3_51644NTP_009526S2.3_58893605.N12.gz43_513823
1588NTN_009848S8.3_51645NTP_009848S8.3_57533590.D03.GZ43_512133
1589NTN_009866S24.3_51646NTP_009866S24.3_56263577.E19.GZ43_509845
1590NTN_010018S2.3_51647NTP_010018S2.3_57033580.P04.GZ43_510000
1591NTN_010164S3.3_81648NTP_010164S3.3_87273583.K08.GZ43_510443
1592NTN_010289S6.3_51649NTP_010289S6.3_57423583.O03.GZ43_510367
1593NTN_010663S1.3_11650NTP_010663S1.3_15063565.N13.GZ43_508204
1594NTN_010757S4.3_21651NTP_010757S4.3_27803590.M09.GZ43_512238
1595NTN_011059S6.3_41652NTP_011059S6.3_44253559.E20.GZ43_507539
1596NTN_011130S2.3_31653NTP_011130S2.3_312263754.C22.gz43_533175
1596NTN_011130S2.3_31653NTP_011130S2.3_312263754.C22.gz43_533175
1597NTN_011266S2.2_31654NTP_011266S2.2_312093666.G12.gz43_521473
1598NTN_011361S6.3_71655NTP_011361S6.3_74283559.H09.GZ43_507366
1599NTN_011430S6.3_61656NTP_011430S6.3_611953665.O19.gz43_521209
1600NTN_011512S51.3_31657NTP_011512S51.3_33333553.K07.GZ43_506569
1601NTN_017582S2.3_61658NTP_017582S2.3_69483614.L13.gz43_514989
1602NTN_019508S1.3_61659NTP_019508S1.3_69563617.C21.gz43_515492
1603NTN_019721S6.3_11660NTP_019721S6.3_11393541.A23.GZ43_505279
1604NTN_022448S1.3_21661NTP_022448S1.3_29433614.G20.gz43_515096
1604NTN_022448S1.3_21661NTP_022448S1.3_29153611.B16.gz43_514643
1604NTN_022448S1.3_21661NTP_022448S1.3_210873646.B20.gz43_519154
1605NTN_022456S1.3_21662NTP_022456S1.3_29493614.M08.gz43_514910
1606NTN_022526S1.3_21663NTP_022526S1.3_210183629.E01.gz43_516933
1606NTN_022526S1.3_21663NTP_022526S1.3_211613664.A11.gz43_520683
1606NTN_022526S1.3_21663NTP_022526S1.3_210363632.N13.gz43_517518
1607NTN_022807S2.3_11664NTP_022807S2.3_110803643.O21.gz43_518799
1608NTN_022948S1.3_11665NTP_022948S1.3_110253629.J03.gz43_516970
1608NTN_022948S1.3_11665NTP_022948S1.3_111973665.O23.gz43_521273
1608NTN_022948S1.3_11665NTP_022948S1.3_112053666.D11.gz43_521454
1608NTN_022948S1.3_11665NTP_022948S1.3_19653617.N10.gz43_515327
1608NTN_022948S1.3_11665NTP_022948S1.3_111053661.A08.gz43_519483
1608NTN_022948S1.3_11665NTP_022948S1.3_111773664.L21.gz43_520854
1608NTN_022948S1.3_11665NTP_022948S1.3_19393614.D14.gz43_514997
1608NTN_022948S1.3_11665NTP_022948S1.3_110983646.K14.gz43_519067
1608NTN_022948S1.3_11665NTP_022948S1.3_19383614.C18.gz43_515060
1609NTN_022948S1.3_51666NTP_022948S1.3_59393614.D14.gz43_514997
1609NTN_022948S1.3_51666NTP_022948S1.3_513103759.L10.gz43_533760
1609NTN_022948S1.3_51666NTP_022948S1.3_59653617.N10.gz43_515327
1610NTN_023142S2.3_41667NTP_023142S2.3_44103556.O08.GZ43_506973
1611NTN_023860S1.3_11668NTP_023860S1.3_111803664.P18.gz43_520810
1612NTN_024001S1.3_41669NTP_024001S1.3_43703556.B09.GZ43_506976
1612NTN_024001S1.3_41669NTP_024001S1.3_43943556.K04.GZ43_506905
1613NTN_024871S1.3_71670NTP_024871S1.3_77003580.O06.GZ43_510031
1614NTN_024882S1.3_31671NTP_024882S1.3_36023574.G07.GZ43_509271
1615NTN_025842S13.2_11672NTP_025842S13.2_19993623.N23.gz43_516526
1616NTN_025864S1.1_11673NTP_025864S1.1_110673643.F07.gz43_518566
1616NTN_025864S1.1_11673NTP_025864S1.1_110673643.F07.gz43_518566
1616NTN_025864S1.1_11673NTP_025864S1.1_113343762.D22.gz43_534328
1617NTN_025907S4.2_31674NTP_025907S4.2_311923665.O06.gz43_521001
1618NTN_026331S1.1_11675NTP_026331S1.1_15663571.H16.GZ43_509032
|
Through contig and consensus sequence assembly and the use of homology searching software programs, the sequence information provided herein can be readily extended to confirm, or confirm a predicted, gene having the sequence of the polynucleotides described in the present invention. Further the information obtained can be used to identify the function of the gene product of the gene corresponding to the polynucleotides described herein. While not necessary to the practice of the invention, identification of the function of the corresponding gene, can provide guidance in the design of therapeutics that target the gene to modulate its activity and modulate the cancerous phenotype (e.g., inhibit metastasis, proliferation, and the like).
Example 20
Results of Public Database Search to Identify Function of Gene Products
SEQ ID NOS:134-1618 were translated in all three reading frames, and the nucleotide sequences and translated amino acid sequences used as query sequences to search for homologous sequences in the GenBank (nucleotide sequences) database. Query and individual sequences were aligned using the TeraBLAST program available from TimeLogic, Crystal Bay, Nev. The sequences were masked to various extents to prevent searching of repetitive sequences or poly-A sequences, using the RepeatMasker masking program for masking low complexity as described above.
Table 17 (inserted prior to claims) provides the alignment summaries having a p value of 1×10e-2 or less indicating substantial homology between the sequences of the present invention and those of the indicated public databases. Specifically, Table 17 provides: 1) the SEQ ID NO (“SEQ ID”) of the query sequence; 2) the sequence name (“SEQ NAME”) used as an internal identifier of the query sequence; 3) the accession number (“ACCESSION”) of the GenBank database entry of the homologous sequence; 4) a description of the GenBank sequences (“GENBANK DESCRIPTION”); and 5) the score of the similarity of the polynucleotide sequence and the GenBank sequence (“GENBANK SCORE”). The alignments provided in Table 8 are the best available alignment to a DNA sequence at a time just prior to filing of the present specification. Incorporated by reference is all publicly available information regarding the sequence listed in Table 17 and their related sequences. The search program and database used for the alignment, as well as the calculation of the p value are also indicated. Full length sequences or fragments of the polynucleotide sequences can be used as probes and primers to identify and isolate the full length sequence of the corresponding polynucleotide.
TABLE 17
|
|
SEQGENBANK
IDSEQ NAMEACCESSIONGENBANK DESCRIPTIONSCORE
|
|
1343538.O24.GZ43_504925AF047717Streptomyces chrysomallus actinomycin1.17E−04
synthetase II (acmB) gene, complete cds
1353538.P11.GZ43_504718AF111848Homo sapiens PRO0529 mRNA,2.00E−06
complete cds
1363541.A04.GZ43_504975X58178S. pyogenes for emm41 gene5.00E−06
1373541.A05.GZ43_504991AF190638Mus musculus nephrin NPHS1 (Nphs1)2.00E−06
gene, partial cds
1383541.A16.GZ43_505167AB024689Mus musculus gene, exon 3, partial6.00E−06
sequence
1393541.A23.GZ43_505279M14155Human insulin-like growth factor (IGF-I)3.00E−06
IB gene, exon 4
1403541.B04.GZ43_504976U32801Haemophilus influenzae Rd section 1161.10E−05
of 163 of the complete genome
1413541.B17.GZ43_505184X89398H. sapiens ung gene for uracil DNA-1.21E−04
glycosylase
1423538.G08.GZ43_504661AF270390Staphylococcus epidermidis strain SR13.00E−06
clone step.4045d08 genomic sequence
1433538.G17.GZ43_504805AC006623Caenorhabditis elegans clone C52E2,4.00E−06
complete sequence
1443538.G19.GZ43_504837AB042425Homo sapiens Pim-2h, hUGT2, hUGT1,6.60E−11
genes for pim-2 protooncogene homolog,
UDP-galactose transporter 1, UDP-
galactose transporter 2, complete cds
1453538.G22.GZ43_504885L08338Human immunodeficiency virus type 13.10E−07
proviral envelope glycoprotein gene V3
region from A196/4537, clone 6 (from
adult)
1463538.H05.GZ43_504614AE006731Sulfolobus solfataricus section 90 of 2722.00E−06
of the complete genome
1473538.H21.GZ43_504870AL121807S. pombe chromosome III cosmid c1321.30E−05
1483538.I08.GZ43_504663AF186379Homo sapiens ligand effect modulator-68.00E−10
(LEM6) mRNA, complete cds
1493538.I13.GZ43_504743AC007658Arabidopsis thaliana chromosome II3.30E−08
section 216 of 255 of the complete
sequence. Sequence from clones F27I1
1503538.J22.GZ43_504888X04616Anacystis nidulans R2 psbAI gene for8.90E−07
photosystem II Q(B) protein
1513538.K12.GZ43_504729X91656M. musculus Srp20 gene4.40E−05
1523538.K23.GZ43_504905M62849Human papillomavirus ORFs4.40E−07
1533538.L16.GZ43_504794AE001382Plasmodium falciparum chromosome 2,7.00E−06
section 19 of 73 of the complete
sequence
1543538.M02.GZ43_504571U07976Human T cell receptor beta7.00E−06
(TCRBV7S2, TCRBV13S2-1,
TCRBV6S7-1) genes, TCRBV deleted 2
haplotype, partial cds
1553538.M05.GZ43_504619AC079878Homo sapiens BAC clone RP11-343P211.40E−07
from 7, complete sequence
1563538.M08.GZ43_504667AF182668Zenaida galapagoensis beta-fibrinogen4.70E−08
gene, partial sequence
1573538.N20.GZ43_504860AB033411Taenia crassiceps mitochondrial gene for6.80E−07
cytochrome c oxidase subunit 1, partial
cds
1583538.O07.GZ43_504653X68019Feline Immunodeficiency Virus GAG4.00E−06
gene
1593541.E11.GZ43_505091M73447Human repeat polymorphism at locus3.00E−08
D9S59
1603541.E14.GZ43_505139AJ243419Acaulospora trappei partial 18S rRNA,1.10E−07
5.8S rRNA and partial 28S rRNA genes
and internal transcribed spacers 1 and 2
(ITS1, ITS2), isolate AU 219
1613541.E15.GZ43_505155U13679Human lactate dehydrogenase-A (LDH-3.50E−10
A) gene, promoter region
1623541.G17.GZ43_505189AE004851Pseudomonas aeruginosa PA01, section1.30E−05
412 of 529 of the complete genome
1633541.H14.GZ43_505142AJ252202Drosophila melanogaster D-COQ7 gene9.00E−06
for putative COQ7 isologue, exons 1-3
1643541.I15.GZ43_505159X98371D. subobscura sex-lethal gene6.00E−06
1653541.I17.GZ43_505191AK023918Homo sapiens cDNA FLJ13856 fis,1.70E−22
clone THYRO1000988
1663541.I18.GZ43_505207AF329081Bos taurus AMP-activated protein kinase5.30E−33
gamma-1 (PRKAG1) gene, partial cds
1673541.J19.GZ43_505224AF002749Psychotria urceolata ribosomal protein3.01E−03
S16 (rps16) gene, chloroplast gene
encoding chloroplast protein, partial
intron
1683541.K09.GZ43_505065AF027607Gallus gallus L-type voltage-gated9.00E−06
calcium channel alpha1D subunit
ChCaChA1D precursor mRNA,
complete intron sequence
1693541.L19.GZ43_505226AE003949Xylella fastidiosa 9a5c, section 95 of 2292.00E−06
of the complete genome
1703541.M02.GZ43_504955BC004556Homo sapiens, Similar to CG7083 gene6.20E−07
product, clone MGC: 10534
IMAGE: 3957147, mRNA, complete cds
1713541.M07.GZ43_505035X05616Kangaroo rat repetitive DNA with4.80E−08
insertion sequence
1723541.M18.GZ43_505211M81888Parvovirus LuII DNA sequence6.60E−05
1733541.O04.GZ43_504989AF081828Ixodes hexagonus mitochondrial DNA,3.00E−06
complete genome
1743541.O13.GZ43_505133AK026465Homo sapiens cDNA: FLJ22812 fis,8.00E−06
clone KAIA2955
1753541.O23.GZ43_505293X54859Porcine TNF-alpha and TNF-beta genes2.90E−05
for tumour necrosis factors alpha and
beta, respectively
1763541.P05.GZ43_505006AE006642Sulfolobus solfataricus section 1 of 2723.50E−05
of the complete genome
1773541.P22.GZ43_505278U10400Saccharomyces cerevisiae chromosome1.80E−05
VIII cosmid L2825
1783544.A09.GZ43_505439X75677C. parapsilosis mt tRNA genes (591 bps)3.70E−08
1793544.A13.GZ43_505503D28811Schistosoma japonicum mRNA for5.40E−05
paramyosin, complete cds
1803544.A14.GZ43_505519M87111Human immunodeficiency virus type 22.90E−05
(FORTC2) reverse transcriptase
fragment
1813544.A17.GZ43_505567L23650Caenorhabditis elegans cosmid C27D11,5.60E−07
complete sequence
1823544.B02.GZ43_505328AF060543Homo sapiens importin alpha 7 subunit1.50E−49
mRNA, complete cds
1833544.B09.GZ43_505440AB051473Homo sapiens mRNA for KIAA16861.80E−05
protein, partial cds
1843544.B18.GZ43_505584AJ224821Loxodonta africana complete4.00E−06
mitochondrial genomic sequence
1853544.E05.GZ43_505379AL451187Human DNA sequence from clone1.30E−07
RP11-49J23 on chromosome 6, complete
sequence [Homo sapiens]
1863544.E18.GZ43_505587L08338Human immunodeficiency virus type 13.30E−07
proviral envelope glycoprotein gene V3
region from A196/4537, clone 6 (from
adult)
1873544.F06.GZ43_505396X60833R. norvegicus TDO2 gene for tryptophan7.80E−07
2,3-dioxygenase, exon 6
1883544.F16.GZ43_505556U72716Drosophila melanogaster D3-100EF2.00E−06
mRNA, complete cds
1893544.G06.GZ43_505397AC002359Homo sapiens Xp22 Cosmid U239B31.60E−05
(from Lawrence Livermore X library)
complete sequence
1903544.G10.GZ43_505461X56015Crithidia oncopelti mitochondrial ND4,4.80E−05
ND5, COI, 12S ribosomal RNA genes
for NADH dehydrogenase subunit 4/5,
cytochrome oxidase subunit I and 12S
ribosomal RNA
1913544.G11.GZ43_505477U80927Dictyostelium discoideum unknown9.00E−08
protein gene, complete cds
1923544.G12.GZ43_505493AF245483Oryza sativa OSE4 (OSE4) gene,1.70E−07
complete cds
1933544.H03.GZ43_505350Y12855Homo sapiens P2X7 gene, exon 12 and2.30E−05
13
1943544.H15.GZ43_505542AF194829Tetragonia tetragonioides NADH2.00E−06
dehydrogenase (ndhF) gene, partial cds;
chloroplast gene for chloroplast product
1953544.H24.GZ43_505686BC008353Homo sapiens, Similar to RIKEN cDNA2.50E−18
0610008P16 gene, clone MGC: 15937
IMAGE: 3537224, mRNA, complete cds
1963544.I07.GZ43_505415AF010533Plasmodium falciparum microsatellite1.80E−08
TA21 sequence
1973544.I15.GZ43_505543D29794Mouse gene for T cell receptor gamma3.00E−06
chain
1983544.I20.GZ43_505623AE000677Aquifex aeolicus section 9 of 109 of the4.00E−06
complete genome
1993544.J04.GZ43_505368Z97015Lactococcus lactis cremoris sucrose gene1.00E−06
cluster
2003544.J11.GZ43_505480M67480Human prothymosin-alpha gene,5.10E−10
complete cds
2013544.J13.GZ43_505512AJ249884Lepeophtheirus salmonis microsatellite5.70E−08
DNA, locus Ls.NUIG.09
2023544.J23.GZ43_505672AJ245823Trypanosoma brucei PK4 gene for6.00E−06
protein kinase
2033544.K16.GZ43_505561U18191Human HLA class I genomic survey2.50E−07
sequence
2043544.L11.GZ43_505482X07127Kluyveromyces lactis killer plasmid k12.90E−05
DNA
2053544.L13.GZ43_505514BC005028Homo sapiens, hypothetical protein1.80E−31
FLJ11323, clone MGC: 12582
IMAGE: 3953383, mRNA, complete cds
2063544.M06.GZ43_505403AC006687Caenorhabditis elegans cosmid T20C7,2.30E−05
complete sequence
2073544.M10.GZ43_505467M92378Mus musculus GABA transporter1.30E−05
mRNA sequence
2083544.N07.GZ43_505420U48705Human receptor tyrosine kinase DDR7.40E−07
gene, complete cds
2093544.N12.GZ43_505500BC007621Homo sapiens, Similar to Orthodenticle5.70E−07
(Drosophila) homolog 1, clone
MGC: 15736 IMAGE: 3355563, mRNA,
complete cds
2103544.N19.GZ43_505612AF270077Staphylococcus epidermidis strain SR12.00E−07
clone step.1047c06 genomic sequence
2113544.O03.GZ43_505357U15681Myrmecia pilosula HI87-1561.00E−06
mitochondrion cytochrome b gene,
partial cds
2123544.O10.GZ43_505469AF056032Homo sapiens kynurenine 3-hydroxylase5.00E−06
mRNA, complete cds
2133544.O15.GZ43_505549U37373Xenopus laevis tail-specific thyroid3.00E−06
hormone up-regulated (gene 5) mRNA,
complete cds
2143544.O20.GZ43_505629D66906Bombyx mori DNA for sorbitol2.00E−06
dehydrogenase, complete cds
2153544.P18.GZ43_505598J04357Red clover necrotic mosaic virus RNA-1,4.00E−06
complete sequence
2163547.A04.GZ43_505743AF118558Mus musculus hitchhiker-3, hitchhiker-4,5.40E−07
and hitchhiker-5 mRNA sequences
2173547.A11.GZ43_505855U93874Bacillus subtilis cysteine synthase4.00E−06
(yrhA), cystathionine gamma-lyase
(yrhB), YrhC (yrhC), YrhD (yrhD),
formate dehydrogenase chain A (yrhE),
YrhF (yrhF), formate dehydrogenase
(yrhG), YrhH (yrhH), regulatory protein
(yrhI), cytochrome P450 102 (yrhJ),>
2183547.A24.GZ43_506063AL157466Homo sapiens mRNA; cDNA8.80E−07
DKFZp761E2423 (from clone
DKFZp761E2423)
2193547.C05.GZ43_505761X52589Bovine rotavirus RNA for virus protein 21.00E−05
(VP2)
2203547.C17.GZ43_505953U67594Methanococcus jannaschii section 136 of3.80E−05
150 of the complete genome
2213547.C23.GZ43_506049AJ250862Bacillus sp. HIL-Y85/54728 mersacidin1.20E−05
biosynthesis gene cluster (mrsK2,
mrsR2, mrsF, mrsG, mrsE, mrsA,
mrsR1, mrsD, mrsM and mrsT genes)
2223547.D19.GZ43_505986AF050491Microgadus tomcod aromatic4.00E−06
hydrocarbon receptor (ahr) gene, exons
8-11, partial cds
2233547.D23.GZ43_506050M33190Rat cytochrome P450 II A3 (CYP2A3)5.80E−05
gene, complete cds
2243547.E04.GZ43_505747L35658Homo sapiens (subclone H8 9_d12 from7.70E−07
P1 35 H5 C8) DNA sequence
2253547.F02.GZ43_505716AF038190Homo sapiens clone 23582 mRNA1.10E−07
sequence
2263547.F10.GZ43_505844AY008833Staphylococcus aureus tcaR-tcaA-tcaB5.00E−06
operon, complete sequences
2273547.F20.GZ43_506004AB037821Homo sapiens mRNA for KIAA14001.00E−06
protein, partial cds
2283547.G02.GZ43_505717M88397Naegleria fowleri virulence-related3.70E−07
protein (NF314) mRNA, complete cds
2293547.G09.GZ43_505829AJ315644Homo sapiens mRNA for proton myoinositol7.90E−07
symporter (Hmit gene)
2303547.G22.GZ43_506037Z33603P. radiata (Pr1.6) microsatellite DNA,1.70E−07
703 bp
2313547.H12.GZ43_505878L04309Shigella flexneri ipgD, ipgE, ipgF genes,3.00E−06
complete cds
2323547.H14.GZ43_505910AL137502Homo sapiens mRNA; cDNA2.90E−07
DKFZp761H171 (from clone
DKFZp761H171); partial cds
2333547.I07.GZ43_505799M15332B. sphaericus ermG gene encoding rRNA7.00E−06
methyltransferase (macrolide-
lincosamide-streptogramin B resistance
element)
2343547.I16.GZ43_505943AF015157Homo sapiens clone HS19.12 Alu-Ya54.70E−10
sequence
2353547.I17.GZ43_505959AE007758Clostridium acetobutylicum ATCC8243.00E−06
section 246 of 356 of the complete
genome
2363547.I20.GZ43_506007L37606Medicago sativa (clone GG16-1)1.50E−05
NADH-dependent glutamate synthase
gene, complete cds
2373547.J05.GZ43_505768Z16911H. sapiens (D20S113) DNA segment2.80E−07
containing (CA) repeat; clone
AFM205th8; single read
2383547.J10.GZ43_505848Z37803HIV-1 DNA V3 region (patient 15,8.80E−07
sample CSF, clone 9)
2393547.J20.GZ43_506008AF013273Candida albicans histidine kinase 1 gene,3.30E−05
complete cds
2403547.J22.GZ43_506040AF289080Lycopersicon esculentum alpha-4.00E−06
galactosidase gene, partial cds
2413547.K01.GZ43_505705AF267863Homo sapiens DC43 mRNA, complete7.30E−22
cds
2423547.L09.GZ43_505834Z22175Caenorhabditis elegans cosmid K01F9,1.40E−05
complete sequence
2433547.L11.GZ43_505866AJ288648Limnodynastes tasmaniensis5.90E−07
mitochondrial partial nadh4 gene for
NADH dehydrogenase subunit 4 and
partial tRNA-His gene, sample 26 from
Australia: Boolara
2443547.L16.GZ43_505946AE001293Chlamydia trachomatis section 20 of 877.10E−07
of the complete genome
2453547.L22.GZ43_506042AF287006Danio rerio T-box brain 1 mRNA, partial7.00E−06
cds
2463547.M02.GZ43_505723AE007788Clostridium acetobutylicum ATCC8241.00E−05
section 276 of 356 of the complete
genome
2473547.M07.GZ43_505803Z46252M. musculus DNA for region6.00E−06
surrounding retrovirus restriction locus
Fv1
2483547.M08.GZ43_505819AB020684Homo sapiens mRNA for KIAA08771.50E−05
protein, partial cds
2493547.M16.GZ43_505947AF335240Petunia x hybrida MADS-box3.00E−06
transcription factor FBP22 (FBP22)
mRNA, complete cds
2503547.N06.GZ43_505788AF299346Renispora flavissima isolate CEH3131.70E−08
18S ribosomal RNA gene, partial
sequence; internal transcribed spacer 1,
5.8S ribosomal RNA gene and internal
transcribed spacer 2, complete sequence;
and 28S ribosomal RNA gene, partial
sequence
2513547.O03.GZ43_505741AE002344Chlamydia muridarum, section 72 of 856.60E−07
of the complete genome
2523547.O07.GZ43_505805D50608Rat gene for cholecystokinin type-A1.60E−05
receptor (CCKAR), complete cds
2533547.O14.GZ43_505917AL137502Homo sapiens mRNA; cDNA2.90E−07
DKFZp761H171 (from clone
DKFZp761H171); partial cds
2543547.P18.GZ43_505982AJ131734Plasmodium berghei DNA including6.10E−07
upstream sequence NTS and 5′ETS of
the 18S rRNA gene (A rRNA gene unit)
2553547.P21.GZ43_506030AC006619Caenorhabditis elegans cosmid C46C11,1.70E−05
complete sequence
2563547.P22.GZ43_506046AJ000871Streptococcus mitis comC, comD, comE2.00E−06
genes, isolate B5
2573550.A12.GZ43_506255M22310Human epidermal growth factor receptor4.80E−07
proto-oncogene downstream enhancer
2583550.A16.GZ43_506319L39435Senecio mikanioides chloroplast NADH2.00E−06
dehydrogenase (ndhF) gene, complete
cds
2593550.B06.GZ43_506160D14161Hordeum vulgare ids-4 mRNA, complete1.10E−08
cds
2603550.C01.GZ43_506081AK025182Homo sapiens cDNA: FLJ21529 fis,4.20E−09
clone COL05981
2613550.C22.GZ43_506417X52028Rattus norvegicus P450 IID3 gene1.41E−04
2623550.D16.GZ43_506322Y10345H. sapiens GalNAc-T3 gene, 3′UTR5.00E−07
2633550.D23.GZ43_506434AF134403Escherichia coli plasmid pAA2 Shf (shf),6.90E−07
hexosyltransferase homolog (capU), and
VirK (virK) genes, complete cds
2643550.E02.GZ43_506099U66074Tritrichomonas foetus putative8.90E−07
superoxide dismutase 2 (SOD2) gene,
complete cds
2653550.E06.GZ43_506163Z23341H. sapiens (D8S528) DNA segment2.30E−08
containing (CA) repeat; clone
AFM080xh7; single read
2663550.F06.GZ43_506164M59447Drosophila melanogaster Sex-lethal3.00E−06
(Sx1) mRNA, complete cds
2673550.F08.GZ43_506196M24901Rabbit pulmonary surfactant-associated3.40E−07
protein (SP-B) mRNA, complete cds
2683550.F20.GZ43_506388AF216169Simicratea welwitschii clone 25.40E−08
phytochrome B (PHYB) gene, exon 1
and partial cds
2693550.F22.GZ43_506420AP000739Arabidopsis thaliana genomic DNA,2.20E−05
chromosome 3, P1 clone: MEK6
2703550.G02.GZ43_506101AL022342Human DNA sequence from clone RP1-7.40E−05
29M10 on chromosome 20, complete
sequence [Homo sapiens]
2713550.G08.GZ43_506197AK021312Mus musculus 13 days embryo stomach3.60E−08
cDNA, RIKEN full-length enriched
library, clone: D530039A21, full insert
sequence
2723550.G10.GZ43_506229M31684D. melanogaster cytoskeleton-like3.00E−06
bicaudalD protein (BicD) mRNA,
complete cds
2733550.G15.GZ43_506309AF087141Mus musculus uncharacterized long4.00E−06
terminal repeat, complete sequence; and
valyl-tRNA synthetase (G7a) gene,
complete cds
2743550.G23.GZ43_506437X02547Trypanosoma brucei mitochondrial2.00E−06
genes for 12S and 9S ribosomal RNA
2753550.H10.GZ43_506230U55711Human ataxia-telangiectasia (ATM)6.10E−08
gene, exon 11
2763550.H21.GZ43_506406Z68755Human DNA sequence from cosmid2.00E−06
L118D5, Huntington's Disease Region,
chromosome 4p16.3
2773550.H23.GZ43_506438AF151388Dermatobia hominis strain Alfenas1.20E−07
tRNA-Ile gene, partial sequence; D-loop,
complete sequence; and 12S ribosomal
RNA, partial sequence; mitochondrial
genes for mitochondrial products
2783550.I03.GZ43_506119AF117258Staphylococcus aureus plasmid pIP6806.50E−08
replication protein RepE (repE) gene,
partial cds; resolvase (res),
acetyltransferase Vat (vat), and
hydrolase VgB (vgb) genes, complete
cds; and unknown gene
2793550.I19.GZ43_506375AE002781Drosophila melanogaster genomic3.90E−05
scaffold 142000013385442, complete
sequence
2803550.I21.GZ43_506407AE001002Archaeoglobus fulgidus section 105 of4.20E−05
172 of the complete genome
2813550.J05.GZ43_506152AF080689Homo sapiens protein kinase PITSLRE5.50E−10
(CDC2L2) gene, exons 8 and 9
2823550.J11.GZ43_506248Z82761R. prowazekii genomic DNA fragment1.00E−06
(clone A793R)
2833550.K05.GZ43_506153X15407Maize pseudo-Gpa2 pseudogene for3.20E−05
glyceraldehyde-3-phosphate
dehydrogenase subunit A
2843550.K09.GZ43_506217X62631S. pombe wis1 gene for protein kinase1.50E−07
2853550.K14.GZ43_506297M59743Rabbit cardiac muscle Ca-2+ release1.00E−06
channel (ryanodine receptor) mRNA,
complete cds
2863550.L16.GZ43_506330AF201383Buchnera aphidicola isopropylmalate1.00E−06
dehydratase subunit (leuC) gene, partial
cds
2873550.L19.GZ43_506378M77244H. sapiens erythropoietin receptor4.00E−09
(EPOR) gene, 5′ end
2883550.L23.GZ43_506442L76259Homo sapiens PTS gene, complete cds8.00E−06
2893550.M21.GZ43_506411M87339Human replication factor C, 37-kDa5.00E−06
subunit mRNA, complete cds
2903550.N01.GZ43_506092AF191009Helicobacter pylori strain ChinaF30A1.10E−07
cag pathogenicity island polymorphic
right end, type IIIa motif
2913550.N07.GZ43_506188AF235499Mus musculus SH2-containing inositol1.55E−04
5-phosphatase (Ship) gene, exons 3
through 6
2923550.O03.GZ43_506125D14813Human DNA for osteopontin, complete4.50E−05
cds
2933550.O04.GZ43_506141U08596Canis familiaris delayed rectifier K+6.00E−06
channel mRNA, partial cds
2943550.O08.GZ43_506205XM_017044Homo sapiens similar to diaphanous6.40E−09
(Drosophila, homolog) 2 (H. sapiens)
(LOC91459), mRNA
2953550.O15.GZ43_506317U15977Mus musculus long chain fatty acyl CoA2.80E−05
synthetase mRNA, complete cds
2963550.O17.GZ43_506349X62578C. caldarium plastid genes ompR′, psbD,2.80E−05
psbC, rps16 and groEL
2973550.O18.GZ43_506365L34363Human X-linked nuclear protein (XNP)4.00E−06
gene, complete cds
2983550.O21.GZ43_506413AB056784Macaca fascicularis brain cDNA5.20E−07
clone: QnpA-11501, full insert sequence
2993550.P18.GZ43_506366AK002041Homo sapiens cDNA FLJ11179 fis,5.30E−07
clone PLACE1007450
3003550.P23.GZ43_506446AF200361Rattus norvegicus cytochrome P450 4F11.40E−05
(Cyp4F1) gene, complete cds
3013553.A09.GZ43_506591AL109980Human DNA sequence from clone RP4-3.50E−12
697G8 on chromosome 22, complete
sequence [Homo sapiens]
3023553.B07.GZ43_506560L37056Strongylocentrotus purpuratus myc4.60E−07
protein mRNA, complete cds
3033553.B16.GZ43_506704U43542Nicotiana tabacum diphenol oxidase2.00E−06
mRNA, complete cds
3043553.B22.GZ43_506800L34040Homo sapiens stromelysin gene,6.00E−06
promoter region
3053553.D04.GZ43_506514Y07599S. pombe mRNA for dmf1 gene9.40E−07
3063553.D07.GZ43_506562X13835R. norvegicus CaMII gene, exons 3, 4 & 52.00E−06
3073553.D14.GZ43_506674L38424Bacillus subtilis dihydropicolinate1.80E−05
reductase (jojE) gene, complete cds;
poly(A) polymerase (jojI) gene,
complete cds; biotin acetyl-CoA-
carboxylase ligase (birA) gene, complete
cds; jojC, jojD, jojF, jojG, jojH genes,
complete cds's
3083553.D19.GZ43_506754X53431Yeast gene for STE119.00E−06
3093553.E08.GZ43_506579AF062863Arabidopsis thaliana putative1.80E−07
transcription factor (MYB11) mRNA,
partial cds
3103553.E09.GZ43_506595X71067X. laevis XFG 5-1 and XFG 5-2 genes for6.60E−05
zinc finger proteins
3113553.F12.GZ43_506644X63223B. taurus CI-MNLL mRNA for6.90E−08
ubiquinone oxidoreductase complex
3123553.F13.GZ43_506660L81869Homo sapiens (subclone 1_c4 from P13.00E−08
H55) DNA sequence, complete sequence
3133553.F19.GZ43_506756U97190Caenorhabditis elegans cosmid B0025,3.00E−06
complete sequence
3143553.G05.GZ43_506533S76404beta-HKA = H, K-ATPase beta-subunit8.00E−06
[rats, Genomic, 8983 nt, segment 2 of 2]
3153553.G06.GZ43_506549X68048Phaseolus vulgaris chloroplast DNA for5.00E−06
tRNA-His gene region
3163553.G07.GZ43_506565AF068289Homo sapiens HDCMD34P mRNA,4.40E−12
complete cds
3173553.G21.GZ43_506789Z33603P. radiata (Pr1.6) microsatellite DNA,1.70E−07
703 bp
3183553.H06.GZ43_506550AF090901Homo sapiens clone HQ0195$ PRO01958.00E−07
mRNA, complete cds
3193553.H09.GZ43_506598AF270105Staphylococcus epidermidis strain SR19.80E−07
clone step.1049c09 genomic sequence
3203553.H21.GZ43_506790Z18359Glycine max seed-specific low molecular2.00E−06
weight sulfur-rich protein
3213553.I13.GZ43_506663AF155115Homo sapiens NY-REN-58 antigen1.70E−07
mRNA, complete cds
3223553.I16.GZ43_506711AF270229Staphylococcus epidermidis strain SR11.20E−05
clone step.1055d10 genomic sequence
3233553.J12.GZ43_506648U53400Rattus norvegicus chromosome 101.55E−01
microsatellite sequence D10Mco21
3243553.J14.GZ43_506680M10014Homo sapiens map 4q28 fibrinogen9.00E−06
(FGG) gene, alternative splice products,
complete cds
3253553.J16.GZ43_506712Z23341H. sapiens (D8S528) DNA segment2.30E−08
containing (CA) repeat; clone
AFM080xh7; single read
3263553.J17.GZ43_506728AK021312Mus musculus 13 days embryo stomach3.60E−08
cDNA, RIKEN full-length enriched
library, clone: D530039A21, full insert
sequence
3273553.J22.GZ43_506808AF120279Mus musculus proline dehydrogenase5.00E−06
mRNA, complete cds
3283553.J24.GZ43_506840Z18359Glycine max seed-specific low molecular2.00E−06
weight sulfur-rich protein
3293553.K01.GZ43_506473U31465Kluyveromyces lactis telomerase RNA2.00E−06
component (TER1) gene, complete
sequence
3303553.K02.GZ43_506489X60672M. musculus mRNA for radixin1.00E−06
3313553.K03.GZ43_506505Z71943G. hyalina (92-89) DNA for internal1.06E−02
transcribed spacer 1
3323553.K05.GZ43_506537M80596Saccharomyces cerevisiae VAC1 gene6.00E−06
(required for vacuole inheritance and
vacuole protein sorting), complete cds
3333553.K07.GZ43_506569AJ275317Cicer arietinum partial mRNA for malate7.60E−07
dehydrogenase
3343553.K15.GZ43_506697X57377Mouse dilute myosin heavy chain gene2.40E−05
for novel heavy chain with unique C-
terminal region
3353538.A11.GZ43_504703Z75199S. cerevisiae chromosome XV reading6.00E−06
frame ORF YOR291w
3363538.A24.GZ43_504911AF270077Staphylococcus epidermidis strain SR12.00E−07
clone step.1047c06 genomic sequence
3373538.B01.GZ43_504544AF368255Arabidopsis thaliana small zinc finger-4.10E−07
like protein TIM13 mRNA, complete
cds; nuclear gene for mitochondrial
product
3383538.B20.GZ43_504848AB069994Macaca fascicularis testis cDNA1.40E−07
clone: QtsA-10636, full insert sequence
3393538.C01.GZ43_504545AF072375Pseudoalteromonas sp. S9 beta-1.50E−04
hexosaminidase (chiP) gene, complete
cds
3403538.C02.GZ43_504561AJ011271Human immunodeficiency virus type 27.10E−08
partial env gene, isolate b1286
3413538.D06.GZ43_504626AF100765Oryza sativa receptor-like kinase3.00E−06
(8ARK1) gene, complete cds
3423538.D09.GZ43_504674Z47784M. musculus mRNA expressed in islet3.40E−08
cells (clone 58)
3433538.D21.GZ43_504866AE006568Streptococcus pyogenes M1 GAS strain6.00E−07
SF370, section 97 of 167 of the complete
genome
3443538.E15.GZ43_504771AB027966Schizosaccharomyces pombe gene for2.30E−08
Hypothetical protein, partial cds,
clone: TB89
3453538.F02.GZ43_504564AK001871Homo sapiens cDNA FLJ11009 fis,5.90E−09
clone PLACE1003108
3463538.F08.GZ43_504660U39161Human phosphodiesterase (PDEA) gene,7.40E−07
intron 8, 5′ end
3473553.K23.GZ43_506825Y12052Homo sapiens gene encoding guanine3.00E−06
nucleotide-binding protein beta3 subunit,
exon 5
3483553.K24.GZ43_506841AP001419Homo sapiens genomic DNA,1.00E−06
chromosome 21q22.2, clone: PAC24K9,
LB7T-ERG region, complete sequence
3493553.L02.GZ43_506490X15028Chicken hsp90 gene for 90 kDa-heat3.60E−05
shock protein 5′-end
3503553.L04.GZ43_506522L34665Rattus norvegicus H+/K+-ATPase beta1.20E−09
subunit (HKB) gene, exon 6
3513553.L21.GZ43_506794M87843Human transforming growth factor beta-2.30E−05
2 gene, 5′ end
3523553.M12.GZ43_506651AB026592Limnoporus esakii mitochondrial gene1.10E−07
for 16S ribosomal RNA, partial sequence
3533553.M23.GZ43_506827AE006349Lactococcus lactis subsp. lactis IL14038.00E−07
section 111 of 218 of the complete
genome
3543553.N01.GZ43_506476U80457Human transcription factor SIM2 short2.00E−06
form mRNA, complete cds
3553553.N02.GZ43_506492U81144Caenorhabditis elegans non-alpha3.20E−07
nicotinic acetylcholine receptor subunit
precursor (unc-29) gene, complete cds
3563553.N04.GZ43_506524AE006296Lactococcus lactis subsp. lactis IL14032.00E−06
section 58 of 218 of the complete
genome
3573553.N07.GZ43_506572AK026258Homo sapiens cDNA: FLJ22605 fis,1.00E−06
clone HSI04743
3583553.N08.GZ43_506588U05822Human proto-oncogene BCL3 gene,1.90E−14
exon 2
3593553.O07.GZ43_506573X97196D. melanogaster X gene4.00E−06
3603553.O18.GZ43_506749AE001146Borrelia burgdorferi (section 32 of 70) of1.60E−05
the complete genome
3613553.O23.GZ43_506829X63509Mus musculus partial L1 gene, exons 2-46.00E−06
3623553.P03.GZ43_506510M24901Rabbit pulmonary surfactant-associated4.20E−07
protein (SP-B) mRNA, complete cds
3633553.P05.GZ43_506542AF239156Homo sapiens peptide deformylase-like1.00E−06
protein mRNA, complete cds
3643553.P12.GZ43_506654AF283753Acipenser persicus isolate cw2033.90E−07
cytochrome b gene, partial cds;
mitochondrial gene for mitochondrial
product
3653553.P18.GZ43_506750AK021312Mus musculus 13 days embryo stomach3.50E−08
cDNA, RIKEN full-length enriched
library, clone: D530039A21, full insert
sequence
3663553.P21.GZ43_506798AB044136Homo sapiens genomic DNA, clone: #74.00E−06
3673556.A03.GZ43_506879X61084C. griseus rhodopsin gene for opsin4.30E−05
protein
3683556.A06.GZ43_506927L46904Homo sapiens (subclone 4_c6 from P11.20E−08
H22) DNA sequence
3693556.B06.GZ43_506928AK002041Homo sapiens cDNA FLJ11179 fis,1.40E−07
clone PLACE1007450
3703556.B09.GZ43_506976U88832Human groucho protein homolog (AES)7.00E−07
gene, exons 2-7 and complete cds
3713556.B10.GZ43_506992M11925Influenza5.00E−06
A/chicken/Pennsylvania/8125/83
(H5N2) neuraminidase (NA) gene,
complete cds
3723556.B14.GZ43_507056Z80218Caenorhabditis elegans cosmid F52D4,2.20E−05
complete sequence
3733556.C13.GZ43_507041AF348512Mus musculus polyamine-modulated8.00E−06
factor-1 gene, exons 2 through 5 and
complete cds
3743556.C15.GZ43_507073X82013S. cerevisiae mRNA for SUL13.00E−06
3753556.C18.GZ43_507121Z23973H. sapiens (D7S660) DNA segment5.00E−06
containing (CA) repeat; clone
AFM277vd5; single read
3763556.C24.GZ43_507217AE001381Plasmodium falciparum chromosome 2,6.90E−07
section 18 of 73 of the complete
sequence
3773556.D15.GZ43_507074U48288Rattus norvegicus A-kinase anchoring5.50E−07
protein AKAP 220 mRNA, complete cds
3783556.D20.GZ43_507154AF092684Neochlamisus scabripennis haplotype4.00E−07
113 cytochrome oxidase I (COI) gene,
mitochondrial gene encoding
mitochondrial protein, partial cds
3793556.D23.GZ43_507202X16416Human c-abl mRNA encoding p1502.25E−04
protein
3803556.E13.GZ43_507043AL049948Homo sapiens mRNA; cDNA6.60E−08
DKFZp564K0222 (from clone
DKFZp564K0222)
3813556.E24.GZ43_507219Z57634H. sapiens CpG island DNA genomic8.70E−07
Msel fragment, clone 187e9, forward
read cpg187e9.ft1a
3823556.F10.GZ43_506996AF025409Homo sapiens zinc transporter 4 (ZNT4)3.90E−34
mRNA, complete cds
3833556.G15.GZ43_507077X15407Maize pseudo-Gpa2 pseudogene for3.40E−05
glyceraldehyde-3-phosphate
dehydrogenase subunit A
3843556.H01.GZ43_506854AF269443Staphylococcus epidermidis strain SR13.00E−06
clone step.1003h04 genomic sequence
3853556.H02.GZ43_506870U31465Kluyveromyces lactis telomerase RNA3.00E−06
component (TER1) gene, complete
sequence
3863556.H12.GZ43_507030Z68886Human DNA sequence from cosmid1.70E−07
L21F12, Huntington's Disease Region,
chromosome 4p16.3
3873556.H20.GZ43_507158AB034628Equus caballus microsatellite TKY319,1.70E−07
TKY320 DNA
3883556.I02.GZ43_506871AL390767Human DNA sequence from clone RP1-2.00E−06
68P15 on chromosome 11p13-14.2
Contains GSSs and ESTs. Contains part
of a novel gene, complete sequence
[Homo sapiens]
3893556.I14.GZ43_507063U34042Mus musculus mammalian tolloid-like1.50E−05
protein mRNA, complete cds
3903556.J05.GZ43_506920U31465Kluyveromyces lactis telomerase RNA2.00E−06
component (TER1) gene, complete
sequence
3913556.J07.GZ43_506952AL359621Homo sapiens mRNA; cDNA2.00E−06
DKFZp434M1631 (from clone
DKFZp434M1631)
3923556.J14.GZ43_507064M81830Human somatostatin receptor isoform 21.00E−06
(SSTR2) gene, complete cds
3933556.J16.GZ43_507096Y15484Canis familiaris gene encoding retinal2.90E−08
guanylate cyclase E
3943556.K04.GZ43_506905U88832Human groucho protein homolog (AES)8.00E−07
gene, exons 2-7 and complete cds
3953556.K12.GZ43_507033AP001419Homo sapiens genomic DNA,1.00E−06
chromosome 21q22.2, clone: PAC24K9,
LB7T-ERG region, complete sequence
3963556.K13.GZ43_507049AK023589Homo sapiens cDNA FLJ13527 fis,2.00E−06
clone PLACE1006076
3973556.K17.GZ43_507113X71634D. bifasciata P-Transposon3.00E−06
3983556.L08.GZ43_506970X02367Glaucoma chattoni rDNA 3′ NTS8.20E−08
3993556.L09.GZ43_506986AF154329Pisum sativum MAP kinase PsMAPK24.10E−07
(Mapk2) mRNA, complete cds
4003556.L16.GZ43_507098AB041791Homo sapiens HSPDE10A gene for3.10E−08
phosphodiesterase 10A1 (PDE10A1),
exon 17
4013556.L23.GZ43_507210M23720Rat carboxypeptidase (CA2) gene, exon5.00E−06
10
4023556.M02.GZ43_506875U91963Human tolloid-like protein (TLL)1.40E−05
mRNA, complete cds
4033556.M11.GZ43_507019X16353R. rickettsii ompB gene for outer7.60E−05
membrane protein B
4043556.M23.GZ43_507211X93496H. sapiens TRAP gene, 5′ flanking region5.60E−23
4053556.N02.GZ43_506876U26458Snakehead retrovirus (SnRV), complete3.20E−05
genome
4063556.N04.GZ43_506908L39064Homo sapiens interleukin 9 receptor4.00E−09
precursor (IL9R) gene, complete cds
4073556.N05.GZ43_506924M63437Chicken KLG gene, complete cds2.00E−06
4083556.N06.GZ43_506940AF327424Arabidopsis thaliana unknown protein2.00E−07
(T14P1.19/At2g45010) mRNA, partial
cds
4093556.N21.GZ43_507180AB022157Mus musculus Cctd gene for chaperonin4.00E−06
containing TCP-1 delta subunit,
complete cds
4103556.O08.GZ43_506973X00171Vibrio cholera toxin (ctx) operon DNA7.00E−06
sequence from strain 2125
4113556.O13.GZ43_507053U41106Caenorhabditis elegans cosmid W06A111.20E−05
4123556.P07.GZ43_506958M15085T. brucei expressed copy of the ILTat 1.31.10E−07
variable surface glycoprotein gene, 5′
flank
4133559.A04.GZ43_507279AE006824Sulfolobus solfataricus section 183 of4.70E−05
272 of the complete genome
4143559.A20.GZ43_507535X71787A. thaliana AAP2 mRNA for amino acid2.00E−06
permease
4153559.A24.GZ43_507599X56494H. sapiens M gene for M1-type and M2-1.80E−05
type pyruvate kinase
4163559.B04.GZ43_507280AJ251550Homo sapiens partial AK155 gene for2.50E−05
AK155 protein, exons 1-3 and joined
CDS
4173559.B06.GZ43_507312AF077344Homo sapiens cartilage-derived C-type5.80E−05
lectin (CLECSF1) gene, exons 1 and 2
4183559.B08.GZ43_507344D50552Xenopus laevis xSox12 mRNA for4.00E−07
XSOX12, complete cds
4193559.B10.GZ43_507376L76259Homo sapiens PTS gene, complete cds9.00E−06
4203559.B18.GZ43_507504M29109D. discoideum actin M6 gene, 5′ flank3.40E−07
4213559.C06.GZ43_507313X99910C. carpio mRNA transcription factor,1.60E−05
ovx1
4223559.D21.GZ43_507554AK022877Homo sapiens cDNA FLJ12815 fis,2.00E−06
clone NT2RP2002546
4233559.E06.GZ43_507315U97408Caenorhabditis elegans cosmid F48A93.00E−06
4243559.E09.GZ43_507363L40489Ureaplasma urealyticum UreA (ureA),3.00E−07
UreB (ureB), UreC (ureC), UreE (ureE),
UreF (ureF), and UreG (ureG) genes,
complete cds; UreD (ureD) gene, partial
cds; and unknown gene
4253559.E20.GZ43_507539AF113521Zea mays putative transcription factor8.20E−08
mRNA sequence
4263559.F07.GZ43_507332AF109377Mus musculus ldlBp (LDLB) mRNA,4.30E−05
complete cds
4273559.F17.GZ43_507492U11292Human Ki nuclear autoantigen mRNA,6.40E−07
complete cds
4283559.H09.GZ43_507366X13414Murine I gene for MHC class II(Ia)9.00E−06
associated invariant chain
4293559.H22.GZ43_507574U61402Streptococcus thermophilus GalR (galR),1.00E−06
galactokinase (galK) and gal-1-P
uridylyltransferase (galT) genes,
complete cds
4303559.H24.GZ43_507606U67594Methanococcus jannaschii section 136 of3.40E−05
150 of the complete genome
4313559.I05.GZ43_507303X97289S. salar genes encoding alpha-globin and7.00E−06
beta-globin, clone 6
4323559.J04.GZ43_507288L10709Human constitutive endothelial nitric8.90E−12
oxide synthase gene, exons 25 and 26
and complete cds
4333559.J20.GZ43_507544U67559Methanococcus jannaschii section 101 of5.70E−05
150 of the complete genome
4343559.K16.GZ43_507481Z48955D. virginiana partial LINE-1 repetitive2.40E−08
DNA and putative RT
4353559.K17.GZ43_507497AC004497Homo sapiens chromosome 21, P1 clone4.00E−06
LBNL#6 (LBNL H10), complete
sequence
4363559.L01.GZ43_507242X58774Herpesvirus saimiri sRNA1, sRNA2,1.00E−06
sRNA3 and sRNA4 genes for small
viral RNAs
4373559.L14.GZ43_507450X67774C. upsaliensis (LMG 8854) 23S rRNA1.30E−05
gene
4383559.L19.GZ43_507530Z57634H. sapiens CpG island DNA genomic7.70E−07
Mse1 fragment, clone 187e9, forward
read cpg187e9.ft1a
4393559.M02.GZ43_507259AF042834Homo sapiens phosphodiesterase delta1.30E−05
subunit gene, exons 2, 3 and 4
4403559.M09.GZ43_507371U07628Caenorhabditis elegans N2 APX-1 (apx-2.00E−06
1) mRNA, complete cds
4413559.N05.GZ43_507308Z24259H. sapiens (D19S417) DNA segment3.70E−07
containing (CA) repeat; clone
AFM304zg1; single read
4423559.N18.GZ43_507516S75829{dinucleotide repeats, microsatellite1.90E−07
marker} [Dryobalanops lanceolata,
Genomic, 230 nt]
4433559.N21.GZ43_507564AL353948Homo sapiens mRNA; cDNA5.30E−07
DKFZp761P0114 (from clone
DKFZp761P0114)
4443559.O01.GZ43_507245AL110269Homo sapiens mRNA; cDNA1.60E−17
DKFZp564A122 (from clone
DKFZp564A122); partial cds
4453559.O05.GZ43_507309Y08695Clostridium tertium nanH gene7.40E−07
4463559.O07.GZ43_507341AJ249489Xenopus laevis partial mRNA for5.40E−07
putative olfactory receptor (xb6 gene)
4473559.O20.GZ43_507549X02886Human gene for T-cell receptor alpha2.00E−06
chain J region
4483559.P10.GZ43_507390X66030Homo sapiens partial ufo gene encoding4.90E−07
tyrosine kinase receptor
4493559.P15.GZ43_507470Z16777H. sapiens (D2S139) DNA segment4.00E−06
containing (CA) repeat; clone
AFM177xh4; single read
4503559.P18.GZ43_507518AJ228072Nicotiana benthamiana DNA for Tnt12.80E−07
retrotransposable element, isolate ben15
4513559.P24.GZ43_507614U32372Rattus norvegicus tyrosine-ester4.90E−07
sulfotransferase mRNA, complete cds
4523562.A01.GZ43_507615AE000496Escherichia coli K12 MG1655 section1.56E−04
386 of 400 of the complete genome
4533562.A15.GZ43_507839AF068289Homo sapiens HDCMD34P mRNA,6.60E−11
complete cds
4543562.B22.GZ43_507952AK014534Mus musculus 0 day neonate skin1.10E−07
cDNA, RIKEN full-length enriched
library, clone: 4631424J17, full insert
sequence
4553562.C23.GZ43_507969L01787Ascaris suum phosphoenolpyruvate3.70E−07
carboxykinase (PEPCK) gene, complete
cds
4563562.D10.GZ43_507762X54061D. melanogaster mRNA coding for a6.60E−07
205K microtubule-associated protein
(MAP)
4573562.E01.GZ43_507619M29812Homo sapiens Ig H-chain V71-41.50E−05
(IGH@) gene, partial cds
4583562.E03.GZ43_507651X03729Vaccinia virus late gene cluster from2.63E−04
central portion of genome containing the
L65 gene locus
4593562.E12.GZ43_507795M29694B. licheniformis RNA polymerase sigma-1.60E−05
30 factor (spo0H) gene, complete cds
4603562.F19.GZ43_507908AE006216Pasteurella multocida PM70 section 1832.40E−05
of 204 of the complete genome
4613562.F20.GZ43_507924M91004Rabbit endothelial leukocyte adhesion2.00E−06
molecule I (ELAM1), complete cds
4623562.G13.GZ43_507813X69818E. muelleri COLF1 gene for extracellular1.00E−06
matrix protein
4633562.G19.GZ43_507909AK019034Mus musculus 10 day old male pancreas1.00E−05
cDNA, RIKEN full-length enriched
library, clone: 1810049K24, full insert
sequence
4643562.H11.GZ43_507782AF206598Algyroides fitzingeri 12S ribosomal1.40E−07
RNA gene, partial sequence; tRNA-Val
gene, complete sequence; and 16S
ribosomal RNA gene, partial sequence;
mitochondrial genes for mitochondrial
products
4653562.H12.GZ43_507798AK002041Homo sapiens cDNA FLJ11179 fis,5.30E−07
clone PLACE 1007450
4663562.I01.GZ43_507623S39048knob associated histidine-rich protein2.00E−06
KAHRP {5′region} [Plasmodium
falciparum, Genomic, 2215 nt]
4673562.I02.GZ43_507639AF129501Buchnera aphidicola natural-host1.60E−07
Diuraphis noxia acetohydroxy acid
synthase large subunit (ilvI) and
acetohydroxy acid synthase small
subunit (ilvH) genes, complete cds; and
unknown genes
4683562.I13.GZ43_507815M26049Yeast (S. cerevisiae) RAD9 protein4.00E−06
(required for cell cycle arrest during
DNA repair) gene, complete cds
4693562.I15.GZ43_507847AF310880Barbatula barbatula microsatellite Bbar51.60E−07
sequence
4703562.J09.GZ43_507752AF236642Calothrix parietina clone 102-2A 16S-23S3.30E−07
internal transcribed spacer, complete
sequence; and tRNA-Ile and tRNA-Ala
genes, complete sequence
4713562.J13.GZ43_507816AC010728Homo sapiens BAC clone RP11-258E221.30E−05
from Y, complete sequence
4723562.K04.GZ43_507673S79777{specific DNA probe for Plasmodium5.40E−07
vivax pARC 1153} [Plasmodium vivax,
host = human, Genomic, 665 nt]
4733562.K08.GZ43_507737AJ403240M. musculus DNA for vimentin-binding2.00E−06
fragment VimE8
4743562.L12.GZ43_507802AE007840Clostridium acetobutylicum ATCC8245.80E−07
section 328 of 356 of the complete
genome
4753562.N24.GZ43_507996AF255609Homo sapiens high mobility group2.00E−07
protein HMG1 gene, exons 1 and 2,
partial cds
4763562.O11.GZ43_507789M15027Human myelin proteolipid protein gene,1.00E−06
exon 2
4773562.O18.GZ43_507901AL050208Homo sapiens mRNA; cDNA2.40E−07
DKFZp586F2323 (from clone
DKFZp586F2323)
4783562.O20.GZ43_507933AY020756Oryza sativa microsatellite MRG30814.90E−08
containing (TA)X13, genomic sequence
4793562.P21.GZ43_507950AF036318Skeletonema costatum cyclin (CYCL)7.20E−07
gene, partial cds
4803562.P23.GZ43_507982AF126719Plasmodium falciparum cAMP-3.00E−06
dependent protein kinase (pka) gene,
complete cds
4813565.A23.GZ43_508351AL122065Homo sapiens mRNA; cDNA1.50E−07
DKFZp434N011 (from clone
DKFZp434N011)
4823565.B05.GZ43_508064AF163325Trichoderma harzianum mitochondrial1.50E−07
plasmid pThr1, complete plasmid
sequence
4833565.B13.GZ43_508192X62689T. retusa DNA for brachiopod cubitus-9.00E−06
interruptus dominant (ciD) homologue
4843565.B14.GZ43_508208M29929Human insulin receptor (allele 1) gene,4.30E−12
exons 14, 15, 16 and 17
4853565.C04.GZ43_508049AE006183Pasteurella multocida PM70 section 1502.00E−06
of 204 of the complete genome
4863565.C06.GZ43_508081S79836SCPx/SCP2 = sterol carrier protein3.00E−06
x/sterol carrier protein 2 {promoter}
[human, Genomic, 3575 nt]
4873565.C17.GZ43_508257L13937Bovine phospholipase C mRNA,3.00E−07
complete cds
4883565.D14.GZ43_508210M37818Human keratin (psi-K-alpha)3.50E−08
pseudogene, exons 4, 5, 6, 7 and 8, and
keratin (psi-K-beta) pseudogene,
complete cds
4893565.D17.GZ43_508258Z19005C. pasteurianum gene for ferredoxin1.00E−06
4903565.D19.GZ43_508290AE007758Clostridium acetobutylicum ATCC8243.00E−06
section 246 of 356 of the complete
genome
4913565.E16.GZ43_508243L42813Protopterus dolloi complete2.49E−04
mitochondrial genome
4923565.G07.GZ43_508101U97500Homo sapiens butyrophilin (BT3.3)1.30E−05
gene, exons 1-4
4933565.G09.GZ43_508133M95098Bos taurus lysozyme gene (cow 2),1.26E−04
complete cds
4943565.G22.GZ43_508341AJ400873Homo sapiens partial GPLD1 gene for1.40E−09
glycosylphosphatidylinositol
phospholipase D, exons 15-20
4953565.H06.GZ43_508086U67465Methanococcus jannaschii section 7 of6.10E−07
150 of the complete genome
4963565.H10.GZ43_508150M15350Bacillus sp. strain 170 beta-lactamase5.70E−08
gene, complete cds
4973565.H11.GZ43_508166AB044878Equus caballus DNA, microsatellite3.20E−09
TKY378
4983565.H15.GZ43_508230AL122122Homo sapiens mRNA; cDNA5.00E−06
DKFZp434L098 (from clone
DKFZp434L098)
4993565.H23.GZ43_508358J05492E. coli cytochrome O ubiquinol oxidase1.00E−06
(cyoA, cyoB, cyoC, cyoD and cyoE
genes, complete cds
5003565.H24.GZ43_508374AE001417Plasmodium falciparum chromosome 2,1.70E−10
section 54 of 73 of the complete
sequence
5013565.K15.GZ43_508233AB062985Macaca fascicularis brain cDNA6.90E−105
clone: QmoA-10670, full insert sequence
5023565.L22.GZ43_508346L81801Homo sapiens (subclone 1_a2 from P11.30E−05
H31) DNA sequence, complete sequence
5033565.M15.GZ43_508235X08038Methanobacterium thermoautotrophicum1.10E−05
rpoT, rpoU, rpoV and rpoX genes for
RNA polymerase subunits A, B′, B″ and C
5043565.M20.GZ43_508315Z93381Caenorhabditis elegans cosmid F28G4,1.20E−05
complete sequence
5053565.N12.GZ43_508188M21573Salmon (S. salar) growth hormone gene,5.70E−05
complete cds
5063565.N13.GZ43_508204AK001163Homo sapiens cDNA FLJ10301 fis,5.20E−08
clone NT2RM2000032
5073565.N19.GZ43_508300AF321321Homo sapiens dopamine transporter2.00E−06
(SLC6A3) gene, exon 15 and complete
cds
5083565.O02.GZ43_508029X59773Pisum sativum mRNA for P protein, a1.30E−05
part of glycine cleavage complex
5093565.O03.GZ43_508045Z27113H. sapiens gene for RNA polymerase II2.00E−15
subunit 14.4 kD
5103565.O07.GZ43_508109X96607M. musculus IgH 3′ alpha enhancer DNA6.40E−05
5113565.O15.GZ43_508237Z35484Thermoanaerobacter sp. ATCC536273.00E−06
cgtA gene
5123565.P03.GZ43_508046U11292Human Ki nuclear autoantigen mRNA,6.40E−07
complete cds
5133565.P09.GZ43_508142X56261Yeast PPH1 gene for protein1.00E−06
phosphatase 2A
5143565.P22.GZ43_508350AE007790Clostridium acetobutylicum ATCC8243.00E−06
section 278 of 356 of the complete
genome
5153565.P24.GZ43_508382X61146N. tabacum NTP303 pollen specific2.70E−05
mRNA
5163568.A10.GZ43_508545U46925Arabidopsis thaliana GTP-binding3.00E−06
protein ATGB2 mRNA, complete cds
5173568.B02.GZ43_508418U83640Mus caroli Sp100 gene, exons 3 and 41.90E−08
5183568.B05.GZ43_508466BC008293Homo sapiens, Similar to RIKEN cDNA3.20E−16
A430101B06 gene, clone MGC: 13017
IMAGE: 3537789, mRNA, complete cds
5193568.C22.GZ43_508739AF280797Homo sapiens NPC-related protein1.00E−06
NAG73 mRNA, complete cds
5203568.D23.GZ43_508756AK022922Homo sapiens cDNA FLJ12860 fis,8.00E−06
clone NT2RP2003559
5213568.E17.GZ43_508661AF068294Homo sapiens HDCMB45P mRNA,5.30E−09
partial cds
5223568.E20.GZ43_508709AE006417Lactococcus lactis subsp. lactis IL14031.10E−05
section 179 of 218 of the complete
genome
5233568.F06.GZ43_508486U52198Vibrio anguillarum flagellin E (flaE),2.20E−05
flagellin D (flaD), and flagellin B (flaB)
genes, complete cds, and (flaG) gene,
partial cds
5243568.F07.GZ43_508502Z23599H. sapiens (D13S263) DNA segment1.90E−08
containing (CA) repeat; clone
AFM210yg11; single read
5253568.F11.GZ43_508566AE007525Clostridium acetobutylicum ATCC8244.20E−07
section 13 of 356 of the complete
genome
5263568.F12.GZ43_508582D50416Mouse mRNA for AREC3, complete cds1.90E−05
5273568.F22.GZ43_508742AF025900Histrionicus histrionicus CA7.80E−07
dinucleotide repeat locus Hhimicro1
5283568.G10.GZ43_508551U66074Tritrichomonas foetus putative9.70E−07
superoxide dismutase 2 (SOD2) gene,
complete cds
5293568.G12.GZ43_508583AB062941Macaca fascicularis brain cDNA9.50E−47
clone: QflA-14927, full insert sequence
5303568.G24.GZ43_508775L27221Giardia intestinalis pyruvate:flavodoxin3.20E−05
oxidoreductase and flanking genes
5313568.H20.GZ43_508712X75887B. taurus Brevican mRNA4.70E−05
5323568.J10.GZ43_508554AF194829Tetragonia tetragonioides NADH2.00E−06
dehydrogenase (ndhF) gene, partial cds;
chloroplast gene for chloroplast product
5333568.J22.GZ43_508746Y11031C. coli pldA gene1.00E−06
5343568.K01.GZ43_508411AL137751Homo sapiens mRNA; cDNA3.00E−06
DKFZp434I0812 (from clone
DKFZp434I0812); partial cds
5353568.K04.GZ43_508459Z82295R. prowazekii genomic DNA fragment7.20E−08
(clone A153F)
5363568.L04.GZ43_508460AL050105Homo sapiens mRNA; cDNA1.00E−05
DKFZp586H0519 (from clone
DKFZp586H0519); partial cds
5373568.M03.GZ43_508445L76259Homo sapiens PTS gene, complete cds8.00E−06
5383568.M13.GZ43_508605X61218M. musculus cervicolor (strain CRP)3.10E−09
Tcp-1 gene for t-complex polypeptide 1,
exons 8-10
5393568.N11.GZ43_508574AL079296Homo sapiens mRNA full length insert2.00E−06
cDNA clone EUROIMAGE 609395
5403568.O17.GZ43_508671AF078848Homo sapiens BUP mRNA, complete9.50E−09
cds
5413568.P04.GZ43_508464AB041548Mus musculus brain cDNA, clone5.00E−06
MNCb-3816, similar to AF171875 g1-
related zinc finger protein (Mus
musculus)
5423568.P18.GZ43_508688AL358951Human DNA sequence from clone RP3-3.00E−07
456L16 on chromosome 6, complete
sequence [Homo sapiens]
5433568.P19.GZ43_508704U43542Nicotiana tabacum diphenol oxidase2.00E−06
mRNA, complete cds
5443571.A04.GZ43_508833AF017116Homo sapiens type-2 phosphatidic acid2.40E−07
phosphohydrolase (PAP2) mRNA,
complete cds
5453571.A07.GZ43_508881L81867Homo sapiens (subclone 1_a8 from P19.00E−06
H54) DNA sequence, complete sequence
5463571.A08.GZ43_508897X85041H. sapiens PE5L gene ALU repeat region2.00E−06
5473571.A11.GZ43_508945U19361Petromyzon marinus neurofilament4.70E−08
subunit NF-180 mRNA, complete cds
5483571.A14.GZ43_508993AL022342Human DNA sequence from clone RP1-7.00E−05
29M10 on chromosome 20, complete
sequence [Homo sapiens]
5493571.A22.GZ43_509121U09448Vaucheria bursata protein synthesis7.20E−07
elongation factor Tu (tufA) gene,
chloroplast gene encoding chloroplast
protein, partial cds
5503571.B13.GZ43_508978AE002555Neisseria meningitidis serogroup B strain4.40E−05
MC58 section 197 of 206 of the
complete genome
5513571.B22.GZ43_509122AE002555Neisseria meningitidis serogroup B strain4.50E−05
MC58 section 197 of 206 of the
complete genome
5523571.C08.GZ43_508899AJ010154Saguinus oedipus msp-E1 gene1.10E−17
5533571.D04.GZ43_508836AF125460Caenorhabditis elegans cosmid Y9D1A3.60E−07
5543571.D07.GZ43_508884U51654Barbus barbus × Barbus meridionalis8.72E−02
microsatellite clone no. 37
5553571.E02.GZ43_508805AF329081Bos taurus AMP-activated protein kinase4.40E−33
gamma-1 (PRKAG1) gene, partial cds
5563571.E10.GZ43_508933M96068Madagascar periwinkle3.30E−08
hydroxymethylglutaryl-CoA reductase
(HMGR) mRNA, complete cds
5573571.E16.GZ43_509029AE006429Lactococcus lactis subsp. lactis IL14031.30E−05
section 191 of 218 of the complete
genome
5583571.F06.GZ43_508870AL137296Homo sapiens mRNA; cDNA4.40E−07
DKFZp434M0416 (from clone
DKFZp434M0416)
5593571.F16.GZ43_509030M58478Human cystic fibrosis transmembrane6.30E−05
conductance regulator gene, 5′ end
5603571.F23.GZ43_509142AF038397Mus musculus glutaminase (Gls) gene,4.70E−08
partial 3′ sequence
5613571.G22.GZ43_509127M80596Saccharomyces cerevisiae VAC1 gene7.00E−06
(required for vacuole inheritance and
vacuole protein sorting), complete cds
5623571.G24.GZ43_509159Z75330H. sapiens mRNA for nuclear protein SA-11.00E−46
5633571.H01.GZ43_508792U71144Influenza A virus H3N2 A/Akita/1/941.90E−05
nucleoprotein (NP) gene, complete cds
5643571.H10.GZ43_508936AF038564Homo sapiens atrophin-1 interacting6.60E−53
protein 4 (AIP4) mRNA, partial cds
5653571.H12.GZ43_508968K00131mouse b2 repeat sequence from clone3.00E−08
mm61
5663571.H16.GZ43_509032AF179564Homo sapiens GTF2I-like sequence1.20E−23
within duplicated segment of Williams
syndrome region
5673571.H18.GZ43_509064AE000331Escherichia coli K12 MG1655 section1.45E−04
221 of 400 of the complete genome
5683571.I11.GZ43_508953U20661Dictyostelium discoideum unknown9.00E−06
internal repeat protein gene, complete
cds, and unknown orf1, orf2 and orf3
genes, partial cds
5693571.J07.GZ43_508890M58478Human cystic fibrosis transmembrane6.40E−05
conductance regulator gene, 5′ end
5703571.J08.GZ43_508906AK021312Mus musculus 13 days embryo stomach3.60E−08
cDNA, RIKEN full-length enriched
library, clone: D530039A21, full insert
sequence
5713571.J09.GZ43_508922X66483D. discoideum gp80 gene8.90E−07
5723571.J14.GZ43_509002L77119Methanococcus jannaschii small extra-1.40E−05
chromosomal element, complete
sequence.˜
5733571.L01.GZ43_508796AK005500Mus musculus adult female placenta6.00E−06
cDNA, RIKEN full-length enriched
library, clone: 1600019O04, full insert
sequence
5743571.M17.GZ43_509053AF085681Mus musculus tubby like protein 15.00E−06
(Tulp1) mRNA, complete cds
5753571.M19.GZ43_509085D10487B. thermoglucosidasius gene for oligo-9.00E−06
1,6-glucosidase
5763571.M24.GZ43_509165M97680Bluetongue virus type 2 genomic RNA2.00E−06
sequence
5773571.N09.GZ43_508926X86100R. norvegicus BSP gene3.40E−07
5783571.N14.GZ43_509006D32007Mouse mRNA for a homlogue of1.20E−08
human CBFA2T1(Mtg8a), complete cds
5793571.N17.GZ43_509054Z68755Human DNA sequence from cosmid1.70E−10
L118D5, Huntington's Disease Region,
chromosome 4p16.3
5803571.N22.GZ43_509134D00326Porcine rotavirus (strain Gottfried), VP61.00E−06
gene, complete cds
5813571.O08.GZ43_508911X66483D. discoideum gp80 gene8.20E−07
5823574.A20.GZ43_509473AJ271814Drosophila melanogaster mRNA for1.70E−07
meso18E protein
5833574.B01.GZ43_509170U93261Homo sapiens DESP4P1 pseudogene1.00E−06
sequence
5843574.B04.GZ43_509218Y08207C. elaphus mitochondrial tRNA-Thr,1.40E−14
tRNA-Pro and tRNA-Phe genes
5853574.B10.GZ43_509314AL161991Homo sapiens mRNA; cDNA3.00E−06
DKFZp761C169 (from clone
DKFZp761C169); partial cds
5863574.B14.GZ43_509378D79208Apis mellifera mRNA for alpha-7.00E−06
glucosidase, complete cds
5873574.B24.GZ43_509538AE007758Clostridium acetobutylicum ATCC8243.00E−06
section 246 of 356 of the complete
genome
5883574.C09.GZ43_509299AF057708Populus balsamifera subsp. trichocarpa2.40E−07
PTD protein (PTD) gene, complete cds
5893574.C10.GZ43_509315AE005602Escherichia coli O157:H7 EDL9339.70E−05
genome, contig 3 of 3, section 221 of
290
5903574.C12.GZ43_509347AJ223633Enterococcus faecium genes encoding9.50E−07
enterocin L50A and enterocin L50B plus
5′ and 3′ flanking regions
5913574.C14.GZ43_509379X99710L. lactis ORF, genes homologous to vsf-14.00E−06
and pepF2 and gene encoding protein
homologous to methyltransferase
5923574.C16.GZ43_509411AF092920Chlorohydra viridissima head-activator3.00E−07
binding protein precursor (HAB) mRNA,
complete cds
5933574.C23.GZ43_509523AB047856Oryza sativa Ub-CEP52-2 gene for5.00E−08
ubiquitin fused to ribosomal protein L40,
complete cds
5943574.D02.GZ43_509188AB060225Macaca fascicularis brain cDNA570E−07
clone: QflA-14955, full insert sequence
5953574.D12.GZ43_509348M58478Human cystic fibrosis transmembrane6.00E−05
conductance regulator gene, 5′ end
5963574.E02.GZ43_509189L37347Human integral membrane protein2.00E−06
(Nramp2) mRNA, partial
5973574.E03.GZ43_509205X05817Bovine papillomavirus type 4 (BPV-4)6.00E−06
genome
5983574.E14.GZ43_509381U67507Methanococcus jannaschii section 49 of3.40E−05
150 of the complete genome
5993574.F10.GZ43_509318M24376Mouse zinc finger protein (krox-20)3.80E−08
gene, exon 1
6003574.F18.GZ43_509446AF184170Sparus aurata elongation factor 1-alpha3.40E−07
(EF1-alpha) mRNA, complete cds
6013574.F23.GZ43_509526Z29486R. norvegicus (Sprague Dawley) mRNA9.00E−06
for AMP-activated protein kinase
6023574.G07.GZ43_509271AF064079Plasmodium gallinaceum endochitinase1.40E−07
precursor, mRNA, complete cds
6033574.G11.GZ43_509335AF032872Rattus norvegicus potassium channel7.40E−07
regulatory protein KChAP mRNA,
complete cds
6043574.H07.GZ43_509272J04718Human proliferating cell nuclear antigen3.10E−07
(PCNA) gene, complete cds
6053574.I02.GZ43_509193AF200361Rattus norvegicus cytochrome P450 4F11.50E−05
(Cyp4F1) gene, complete cds
6063574.I07.GZ43_509273M29688S. cerevisiae PMS1 gene encoding DNA1.20E−08
mismatch repair protein, complete cds
6073574.J11.GZ43_509338Z24104H. sapiens (D12S338) DNA segment3.20E−07
containing (CA) repeat; clone
AFM291wd9; single read
6083574.J14.GZ43_509386AB008430Homo sapiens mRNA for CDEP,4.70E−05
complete cds
6093574.J23.GZ43_509530AP000384Arabidopsis thaliana genomic DNA,7.10E−07
chromosome 3, P1 clone: MCE21
6103574.K12.GZ43_509355AB031814Mus musculus oatp2 mRNA for organic1.50E−05
anion transporting polypeptide 2,
complete cds
6113574.K20.GZ43_509483AF126719Plasmodium falciparum cAMP-3.00E−06
dependent protein kinase (pka) gene,
complete cds
6123574.L07.GZ43_509276U53400Rattus norvegicus chromosome 108.94E−02
microsatellite sequence D10Mco21
6133574.M03.GZ43_509213AB000404Rice grassy stunt virus genomic RNA65.60E−07
for 20.6K major nonstructural protein
and 36.4K protein, complete cds
6143574.M23.GZ43_509533U18056Lycopersicon esculentum 1-amino-3.40E−07
cyclopropane-1-carboxylate synthase
(LE-ACS1A) gene, complete cds
6153574.N04.GZ43_509230L48479Homo sapiens (subclone 6_h1 from P13.30E−09
H21) DNA sequence
6163574.N10.GZ43_509326M58150Bovine lactoperoxidase (LPO) mRNA,3.60E−05
complete cds
6173574.N12.GZ43_509358AF182950Homo sapiens HEX (HEX) gene, partial9.00E−06
cds and 5′ flanking sequence
6183574.N20.GZ43_509486AE006904Sulfolobus solfataricus section 263 of3.00E−06
272 of the complete genome
6193574.P07.GZ43_509280U60232Homo sapiens cysteine dioxygenase6.30E−08
(CDO-1) gene, 5′ flanking region and
exons 1 and 2
6203574.P17.GZ43_509440AC002218Homo sapiens (subclone 2_c1 from P15.30E−08
H43) DNA sequence, complete sequence
6213577.A06.GZ43_509633U28328Bos taurus dinucleotide repeat RM154,3.40E−27
tandem repeat region
6223577.A18.GZ43_509825X58774Herpesvirus saimiri sRNA1, sRNA2,1.00E−06
sRNA3 and sRNA4 genes for small
viral RNAs
6233577.B12.GZ43_509730BC008400Homo sapiens, postmeiotic segregation2.50E−05
increased (S. cerevisiae) 2, clone
IMAGE: 4273792, mRNA
6243577.B15.GZ43_509778M61127Drosophila melanogaster GTP-binding1.10E−05
protein (arf-like) gene, complete cds
6253577.B19.GZ43_509842AF135526Homo sapiens clone MINT26 colon1.00E−06
cancer differentially methylated CpG
island genomic sequence
6263577.E19.GZ43_509845AF063864Schizosaccharomyces pombe essential1.00E−06
nuclear protein Mcm3p (mcm3+) gene,
complete cds
6273577.F02.GZ43_509574U37434Danio rerio L-isoaspartate (D-aspartate)5.10E−08
O-methyltransferase (PCMT) mRNA,
complete cds
6283577.G07.GZ43_509655AF001893Human MEN1 region clone epsilon/beta3.00E−06
mRNA, 3′ fragment
6293577.G13.GZ43_509751M83821Xenopus laevis mucin B.1 consensus2.10E−07
repeat mRNA
6303577.H06.GZ43_509640AK007565Mus musculus 10 day old male pancreas8.00E−07
cDNA, RIKEN full-length enriched
library, clone: 1810020K22, full insert
sequence
6313577.H08.GZ43_509672L81912Homo sapiens (subclone 2_g5 from PAC2.40E−07
H74) DNA sequence, complete sequence
6323577.H18.GZ43_509832AL157461Homo sapiens mRNA; cDNA4.00E−06
DKFZp434K152 (from clone
DKFZp434K152)
6333577.I01.GZ43_509561U35006Carcharhinus plumbeus Ig lambda light2.00E−06
chain gene, complete cds
6343577.I17.GZ43_509817AF157252Gongronella butleri translation1.00E−06
elongation factor 1-alpha (EF-1alpha)
gene, partial cds
6353577.J04.GZ43_509610AF338249Sus scrofa thyroid-stimulating hormone2.00E−06
receptor mRNA, complete cds
6363577.K06.GZ43_509643AB000264Bacillus firmus DNA for beta-amylase,5.00E−07
partial cds
6373577.K14.GZ43_509771X15441Aspergillus nidulans mitochondrial ndhC1.00E−06
and oxiB genes for NADH
dehydrogenase subunit 3 and cytochrome
oxidase subunit II
6383577.K23.GZ43_509915X52952Rat mRNA for c-mos3.00E−06
6393577.L10.GZ43_509708X60578Hepatitis C genomic RNA for putative3.70E−07
envelope protein (RE56 isolate)
6403577.N10.GZ43_509710Z75121S. cerevisiae chromosome XV reading4.50E−09
frame ORF YOR213c
6413577.N14.GZ43_509774M90058Human serglycin gene, exons 1, 2, and 35.00E−06
6423577.O17.GZ43_509823L19141Lupinus albus L-asparaginase gene,9.10E−08
complete cds
6433577.O22.GZ43_509903AL031008Human DNA sequence from clone5.60E−08
360A4 on chromosome 16. Contains
ESTs, complete sequence [Homo
sapiens]
6443577.P02.GZ43_509584AK006176Mus musculus adult male testis cDNA,4.60E−08
RIKEN full-length enriched library,
clone: 1700020M10, full insert sequence
6453577.P07.GZ43_509664U05822Human proto-oncogene BCL3 gene,2.40E−14
exon 2
6463577.P23.GZ43_509920AJ010341Homo sapiens PISSLRE gene, exons 1,1.00E−11
2, and 3 and joined CDS
6473580.A04.GZ43_509985AJ010213Mus musculus beta-dystrobrevin gene,8.20E−07
exon 10
6483580.A09.GZ43_510065AB037862Homo sapiens mRNA for KIAA14416.30E−15
protein, partial cds
6493580.A13.GZ43_510129U17832Symploce pallens mitochondrion 16S7.80E−07
ribosomal RNA, partial sequence
6503580.A14.GZ43_510145X89414A. thaliana DNA for pyrroline-5-6.00E−06
carboxylase synthetase gene
6513580.B01.GZ43_509938U67487Methanococcus jannaschii section 29 of9.00E−05
150 of the complete genome
6523580.C01.GZ43_509939X14898Hamster p7 preinsertion DNA2.00E−06
6533580.C03.GZ43_509971X76302H. sapiens RY-1 mRNA for putative3.70E−07
nucleic acid binding protein
6543580.C05.GZ43_510003Z22923M. musculus alpha2 (IX) collagen gene,1.60E−05
complete CDS
6553580.D07.GZ43_510036AB062941Macaca fascicularis brain cDNA9.80E−22
clone: QflA-14927, full insert sequence
6563580.D22.GZ43_510276M84136Flaveria chloraefolia flavonol 4′-4.00E−06
sulfotransferase mRNA, complete cds
6573580.E02.GZ43_509957AE001002Archaeoglobus fulgidus section 105 of3.90E−05
172 of the complete genome
6583580.E08.GZ43_510053U48431Drosophila pseudoobscura alpha-3.00E−06
amylase (Amy3) pseudogene, complete
cds
6593580.E10.GZ43_510085Z64717H. sapiens CpG island DNA genomic9.60E−19
Mse1 fragment, clone 161e9, forward
read cpg161e9.ft1a
6603580.E19.GZ43_510229M64984Candida tropicalis open reading frame2.00E−06
DNA sequence
6613580.E21.GZ43_510261M84136Flaveria chloraefolia flavonol 4′-5.00E−06
sulfotransferase mRNA, complete cds
6623580.E23.GZ43_510293AB033570Eptatretus burgeri hgPTPR5a mRNA,2.00E−06
partial cds
6633580.G03.GZ43_509975Y14277Drosophila melanogaster mRNA for1.10E−05
nuclear protein SA
6643580.G13.GZ43_510135AK018491Mus musculus adult male colon cDNA,4.40E−08
RIKEN full-length enriched library,
clone: 9030408N04, full insert sequence
6653580.G14.GZ43_510151AF142660Lama glama microsatellite LCA902.60E−07
sequence
6663580.G18.GZ43_510215D86226Spinacia oleracea DNA for nitrate2.60E−05
reductase, complete cds
6673580.G19.GZ43_510231U60502Glycine max actin (Soy119) gene, partial7.00E−06
cds
6683580.G20.GZ43_510247D38524Human mRNA for 5′-nucleotidase4.80E−11
6693580.G24.GZ43_510311AF084480Mus musculus Williams-Beuren5.00E−06
syndrome deletion transcript 9 homolog
(Wbscr9) mRNA, complete cds
6703580.H12.GZ43_510120X78423D. carota (Queen Anne's Lace) Inv*Dc34.00E−06
gene, 4444 bp
6713580.H16.GZ43_510184Y13786Homo sapiens mRNA for meltrin-4.50E−10
beta/ADAM 19 homologue
6723580.H22.GZ43_510280X62578C. caldarium plastid genes ompR′, psbD,2.50E−05
psbC, rps16 and groEL
6733580.I06.GZ43_510025X51344Spiroplasma virus (SpV1-R8A2 B)4.70E−07
complete genome
6743580.I08.GZ43_510057X02761Human mRNA for fibronectin (FN1.02E−04
precursor)
6753580.I18.GZ43_510217BC007856Homo sapiens, clone MGC: 143372.60E−10
IMAGE: 4298428, mRNA, complete cds
6763580.J10.GZ43_510090AF068206Rangifer tarandus microsatellite4.40E−11
NVHRT16 sequence
6773580.J12.GZ43_510122AE008323Agrobacterium tumefaciens strain C589.30E−05
linear chromosome, section 127 of 187
of the complete sequence
6783580.J18.GZ43_510218AF222689Homo sapiens protein arginine N-1.50E−05
methyltransferase 1 (HRMT1L2) gene,
complete cds, alternatively spliced
6793580.J20.GZ43_510250M31651Homo sapiens sex hormone-binding3.80E−07
globulin (SHBG) gene, complete cds
6803580.J21.GZ43_510266AB054062Pagrus major lpl mRNA for lipoprotein3.00E−06
lipase, complete cds
6813580.K03.GZ43_509979AE007607Clostridium acetobutylicum ATCC8245.00E−05
section 95 of 356 of the complete
genome
6823580.K05.GZ43_510011Z15027H. sapiens HLA class III DNA3.70E−08
6833580.K21.GZ43_510267AF135826Mus musculus neuronal nitric oxide2.20E−09
synthase (NOS-I) gene, exon 1c and 5′-
flanking sequence
6843580.L09.GZ43_510076AL049333Homo sapiens mRNA; cDNA3.40E−13
DKFZp564M116 (from clone
DKFZp564M116)
6853580.L10.GZ43_510092AF278587Borrelia burgdorferi strain BC-1 outer2.00E−06
surface protein C (ospC) gene, partial
cds
6863580.L12.GZ43_510124D14664Human mRNA for KIAA0022 gene,1.10E−05
complete cds
6873580.L13.GZ43_510140K02269Human ERV3 (endogenous retrovirus 3)3.30E−07
gag gene
6883580.L17.GZ43_510204U60232Homo sapiens cysteine dioxygenase2.00E−07
(CDO-1) gene, 5′ flanking region and
exons 1 and 2
6893580.M01.GZ43_509949U53400Rattus norvegicus chromosome 104.54E−01
microsatellite sequence D10Mco21
6903580.M16.GZ43_510189AE006406Lactococcus lactis subsp. lactis IL14033.00E−06
section 168 of 218 of the complete
genome
6913580.M17.GZ43_510205AF348584Arabidopsis thaliana unknown protein6.70E−07
(T8K14.7) mRNA, complete cds
6923580.M18.GZ43_510221X69908H. sapiens gene for mitochondrial ATP1.00E−05
synthase c subunit (P2 form)
6933580.M23.GZ43_510301M17326Mouse endogenous murine leukemia9.00E−06
virus polytropic provirus DNA, complete
cds
6943580.N10.GZ43_510094AF103970Lasioglossum rohweri cytochrome1.00E−06
oxidase I (COI) gene, mitochondrial
gene encoding mitochondrial protein,
partial cds
6953580.N11.GZ43_510110Z80362H. sapiens HLA-DRB pseudogene, exon6.10E−11
1;
6963580.N14.GZ43_510158AB014462Xenopus laevis XNLRR-1 mRNA,1.60E−05
complete cds
6973580.N15.GZ43_510174AF164381Anomochloa marantoidea maturase1.00E−06
(matK) gene, complete cds; chloroplast
gene for chloroplast product
6983580.N23.GZ43_510302AB047880Macaca fascicularis brain cDNA,2.00E−06
clone: QnpA-14303
6993580.O02.GZ43_509967X55948H. aspersa cytoplasmic intermediate4.00E−06
filament gene exons 2 to 6
7003580.O06.GZ43_510031L34649Homo sapiens platelet/endothelial cell4.00E−06
adhesion molecule-1 (PECAM-1) gene,
exon 14
7013580.O07.GZ43_510047Z30183H. sapiens mig-5 gene3.00E−05
7023580.O08.GZ43_510063AF101385Homo sapiens ribosomal protein L111.80E−08
gene, complete cds
7033580.P04.GZ43_510000AC016707Homo sapiens BAC clone RP11-221K41.80E−08
from Y, complete sequence
7043580.P05.GZ43_510016AF055482Thermotoga neapolitana galactose8.00E−07
utilization operon, complete sequence
7053580.P14.GZ43_510160AF009133Rattus norvegicus CD94 (Cd94) mRNA,7.50E−08
complete cds
7063580.P19.GZ43_510240Y15176Human papillomavirus type 80 E6, E7,7.00E−06
E1, E2, E4, L2, and L1 genes
7073583.B06.GZ43_510402X51398Chlamydomonas moewusii chloroplast3.00E−06
DNA for ORF 563 and transfer RNA-
Thr
7083583.B07.GZ43_510418U39382Hexachaeta amabilis 16S ribosomal5.50E−08
RNA gene, mitochondrial gene encoding
mitochondrial RNA, partial sequence
7093583.B10.GZ43_510466S45332erythropoietin receptor [human,3.90E−10
placental, Genomic, 8647 nt]
7103583.B11.GZ43_510482AC006623Caenorhabditis elegans clone C52E2,4.00E−06
complete sequence
7113583.D15.GZ43_510548AF242297Homo sapiens phosducin-like protein3.80E−08
gene, promoter and exon 1
7123583.D22.GZ43_510660Z23548H. sapiens (D10S540) DNA segment3.20E−07
containing (CA) repeat; clone
AFM205xe11; single read
7133583.E11.GZ43_510485X69737E. esula chloroplast rbcL gene for1.30E−08
ribulose-1,5-biphosphate-carboxylase
and promoter region
7143583.E13.GZ43_510517AB007856Homo sapiens KIAA0396 mRNA,2.20E−05
partial cds
7153583.E15.GZ43_510549X74131H. nelsoni small subunit ribosomal RNA7.00E−06
7163583.E17.GZ43_510581AE006633Streptococcus pyogenes M1 GAS strain2.40E−07
SF370, section 162 of 167 of the
complete genome
7173583.F24.GZ43_510694J02846Human tissue factor gene, complete cds7.40E−07
7183583.G09.GZ43_510455X88789P. sativum mRNA for starch synthase2.10E−05
(2035 bp)
7193583.G16.GZ43_510567AK000735Homo sapiens cDNA FLJ20728 fis,4.70E−07
clone HEP11763
7203583.G17.GZ43_510583AK026822Homo sapiens cDNA: FLJ23169 fis,2.60E−05
clone LNG09957
7213583.G21.GZ43_510647U13044Human nuclear respiratory factor-22.00E−06
subunit alpha mRNA, complete cds
7223583.H03.GZ43_510360M26222African green monkey origin of1.00E−13
replication (ORS9) region
7233583.H12.GZ43_510504X01669Human c-k-ras oncogene exon 2 from3.20E−08
lung carcinoma pr310
7243583.H13.GZ43_510520AK022380Homo sapiens cDNA FLJ12318 fis,2.00E−06
clone MAMMA1002068
7253583.H15.GZ43_510552L77119Methanococcus jannaschii small extra-1.60E−05
chromosomal element, complete
sequence.˜
7263583.J02.GZ43_510346AJ007302Sus scrofa triadin gene1.00E−06
7273583.K08.GZ43_510443D63902Mouse mRNA for estrogen-responsive2.50E−11
finger protein, complete cds
7283583.K10.GZ43_510475U11816Lactobacillus strain 30A ornithine1.00E−05
decarboxylase (odci) gene, complete cds
7293583.K11.GZ43_510491X73416W. suaveolens mitochondrial orf16.00E−06
7303583.K14.GZ43_510539U04367Bacillus thuringiensis dakota HD5111.20E−05
CryIII delta-endotoxin gene, partial cds
7313583.K17.GZ43_510587AE004129Vibrio cholerae chromosome I, section8.00E−06
37 of 251 of the complete chromosome
7323583.K23.GZ43_510683AE001410Plasmodium falciparum chromosome 2,4.00E−06
section 47 of 73 of the complete
sequence
7333583.L05.GZ43_510396X55299C. stercorarium celZ gene for endo-beta-1.00E−05
1,4-glucanase (Avicelase I)
7343583.L08.GZ43_510444AF106953Homo sapiens SOS1 (SOS1) gene,7.50E−09
partial cds
7353583.L09.GZ43_510460L34842Soybean chloroplast phytochrome A2.40E−05
(phyA) gene, complete cds
7363583.L17.GZ43_510588X65223T. rubrum mitochondrion genes for5.00E−06
cytochrome oxidase I, cytochrome
oxidase II, ATPase 9, NADH
dehydrogenase subunit 4L, NADH
dehydrogenase subunit 5, tRNA-Gln,
tRNA-Met and tRNA-Arg
7373583.L21.GZ43_510652AF106661Rattus norvegicus glutathione S-5.00E−06
transferase Yb4 (GstYb4) gene,
complete cds
7383583.M08.GZ43_510445BC005276Homo sapiens, Similar to GRO23.70E−07
oncogene, clone IMAGE: 4071652,
mRNA
7393583.M10.GZ43_510477Y00477Human bone marrow serine protease4.70E−09
gene (medullasin) (leukocyte neutrophil
elastase gene)
7403583.M13.GZ43_510525X73030S. cerevisiae YGP1 gene7.00E−06
7413583.N09.GZ43_510462AK018377Mus musculus 16 days embryo lung4.60E−07
cDNA, RIKEN full-length enriched
library, clone: 8430403M08, full insert
sequence
7423583.O03.GZ43_510367X72698P. pygmaeus ZFY gene for Y-linked Zinc3.00E−06
finger protein, final intron
7433583.O11.GZ43_510495U40161Arabidopsis thaliana type 2A protein2.00E−06
serine/threonine phosphatase 55 kDa B
regulatory subunit mRNA, complete cds
7443583.O17.GZ43_510591U67567Methanococcus jannaschii section 109 of2.00E−06
150 of the complete genome
7453583.P09.GZ43_510464AK021312Mus musculus 13 days embryo stomach3.60E−08
cDNA, RIKEN full-length enriched
library, clone: D530039A21, full insert
sequence
7463583.P19.GZ43_510624U12920Caenorhabditis elegans sex1.60E−05
determination (tra-3) gene, exons 2-6
7473583.P22.GZ43_510672AJ133800Homo sapiens CPNE7 gene (partial),7.60E−07
exon 2
7483590.A12.GZ43_512274AF185661Glomus intraradices strain FL208 18S2.00E−06
ribosomal RNA, partial sequence;
internal transcribed spacer 1, 5.8S
ribosomal RNA and internal transcribed
spacer 2, complete sequence; 26S
ribosomal RNA, partial sequence
7493590.B01.GZ43_512099M96068Madagascar periwinkle7.40E−09
hydroxymethylglutaryl-CoA reductase
(HMGR) mRNA, complete cds
7503590.B16.GZ43_512339V01527Mouse gene coding for major2.40E−12
histocompatibility antigen. This is a class
II antigen, I-A-beta
7513590.B21.GZ43_512419AB028983Homo sapiens mRNA for KIAA10601.70E−05
protein, partial cds
7523590.C20.GZ43_512404D86566Human DNA for NOTCH4, partial cds3.20E−07
7533590.D03.GZ43_512133D10371phocine distemper virus (PDV) genomic2.90E−05
RNA for N, P, V, C, M, F, H and L
protein
7543590.D19.GZ43_512389M96163Mus musculus (clone 2) serum inducible7.80E−10
kinase (SNK) mRNA, mRNA sequence
7553590.D23.GZ43_512453AF086485Homo sapiens full length insert cDNA7.70E−09
clone ZD93E02
7563590.E08.GZ43_512214AF055278Homo sapiens DNA repair protein5.90E−12
XRCC4 (XRCC4) gene, exon 1
7573590.E10.GZ43_512246AE001477Helicobacter pylori, strain J99 section 382.00E−06
of 132 of the complete genome
7583590.F01.GZ43_512103AF080395Entamoeba histolytica actin binding2.00E−06
protein (abp2) mRNA, partial cds
7593590.F16.GZ43_512343X79388B. subtilis (168) prkA gene1.20E−05
7603590.G01.GZ43_512104U32690Haemophilus influenzae Rd section 5 of2.80E−05
163 of the complete genome
7613590.G02.GZ43_512120U68040Cochliobolus heterostrophus polyketide1.25E−04
synthase (PKS1) gene, complete cds
7623590.H04.GZ43_512153X66013T. aestivum gene for cathepsin B (Al16)2.50E−07
7633590.H06.GZ43_512185X66177M. musculus mRNA for Hox 2.7 protein8.00E−06
7643590.H09.GZ43_512233AF012899Sambucus nigra ribosome inactivating3.40E−11
protein precursor mRNA, complete cds
7653590.H12.GZ43_512281Y15724Homo sapiens SERCA3 gene, exons 1-72.00E−06
(and joined CDS)
7663590.H16.GZ43_512345AF064079Plasmodium gallinaceum endochitinase6.70E−09
precursor, mRNA, complete cds
7673590.I16.GZ43_512346L06280Drosophila melanogaster adenine4.40E−07
phosphoribosyltransferase (APRT) gene,
complete cds
7683590.J01.GZ43_512107X69573T. reesei xyn1 gene, complete CDS1.70E−07
7693590.J02.GZ43_512123AF092047Homo sapiens homeobox protein Six34.00E−06
(SIX3) gene, complete cds
7703590.J18.GZ43_512379AB027966Schizosaccharomyces pombe gene for2.60E−08
Hypothetical protein, partial cds,
clone: TB89
7713590.J21.GZ43_512427AK014727Mus musculus 0 day neonate head7.90E−08
cDNA, RIKEN full-length enriched
library, clone: 4833419G08, full insert
sequence
7723590.J22.GZ43_512443AK020136Mus musculus 12 days embryo male5.90E−08
wolffian duct includes surrounding
region cDNA, RIKEN full-length
enriched library, clone: 6720460K10, full
insert sequence
7733590.K06.GZ43_512188AF171890Trimeresurus trigonocephalus3.00E−06
cytochrome b (cytb) gene, partial cds;
mitochondrial gene for mitochondrial
product
7743590.K10.GZ43_512252U16775Human immunodeficiency virus type 16.00E−06
isolate VE6 reverse transcriptase (pol)
gene, partial cds
7753590.K19.GZ43_512396U40454Candida albicans topoisomerase type I3.00E−06
(CATOP1) gene, complete cds
7763590.L08.GZ43_512221U52198Vibrio anguillarum flagellin E (flaE),2.00E−05
flagellin D (flaD), and flagellin B (flaB)
genes, complete cds, and (flaG) gene,
partial cds
7773590.L10.GZ43_512253U01155Xenopus laevis angiotensin II receptor4.00E−06
mRNA, complete cds
7783590.M03.GZ43_512142AF252499Bos taurus clone MNB-88 microsatellite4.60E−08
sequence
7793590.M04.GZ43_512158AE007607Clostridium acetobutylicum ATCC8244.50E−05
section 95 of 356 of the complete
genome
7803590.M09.GZ43_512238L04758Oryctolagus cuniculus cytochrome P-4501.00E−06
(CYP4A4) gene, 5′ end
7813590.N04.GZ43_512159Z82038C. thermosaccharolyticum etfB, etfA,2.00E−06
hbd, thlA and actA genes
7823590.N19.GZ43_512399U15603Saccharomyces cerevisiae Csd3p4.00E−06
(CSD3) gene, complete cds
7833590.N21.GZ43_512431L19535Drosophila subobscura sry alpha gene,6.00E−06
complete cds
7843590.O08.GZ43_512224L36588Homo sapiens intron-encoded U22 small4.30E−07
nucleolar RNA (UHG) gene
7853596.C02.GZ43_512500L14849Drosophila melanogaster cytoplasmic8.90E−09
protein tyrosine phosphatase (PTP61F)
mRNA, complete cds
7863596.C20.GZ43_512788M60286Herpesvirus saimiri immediate early1.30E−07
region protein genes, complete cds
7873596.C22.GZ43_512820X15121Soybean Gy1 gene for glycinin subunit1.00E−06
G1
7883596.D01.GZ43_512485Z78414Caenorhabditis elegans cosmid W09D12,4.00E−06
complete sequence
7893596.D07.GZ43_512581M88242Mouse glucocortoid-regulated1.70E−05
inflammatory prostaglandin G/H
synthase (griPGHS) mRNA, complete
cds
7903596.D09.GZ43_512613X99710L. lactis ORF, genes homologous to vsf-15.00E−06
and pepF2 and gene encoding protein
homologous to methyltransferase
7913596.D17.GZ43_512741AF200361Rattus norvegicus cytochrome P450 4F11.40E−05
(Cyp4F1) gene, complete cds
7923596.E08.GZ43_512598AF111848Homo sapiens PRO0529 mRNA,5.00E−06
complete cds
7933596.E22.GZ43_512822X58178S. pyogenes for emm41 gene5.00E−06
7943596.F10.GZ43_512631AL390161Homo sapiens mRNA; cDNA2.00E−06
DKFZp761P0615 (from clone
DKFZp761P0615)
7953596.G13.GZ43_512680AJ000044Tenebrio molitor LPCP29 gene2.00E−06
7963596.H04.GZ43_512537U65018Dictyostelium discoideum3.60E−07
mannosyltransferase gene, complete cds
7973596.H10.GZ43_512633AF104390Penaeus monodon hyperglycemic2.00E−06
hormone homolog PmSGP-V precursor,
mRNA, complete cds
7983596.H17.GZ43_512745D28915Human gene for hepatitis C-associated1.00E−06
microtubular aggregate protein p44, exon
9 and complete cds
7993596.H22.GZ43_512825AF198250Dictyostelium discoideum lim2 protein7.30E−07
(limB) mRNA, complete cds
8003596.I06.GZ43_512570U32444Solanum lycopersicum phytochrome F1.10E−05
(PHYF) gene, partial cds
8013596.I16.GZ43_512730U32444Solanum lycopersicum phytochrome F8.00E−06
(PHYF) gene, partial cds
8023596.J04.GZ43_512539D28596Chicken gene for c-maf proto-oncogene9.30E−10
product c-Maf, short form complete cds
and long form 1st exon
8033596.J13.GZ43_512683AB007856Homo sapiens KIAA0396 mRNA,2.40E−05
partial cds
8043596.K14.GZ43_512700AC024752Caenorhabditis elegans cosmid Y1B5A,3.00E−06
complete sequence
8053596.K15.GZ43_512716Y00469Yeast mRNA for profilin2.00E−06
8063596.L01.GZ43_512493X79703O. aries gene for beta-casein4.00E−06
8073596.L08.GZ43_512605AJ007313Streptomyces coelicolor sigT, trxB and9.80E−07
trxA genes, and ORF1 and ORF2
8083596.L13.GZ43_512685AK018239Mus musculus adult male medulla1.00E−06
oblongata cDNA, RIKEN full-length
enriched library, clone: 6330563C09, full
insert sequence
8093596.N02.GZ43_512511AE001387Plasmodium falciparum chromosome 2,1.00E−06
section 24 of 73 of the complete
sequence
8103596.N12.GZ43_512671Z12841O. cuniculus mRNA for phospholipase4.00E−06
8113596.N15.GZ43_512719U14186Bos taurus general vesicular transport1.70E−05
factor p115 mRNA, complete cds
8123596.N16.GZ43_512735U41106Caenorhabditis elegans cosmid W06A111.10E−05
8133596.N21.GZ43_512815AF097717Homo sapiens 3′-phosphoadenosine 5′-1.40E−07
phosphosulfate synthetase (PAPSS),
exon 8
8143596.O10.GZ43_512640AE001649Chlamydia pneumoniae section 65 of1.10E−05
103 of the complete genome
8153596.O12.GZ43_512672AC006623Caenorhabditis elegans clone C52E2,4.00E−06
complete sequence
8163596.P03.GZ43_512529X82317C. thummi CpY gene1.49E−03
8173596.P04.GZ43_512545AF111855Agrobacterium tumefaciens RNA2.00E−06
polymerase alpha subunit (rpoA) gene,
complete cds
8183596.P07.GZ43_512593L40817Homo sapiens muscle-specific DNase I-3.00E−06
like (DNL1L) gene, exons 1-9, complete
cds
8193596.P08.GZ43_512609M14505Human (clone PSK-J3) cyclin-dependent5.00E−06
protein kinase mRNA, complete cds.,
8203596.P10.GZ43_512641M73770P. falciparum RNA polymerase III largest2.90E−05
subunit gene, complete cds
8213596.P21.GZ43_512817S82725NPM/ALK = fusion gene {translocation1.00E−07
breakpoint} [human, lymphoma cells
SU-DHL-1, Genomic, 1679 nt]
8223599.A04.GZ43_512914X83212H. sapiens tryptophan hydroxylase gene,5.50E−07
promoter region
8233599.A23.GZ43_513218U05259Human MB-1 gene, complete cds2.10E−05
8243599.B15.GZ43_513091AF277068HIV-1 clone QH0791 from Trinidad and6.10E−07
Tobago, envelope protein (env) gene,
complete cds
8253599.B16.GZ43_513107M60517Chicken vitronectin receptor alpha4.00E−06
subunit mRNA, complete cds
8263599.C03.GZ43_512900AB021267Arabidopsis thaliana copia-like2.00E−06
retrotransposon AtRE2-2 gene for
polyprotein, complete cds
8273599.C17.GZ43_513124U28055Homo sapiens hepatocyte growth factor-3.00E−06
like protein homolog mRNA, partial cds
8283599.D03.GZ43_512901L43550Buchnera aphidicola anthranilate3.00E−06
synthase small subunit (trpG) gene,
anthranilate synthase large subunit (trpE)
gene, complete cds
8293599.D05.GZ43_512933AL023779S. pombe chromosome II cosmid c2442.00E−06
8303599.D07.GZ43_512965AL391223Human chromosome 14 DNA sequence5.00E−06
Partial sequence from BAC R-
325N7_PCR1 of library RPCI-11 from
chromosome 14 of Homo sapiens
(Human), complete sequence
8313599.D10.GZ43_513013AF064079Plasmodium gallinaceum endochitinase1.70E−07
precursor, mRNA, complete cds
8323599.E01.GZ43_512870U09184Buchnera aphidicola ferredoxin-NADP9.60E−07
reductase (fprl) gene, partial cds;
anthranilate synthase large subunit (trpE)
and anthranilate synthase small subunit
(trpG) genes, complete cds; heat shock
protein (hslU) gene, partial cds; and
unknown gene
8333599.E05.GZ43_512934X60145Human J-alpha segment J-alpha FR91.20E−05
mRNA for J-alpha region of T-cell
receptor
8343599.F17.GZ43_513127U27037Fistulina hepatica mitochondrial small2.00E−06
subunit ribosomal RNA, mitochondrial
gene, partial sequence
8353599.F24.GZ43_513239Z78414Caenorhabditis elegans cosmid W09D12,5.00E−06
complete sequence
8363599.H05.GZ43_512937AF032891Camponotus consobrinus microsatellite-2.10E−08
containing sequence Ccon12
8373599.H23.GZ43_513225AB024553Bacillus halodurans DNA, complete and4.70E−07
partial cds, strain: C-125
8383599.J11.GZ43_513035AB025112Xenopus laevis XGC-2 mRNA for3.00E−06
guanylyl cyclase-2, complete cds
8393599.K02.GZ43_512892AJ224474Borrelia burgdorferi left chromosomal3.00E−06
subtelomeric region (truA gene)
8403599.K04.GZ43_512924X99710L. lactis ORF, genes homologous to vsf-15.00E−06
and pepF2 and gene encoding protein
homologous to methyltransferase
8413599.K23.GZ43_513228AF074247Homo sapiens neuronal delayed-rectifier8.00E−07
voltage-gated potassium channel splice
variant (KCNQ2) mRNA, complete cds
8423599.L04.GZ43_512925X59773Pisum sativum mRNA for P protein, a1.40E−05
part of glycine cleavage complex
8433599.L15.GZ43_513101U34282Rattus norvegicus fast skeletal muscle2.00E−06
sarcoplasmic reticulum Ca-ATPase
(SERCA1) gene, 5′-flanking sequence
8443599.M04.GZ43_512926AK018953Mus musculus adult male testis cDNA,2.30E−11
RIKEN full-length enriched library,
clone: 1700111D04, full insert sequence
8453599.M22.GZ43_513214AB052179Macaca fascicularis brain cDNA,4.70E−07
clone: QnpA-21934
8463599.M24.GZ43_513246AE003394Drosophila melanogaster genomic7.30E−07
scaffold 142000013386028, complete
sequence
8473599.N09.GZ43_513007X16362Rat SPI-2 serine protease inhibitor gene1.19E−04
8483599.N16.GZ43_513119X92421X. laevis mRNA for RNA helicase p543.00E−06
8493599.N20.GZ43_513183M59447Drosophila melanogaster Sex-lethal2.00E−06
(Sx1) mRNA, complete cds
8503599.N24.GZ43_513247AC005485Homo sapiens PAC clone RP5-998M22.00E−07
from 7q33-q35, complete sequence
8513599.O06.GZ43_512960AJ131667Escherichia coli plasmid pSFO1572.00E−06
8523599.O17.GZ43_513136X96607M. musculus IgH 3′ alpha enhancer DNA8.10E−05
8533599.P05.GZ43_512945X77111N. tabacum chi-V gene1.50E−07
8543602.A09.GZ43_513378AF015303Xenopus laevis small GTPase Ran1.10E−05
binding protein 1 mRNA, complete cds
8553602.B18.GZ43_513523L18892Tetrahymena thermophila histone5.70E−07
(H2A.1) gene, complete cds
8563602.B21.GZ43_513571BC005233Homo sapiens, clone MGC: 122571.60E−10
IMAGE: 3950129, mRNA, complete cds
8573602.B22.GZ43_513587X71765P. falciparum gene for Ca2+ —ATPase1.00E−06
8583602.C24.GZ43_513620AL080106Homo sapiens mRNA; cDNA2.00E−06
DKFZp566O053 (from clone
DKFZp566O053)
8593602.D06.GZ43_513333AF098970Phaseolus vulgaris NBS-LRR-like1.70E−07
protein cD7 (CO-2) mRNA, partial cds
8603602.D11.GZ43_513413M59770P. falciparum calmodulin gene, complete2.20E−07
cds
8613602.E04.GZ43_513302X53582Zea mays ZMPMS1 gene for 19 kDa1.30E−05
zein protein
8623602.E06.GZ43_513334L38718Providencia stuartii (clone pSK.aarP)7.90E−07
transcriptional activator (aarP) gene,
complete cds
8633602.E13.GZ43_513446U58106Blomia tropicalis allergen mRNA,1.70E−07
complete cds
8643602.E21.GZ43_513574M15085T. brucei expressed copy of the ILTat 1.32.90E−07
variable surface glycoprotein gene, 5′
flank
8653602.F12.GZ43_513431X64802H. sapiens F8 mRNA for Interleukin-1-3.40E−58
like species
8663602.G03.GZ43_513288AF036148Danio rerio NeuroD (nrd) mRNA,2.00E−06
complete cds
8673602.G17.GZ43_513512U41106Caenorhabditis elegans cosmid W06A111.30E−05
8683602.I07.GZ43_513354AF000941Mus musculus DNAse I hypersensitive1.20E−05
sites 2-6 of locus control region (LCR)
for T-cell receptor alpha chain (TCRa)
gene
8693602.I11.GZ43_513418AL133620Homo sapiens mRNA; cDNA3.00E−06
DKFZp434F0621 (from clone
DKFZp434F0621)
8703602.I15.GZ43_513482U23479Dictyostelium discoideum8.00E−07
phosphatidylinositol 4-kinase (PIK4)
mRNA, complete cds
8713602.J13.GZ43_513451AK025319Homo sapiens cDNA: FLJ21666 fis,3.30E−07
clone COL08915
8723602.K03.GZ43_513292X85811S. cerevisiae tRNA-Leu, and ORF's1.10E−05
N2212, N2215, N2219, N2223, N2227,
N2231
8733602.K06.GZ43_513340AF133052Walleye epidermal hyperplasia virus4.00E−06
type 2 long terminal repeat, complete
sequence; gag polyprotein (gag-pol)
gene, complete cds; pol polyprotein
(gag-pol) gene, partial cds; envelope
polyprotein (env) and cyclin D homolog
genes, complete cds; and unkn>
8743602.L20.GZ43_513565M62717Human CSP-B gene flanking sequence1.10E−05
8753602.N03.GZ43_513295Z81126Caenorhabditis elegans cosmid T22E6,5.70E−05
complete sequence
8763602.N06.GZ43_513343U62503Human OBR gene, intron sequence1.00E−06
immediately adjacent to the 5′ end of
coding exon 17
8773605.A15.gz43_513858Z46507Bovine herpesvirus type 4 genomic DNA5.00E−06
region (V.TEST)
8783605.C16.gz43_513876AF282517Homo sapiens clone 10ptel_c6t79.40E−08
sequence
8793605.E19.gz43_513926Z22923M. musculus alpha2 (IX) collagen gene,2.10E−05
complete CDS
8803605.G13.gz43_513832AJ132752Gadus morhua mRNA for beta2-1.30E−05
microglobulin, clone b3
8813605.H10.gz43_513785AF257480Rana temporaria microsatellite SB804.10E−09
sequence
8823605.H21.gz43_513961X63507M. musculus HOX-3.5 gene7.80E−05
8833605.I19.gz43_513930AK002100Homo sapiens cDNA FLJ11238 fis,3.30E−11
clone PLACE1008532
8843605.J16.gz43_513883AF039197Gallus gallus Pax-9 gene, putative 5′1.00E−07
regulatory sequence
8853605.K19.gz43_513932X63853S. cerevisiae MAT locus genes BUD5,8.00E−06
mat-alpha1, mat-alpha2, YCR724 and
YCR725
8863605.M17.gz43_513902M30931Simian immunodeficiency virus (SIV)3.70E−05
proviral, complete genome
8873605.N04.gz43_513695AF169388Mus musculus alpha 4 collagen IV8.90E−05
(Col4a4) mRNA, complete cds
8883605.N09.gz43_513775AF029111Adelius sp. 16S ribosomal RNA gene,2.80E−07
mitochondrial gene for mitochondrial
RNA, partial sequence
8893605.N12.gz43_513823BC000358Homo sapiens, protein kinase, AMP-3.90E−47
activated, gamma 1 non-catalytic
subunit, clone MGC: 8666
IMAGE: 2964434, mRNA, complete cds
8903605.N16.gz43_513887X95301D. rerio mRNA for HER-5 protein1.00E−06
8913608.B06.gz43_514099X00004.taurus gene encoding pituitary6.30E−08
glycoprotein hormone alpha subunit,
exons 3 & 4
8923608.B12.gz43_514195X00525Mouse 28S ribosomal RNA3.10E−13
8933608.B24.gz43_514387AF269848Staphylococcus epidermidis strain SR12.00E−06
clone step.1026e06 genomic sequence
8943608.C18.gz43_514292BC000387Homo sapiens, U6 snRNA-associated2.50E−10
Sm-like protein, clone MGC: 8433
IMAGE: 2821171, mRNA, complete cds
8953608.E17.gz43_514278BC008245Homo sapiens, clone IMAGE: 3875012,1.00E−06
mRNA
8963608.E20.gz43_514326U86646Ailurus fulgens beta casein gene, exon 7,4.70E−07
partial cds
8973608.F13.gz43_514215AF125672Homo sapiens silencing mediator of2.00E−06
retinoic acid and thyroid hormone
receptor extended isoform (SMRTE)
mRNA, complete cds
8983608.G09.gz43_514152AE001066Archaeoglobus fulgidus section 41 of4.00E−06
172 of the complete genome
8993608.H05.gz43_514089AJ224981Mus musculus calpain 3 gene, exon 13.00E−06
9003608.H14.gz43_514233AE007394Streptococcus pneumoniae section 77 of3.20E−05
194 of the complete genome
9013608.H18.gz43_514297Z36046S. cerevisiae chromosome II reading7.00E−06
frame ORF YBR177c
9023608.J17.gz43_514283AF024648Arabidopsis thaliana receptor-like8.00E−06
serine/threonine kinase (RKF1) mRNA,
complete cds
9033608.J24.gz43_514395AJ002258Rattus Norvegicus mRNA for Prx3A3.60E−07
protein
9043608.K03.gz43_514060M83199Simmondsia chinensis stearoyl-acyl2.50E−07
carrier protein desaturase mRNA,
complete cds
9053608.K14.gz43_514236AK026999Homo sapiens cDNA: FLJ23346 fis,2.00E−06
clone HEP13716
9063608.L07.gz43_514125M32684Homo sapiens ITGB3 gene, intron 13,3.60E−07
fragment B, partial sequence
9073608.L14.gz43_514237Z34845H. sapiens serotonin transporter gene8.60E−07
9083608.N09.gz43_514159AK022341Homo sapiens cDNA FLJ12279 fis,2.00E−06
clone MAMMA1001743, weakly similar
to Y BOX BINDING PROTEIN-1
9093608.N19.gz43_514319M15085T. brucei expressed copy of the ILTat 1.37.80E−08
variable surface glycoprotein gene, 5′
flank
9103608.N20.gz43_514335AF026169Homo sapiens SALF (SALF) mRNA,1.00E−05
complete cds
9113608.O04.gz43_514080U85193Human nuclear factor I-B2 (NFIB2)7.10E−07
mRNA, complete cds
9123608.P22.gz43_514369AF124241Callerya australis chloroplast tRNA-Leu3.90E−07
(trnL) gene, intron sequence
9133611.A17.gz43_514658X01412Drosophila melanogaster genes for2.00E−06
tRNA-Val and tRNA-Pro (90BC tRNA
locus)
9143611.B11.gz43_514563AL049938Homo sapiens mRNA; cDNA9.80E−10
DKFZp564P1916 (from clone
DKFZp564P1916); partial cds
9153611.B16.gz43_514643M86514Rat proline-rich protein mRNA, 3′ end1.30E−05
9163611.C09.gz43_514532U55950Pleurodeles waltl cytochrome b (CYT-b)2.00E−06
gene, mitochondrial gene encoding
mitochondrial protein, partial cds
9173611.E07.gz43_514502AF261009Lethrinus miniatus clone 89rte,1.70E−12
microsatellite sequence
9183611.E12.gz43_514582M60200Rat vitamin D binding protein gene,1.50E−05
exons 5 and 6
9193611.E20.gz43_514710BC002458Homo sapiens, clone IMAGE: 3343171,2.00E−06
mRNA, partial cds
9203611.F15.gz43_514631U28328Bos taurus dinucleotide repeat RM154,4.30E−27
tandem repeat region
9213611.H10.gz43_514553AE003147Drosophila melanogaster genomic6.00E−07
scaffold 142000013385388, complete
sequence
9223611.H22.gz43_514745X16135Human mRNA for novel heterogeneous7.00E−06
nuclear RNP protein, L protein
9233611.I04.gz43_514458AK001460Homo sapiens cDNA FLJ10598 fis,5.10E−44
clone NT2RP2004841
9243611.I13.gz43_514602M58380Arabidopsis thaliana peroxidase (neutral,3.00E−06
prxCa) gene, complete cds
9253611.J04.gz43_514459S81486p53 {alternatively spliced, intron 9}1.20E−07
[human, Genomic Mutant, 133 nt]
9263611.J15.gz43_514635AC008240Leishmania major chromosome 22 clone4.90E−05
L9259 strain Friedlin, complete sequence
9273611.J17.gz43_514667Z17425Lilium speciosum for two putative cds's8.90E−07
9283611.J22.gz43_514747U60736Human IgHC locus intergenic sequence4.60E−07
9293611.K01.gz43_514412AE001377Plasmodium falciparum chromosome 2,3.00E−06
section 14 of 73 of the complete
sequence
9303611.K12.gz43_514588X02367Glaucoma chattoni rDNA 3′ NTS9.80E−08
9313611.L22.gz43_514749U19361Petromyzon marinus neurofilament5.40E−08
subunit NF-180 mRNA, complete cds
9323611.M18.gz43_514686X95301D. rerio mRNA for HER-5 protein1.00E−06
9333611.M24.gz43_514782AF010239Caenorhabditis elegans glutathione S-7.70E−07
transferase (CeGST1) mRNA, complete
cds
9343611.N01.gz43_514415L19300Staphylococcus aureus DNA sequence1.00E−06
encoding three ORFs, complete cds;
prophage phi-11 sequence homology, 5′
flank
9353611.N09.gz43_514543U50382Danio rerio beta and alpha globin genes,7.00E−06
partial cds
9363611.O16.gz43_514656AB056785Macaca fascicularis brain cDNA6.60E−07
clone: QnpA-11655, full insert sequence
9373611.P08.gz43_514529AK026905Homo sapiens cDNA: FLJ23252 fis,8.00E−06
clone COL04668
9383614.C18.gz43_515060AF239178Paracoccidioides brasiliensis lon5.00E−06
proteinase gene, complete cds; nuclear
gene for mitochondrial product
9393614.D14.gz43_514997AB017511Hydra magnipapillata mRNA for PLC-1.20E−05
betaH1, complete cds
9403614.D21.gz43_515109L10713Pig trinucleotide repeat1.80E−05
9413614.E06.gz43_514870X99739M. musculus mRNA for UBC9 protein,9.10E−07
containing ubiquitin box
9423614.F22.gz43_515127AK021490Homo sapiens cDNA FLJ11428 fis,2.00E−06
clone HEMBA1001071, highly similar
to PROCOLLAGEN ALPHA 1(III)
CHAIN PRECURSOR
9433614.G20.gz43_515096M86514Rat prolin-rich protein mRNA, 3′ end1.30E−05
9443614.H09.gz43_514921AF068289Homo sapiens HDCMD34P mRNA,6.60E−11
complete cds
9453614.H22.gz43_515129X62423P. falciparum pol delta gene for DNA4.00E−06
polymerase delta
9463614.J07.gz43_514891X81027H. sapiens tal-1 DNA1.30E−05
9473614.K22.gz43_515132X63073Pseudanabaena sp. cpeBA operon1.60E−05
encoding phycoerythrin beta and alpha
subunits
9483614.L13.gz43_514989V01561Mouse dispersed repetitive DNA3.00E−06
sequences of the R-family and simple
sequence DNA; member of the B1
family of mouse dispersed repetitive
DNA sequences
9493614.M08.gz43_514910AF272983Homo sapiens SRC tyrosine kinase gene,4.00E−06
exons 1alpha and 1a, alternatively
spliced
9503614.O02.gz43_514816X58913Mitochondrion Drosophila eugracilis8.50E−08
ND2 and COI genes (partial) and genes
for tRNA-Trp, tRNA-Tyr, and tRNA-
Cys
9513614.O07.gz43_514896AL031538S. pombe chromosome III cosmid c19069.80E−07
9523614.O16.gz43_515040AB056785Macaca fascicularis brain cDNA2.00E−06
clone: QnpA-11655, full insert sequence
9533614.P11.gz43_514961X91656M. musculus Srp20 gene4.60E−05
9543614.P16.gz43_515041Z58907H. sapiens CpG island DNA genomic3.20E−70
Mse1 fragment, clone 116a6, forward
read cpg116a6.ft1a
9553617.B16.gz43_515411AF098275Homo sapiens PSI2TOM20 pseudogene,1.10E−67
complete sequence
9563617.C21.gz43_515492AJ009913Bos taurus plp gene3.40E−05
9573617.F10.gz43_515319L07487Bradyrhizobium japonicum heme-copper6.70E−05
oxidase subunit I homolog (fixN),
cytochrome c (fixO), transmembrane
proteins (fixO and fixQ) diheme
cytochrome c (fixP) and fixG genes,
complete cds
9583617.H16.gz43_515417X54192O. sativa GluB-2 gene for glutelin2.00E−06
9593617.I01.gz43_515178AL513316Human DNA sequence from clone7.20E−08
RP11-522O3 on chromosome 10,
complete sequence [Homo sapiens]
9603617.L16.gz43_515421AE007662Clostridium acetobutylicum ATCC8243.00E−06
section 150 of 356 of the complete
genome
9613617.L21.gz43_515501AL031538S. pombe chromosome III cosmid c19061.00E−06
9623617.M08.gz43_515294X64802H. sapiens F8 mRNA for Interleukin-1-3.40E−58
like species
9633617.M13.gz43_515374Z79239H. sapiens flow-sorted chromosome 61.10E−07
TaqI fragment, SC6pA26F6
9643617.N05.gz43_515247AF387666Mandrillus cytomegalovirus strain1.00E−06
OCOM6-2 glycoprotein B (gB) gene,
partial cds
9653617.N10.gz43_515327AB017511Hydra magnipapillata mRNA for PLC-1.10E−05
betaH1, complete cds
9663617.N14.gz43_515391AJ249346Mus musculus Ankrd2 gene for ankyrin1.00E−05
repeat domain 2 (stretch responsive
muscle), exons 1-9
9673617.N19.gz43_515471U27037Fistulina hepatica mitochondrial small2.00E−06
subunit ribosomal RNA, mitochondrial
gene, partial sequence
9683617.P11.gz43_515345AK002100Homo sapiens cDNA FLJ11238 fis,1.20E−13
clone PLACE1008532
9693617.P12.gz43_515361U04860Rattus norvegicus Sprague-Dawley Ah8.00E−05
receptor mRNA, complete cds
9703617.P13.gz43_515377AE007356Streptococcus pneumoniae section 39 of3.80E−05
194 of the complete genome
9713620.B03.gz43_515810AF238884Botrytis virus F, complete genome6.00E−06
9723620.B24.gz43_516146AF244812Homo sapiens SCAN domain-containing1.30E−07
protein 2 (SCAND2) gene, complete cds,
alternatively spliced
9733620.E12.gz43_515957X95301D. rerio mRNA for HER-5 protein1.00E−06
9743620.E13.gz43_515973X52289Human (D21S167) DNA segment2.50E−19
containing (GT)19 repeat
9753620.E17.gz43_516037AJ002414Arabidosis thaliana mRNA for a hnRNP-9.70E−08
like protein
9763620.E19.gz43_516069X16982Drosophila melanogaster micropia-2.70E−07
Dm11 3′flanking DNA
9773620.E23.gz43_516133Z49438S. cerevisiae chromosome X reading3.00E−06
frame ORF YJL163c
9783620.E24.gz43_516149M75883Human sterol carrier protein X/sterol8.00E−06
carrier protein 2 mRNA, complete cds
9793620.G17.gz43_516039U92971Human protease-activated receptor 33.80E−07
(PAR3) mRNA, complete cds
9803620.G23.gz43_516135X66979X. laevis mRNA XLFLI1.60E−05
9813620.J18.gz43_516058U37373Xenopus laevis tail-specific thyroid3.00E−06
hormone up-regulated (gene 5) mRNA,
complete cds
9823620.K19.gz43_516075U31780Human papillomavirus type 22, complete5.00E−06
genome
9833620.K24.gz43_516155M95627Homo sapiens angio-associated6.00E−06
migratory cell protein (AAMP) mRNA,
complete cds
9843620.O23.gz43_516143L11172Plasmodium falciparum RNA1.00E−05
polymerase I gene, complete cds
9853623.B07.gz43_516258AF132745Mus musculus Sox2 gene, regulatory7.70E−07
region sequence
9863623.E03.gz43_516197X82566M. musculus glyT1 gene (exon 0a)1.80E−09
9873623.E15.gz43_516389AF104420Porcine transmissible gastroenteritis2.90E−05
virus RNA dependent RNA polymerase
gene, partial cds; virus envelope protein
spike (S), envelope protein (sM),
envelope protein (M), and nucleoprotein
(N) genes, complete cds; and unknown
genes
9883623.F03.gz43_516198AJ009936Homo sapiens mRNA for nuclear1.70E−05
hormone receptor PRR1
9893623.F20.gz43_516470U22657Mus musculus genomic locus related to5.80E−05
cellular morphology
9903623.G14.gz43_516375AB035309Paramecium caudatum PcTERT mRNA3.00E−06
for telomerase reverse transcriptase,
complete cds
9913623.H07.gz43_516264Z17324Homo sapiens of MUC1 gene encoding1.80E−07
Mucin
9923623.H10.gz43_516312AB033070Homo sapiens mRNA for KIAA12442.80E−05
protein, partial cds
9933623.H23.gz43_516520AF131763Homo sapiens clone 25232 mRNA1.70E−05
sequence
9943623.I08.gz43_516281M60421Human cytochrome P450scc gene, 5′ end2.80E−05
and promoter region
9953623.I11.gz43_516329AK013191Mus musculus 10, 11 days embryo3.00E−06
cDNA, RIKEN full-length enriched
library, clone: 2810429I04, full insert
sequence
9963623.L05.gz43_516236AJ131991Linum usitatissimum target sequence for3.00E−06
LIS-1 insertion in P1
9973623.L24.gz43_516540U09377Arabidopsis thaliana GF14chi isoform3.00E−06
(GRF1) gene, complete cds
9983623.M10.gz43_516317AF071743Homo sapiens topoisomerase II alpha4.00E−06
(TOP2A) gene, exons 25, 26, and 27
9993623.N23.gz43_516526U57489Eubacterium sp. VPI 12708 bile acid-3.70E−05
inducible operon bile acid-coenzyme A
ligase (baiB), BaiC, BaiD, bile acid 7-
alpha dehydratase (baiE), 3-alpha
hydroxysteroid dehydrogenase (baiA2),
BaiF, bile acid transporter (baiG),
NADH: flavin oxidoreductase (bai>
10003623.P22.gz43_516512U37761Human H1 histamine receptor gene, 5′-1.40E−12
flanking region
10013626.A10.gz43_516689D30745Xenopus laevis MRP RNA gene2.00E−07
10023626.C16.gz43_516787AF241271Bos taurus ZFY gene, intron1.60E−08
10033626.E07.gz43_516645AF053496Caenorhabditis elegans beta chain2.00E−06
spectrin homolog Sma1 (sma1) mRNA,
complete cds
10043626.F03.gz43_516582AJ009771Homo sapiens mRNA for putative RING2.00E−06
finger protein, partial
10053626.G01.gz43_516551BC010926Homo sapiens, Similar to H4 histone1.00E−43
family, member A, clone MGC: 13512
IMAGE: 4273904, mRNA, complete cds
10063626.I20.gz43_516857AK025762Homo sapiens cDNA: FLJ22109 fis,5.80E−07
clone HEP18091
10073626.I23.gz43_516905S55615(156) = G surface antigen {3′ region,3.40E−07
restriction fragment EG4} [Paramecium
primaurelia, Genomic, 407 nt]
10083626.M13.gz43_516749AE001398Plasmodium falciparum chromosome 2,4.00E−06
section 35 of 73 of the complete
sequence
10093626.M15.gz43_516781AF090925Homo sapiens clone HQ0452 PRO04523.10E−07
mRNA, partial cds
10103626.N07.gz43_516654Z58907H. sapiens CpG island DNA genomic2.90E−70
Mse1 fragment, clone 116a6, forward
read cpg116a6.ft1a
10113626.N24.gz43_516926AF041373Rattus norvegicus clathrin assembly8.90E−08
protein short form (CALM) mRNA,
complete cds
10123626.O08.gz43_516671D10445Mouse mRNA for protein C, complete5.00E−06
cds
10133626.P11.gz43_516720L48479Homo sapiens (subclone 6_h1 from P12.20E−07
H21) DNA sequence
10143626.P14.gz43_516768X15028Chicken hsp90 gene for 90 kDa-heat3.80E−05
shock protein 5′-end
10153629.A16.gz43_517169U16958Mus musculus pre-T cell receptor alpha-4.00E−06
type chain precursor mRNA, complete
cds
10163629.B14.gz43_517138X16982Drosophila melanogaster micropia-2.50E−07
Dm11 3′flanking DNA
10173629.C14.gz43_517139Z22537C. parvum precursor of oocyst wall5.00E−06
protein
10183629.E01.gz43_516933D00621Sus scrofa gene for follicle stimulation3.50E−05
hormone beta subunit, exons 1, 2, 3,
complete cds
10193629.E20.gz43_517237AE006900Sulfolobus solfataricus section 259 of9.00E−06
272 of the complete genome
10203629.F24.gz43_517302Y10531Clostridium perfringens sod gene for2.00E−06
superoxide dismutase
10213629.H10.gz43_517080J03654Human immunodeficiency virus type 2,8.00E−06
isolate HIV2FG
10223629.H12.gz43_517112AF017266Danio rerio glutamate decarboxylase6.50E−07
(GAD67) mRNA, partial cds
10233629.I11.gz43_517097AF020810Salmonella enterica VirK (virK), Mig-143.00E−06
(mig-14), NxiA (nxiA), TctE (tctE),
TctD (tctD), TctC (tctC), TctB (tctB),
and TctA (tctA) genes, complete cds;
and O360 (o360) gene, partial cds
10243629.I16.gz43_517177AE007643Clostridium acetobutylicum ATCC8244.40E−05
section 131 of 356 of the complete
genome
10253629.J03.gz43_516970AB017511Hydra magnipapillata mRNA for PLC-1.10E−05
betaH1, complete cds
10263629.J07.gz43_517034M20782Human alpha-2-plasmin inhibitor gene,2.90E−11
exons 2 to 5
10273632.C11.gz43_517475AF026148Perilla frutescens beta-ketoacyl-ACP1.00E−06
synthase I (KAS I) mRNA, complete cds
10283632.C17.gz43_517571U50534Human BRCA2 region, mRNA sequence1.00E−05
CG003
10293632.F07.gz43_517414M12036Human tyrosine kinase-type receptor4.70E−10
(HER2) gene, partial cds
10303632.G01.gz43_517319AC006621Caenorhabditis elegans cosmid C52A10,3.40E−05
complete sequence
10313632.I20.gz43_517625AK024381Homo sapiens cDNA FLJ14319 fis,9.00E−06
clone PLACE3000406
10323632.K20.gz43_517627M27634Vaccinia virus P4a major core protein9.60E−05
gene, complete cds
10333632.M08.gz43_517437X75304H. sapiens giantin mRNA8.00E−06
10343632.M13.gz43_517517U18191Human HLA class I genomic survey3.20E−07
sequence
10353632.M19.gz43_517613AF012131Homo sapiens brachyury variant B3.70E−07
(TBX1) mRNA, complete cds
10363632.N13.gz43_517518AF287491Oncorhynchus mykiss MHC class I2.00E−06
heavy chain precursor (Onmy-UBA)
mRNA, Onmy-UBA*601 allele,
complete cds
10373632.N21.gz43_517646X62423P. falciparum pol delta gene for DNA4.00E−06
polymerase delta
10383632.O06.gz43_517407BC009868Homo sapiens, replication protein A31.40E−18
(14 kD), clone MGC: 16404
IMAGE: 3940438, mRNA, complete cds
10393632.P07.gz43_517424AE001066Archaeoglobus fulgidus section 41 of3.00E−06
172 of the complete genome
10403635.A06.gz43_517777AK005546Mus musculus adult female placenta1.40E−07
cDNA, RIKEN full-length enriched
library, clone: 1600027G01, full insert
sequence
10413635.A08.gz43_517809Z49280S. cerevisiae chromosome X reading6.00E−06
frame ORF YJL005w
10423635.A13.gz43_517889AF143236Homo sapiens apoptosis related protein2.00E−06
APR-2 mRNA, complete cds
10433635.D07.gz43_517796M58150Bovine lactoperoxidase (LPO) mRNA,3.10E−05
complete cds
10443635.F01.gz43_517702Y19128Homo sapiens enteropeptidase gene,3.00E−09
exon 6
10453635.F06.gz43_517782X63073Pseudanabaena sp. cpeBA operon1.50E−05
encoding phycoerythrin beta and alpha
subunits
10463635.F10.gz43_517846AF107688Aedes aegypti clone 431 Feilai family of3.50E−05
SINES
10473635.H20.gz43_518008AE000613Helicobacter pylori 26695 section 91 of1.10E−05
134 of the complete genome
10483635.J06.gz43_517786U15018Dugbe virus L protein gene, complete1.10E−05
cds
10493635.J09.gz43_517834X85444G. pallida repetitive DNA element2.10E−08
10503635.K05.gz43_517771AF090432Danio rerio serrateB mRNA, complete4.00E−06
cds
10513635.K06.gz43_517787AJ276631Capsicum annuum partial kn gene for6.10E−07
Knolle protein, promoter region
10523635.M18.gz43_517981AL591498Human DNA sequence from clone1.40E−05
RP11-113L12 on chromosome 13,
complete sequence [Homo sapiens]
10533635.O01.gz43_517711AF081788Homo sapiens putative spliceosome3.70E−30
associated protein mRNA, complete cds
10543635.O14.gz43_517919X72224S. cerevisiae genes HSS1, NPL4 and HSP6.00E−06
10553635.P17.gz43_517968AF242307Euphorbia esula sucrose transport protein2.90E−10
mRNA, complete cds
10563635.P18.gz43_517984AF078780Caenorhabditis elegans cosmid C04F2,1.74E−04
complete sequence
10573638.A02.gz43_518097M17988Spiroplasma virus 4 (SpV4) replicative4.00E−06
form, complete genome
10583638.A24.gz43_518449AF064079Plasmodium gallinaceum endochitinase1.60E−07
precursor, mRNA, complete cds
10593638.F15.gz43_518310AJ297538Homo sapiens partial RARA gene, intron 24.00E−06
10603638.H07.gz43_518184AK026258Homo sapiens cDNA: FLJ22605 fis,2.00E−06
clone HSI04743
10613638.J09.gz43_518218U89651Homo sapiens matrix metalloproteinase8.10E−08
MMP Rasi-1 gene, promoter region
10623638.K06.gz43_518171AL139329Human DNA sequence from clone4.40E−11
RP11-228P1 on chromosome 6,
complete sequence [Homo sapiens]
10633638.L10.gz43_518236D26532Mouse mRNA for transcription factor2.00E−08
PEBP2aB2, complete cds
10643638.N05.gz43_518158X62294B. taurus mRNA for adrenal angiotensin9.00E−06
II type-1 receptor
10653643.D21.gz43_518788U17010Allomyces macrogynus mitochondrion1.80E−05
NADH dehydrogenase subunit 5 (nad5)
gene, complete cds
10663643.E24.gz43_518837AL022342Human DNA sequence from clone RP1-6.70E−05
29M10 on chromosome 20, complete
sequence [Homo sapiens]
10673643.F07.gz43_518566M73962Bovine pregnancy-associated6.00E−06
glycoprotein 1 mRNA, complete cds
10683643.G20.gz43_518775AF191214Homo sapiens isovaleryl dehydrogenase1.00E−05
(IVD) gene, exons 1-3
10693643.G24.gz43_518839AK025682Homo sapiens cDNA: FLJ22029 fis,6.00E−06
clone HEP08661
10703643.H09.gz43_518600AK024381Homo sapiens cDNA FLJ14319 fis,1.70E−05
clone PLACE3000406
10713643.I01.gz43_518473AF000306Brassica napus steroid sulfotransferase 23.00E−06
gene, complete cds
10723643.I02.gz43_518489X58433B. subtillis cad gene for lysine2.30E−05
decarboxylase
10733643.I18.gz43_518745M14872Mouse GnRH-GAP gene encoding4.00E−06
gonadotropin-releasing hormone and GnRH-
associated peptide (GAP)
10743643.I24.gz43_518841BC003813Mus musculus, clone MGC: 61392.30E−07
IMAGE: 3487295, mRNA, complete cds
10753643.K06.gz43_518555AL050124Homo sapiens mRNA; cDNA1.60E−07
DKFZp586E151 (from clone
DKFZp586E151)
10763643.L01.gz43_518476AJ278429Mus musculus partial Prkar1a gene for3.00E−06
cAMP-dependent protein kinase
regulatory subunit RIalpha, exons 8-10
and 3′UTR
10773643.N24.gz43_518846BC006511Homo sapiens, clone IMAGE: 3010441,1.00E−05
mRNA
10783643.O16.gz43_518719AE002303Chlamydia muridarum, section 34 of 851.10E−05
of the complete genome
10793643.O18.gz43_518751V00248Drosophila gene for yolk protein I2.00E−06
(vitellogenin)
10803643.O21.gz43_518799AE000614Helicobacter pylori 26695 section 92 of1.40E−05
134 of the complete genome
10813643.P13.gz43_518672Y17693Bungarus multicinctus gene encoding2.00E−07
alpha-bungarotoxin, V31 variant
10823643.P14.gz43_518688AF109352Euperipatoides rowelli microsatellite P188.80E−10
sequence
10833646.A07.gz43_518945X55137H. giganteus type II restriction-3.00E−06
modification system HgiBI
10843646.A09.gz43_518977AF074963Rattus norvegicus endothelin-B receptor2.10E−07
(EDNRB) gene, partial cds
10853646.A12.gz43_519025AF176208Homo sapiens EcoRI-HindIII fragment1.60E−05
upstream of exon 1 of the c-myc gene
10863646.A13.gz43_519041X89445O. chalybea DNA for narB gene and4.00E−05
partial ORFs
10873646.B20.gz43_519154M86514Rat proline-rich protein mRNA, 3′ end1.60E−05
10883646.C06.gz43_518931Z71180Caenorhabditis elegans cosmid F22E12,2.03E−04
complete sequence
10893646.C16.gz43_519091U73608Hepatitis B virus, genome 7648 with G-2.30E−05
>A hypermutations
10903646.E02.gz43_518869U11683Trypanoplasma borreli Tt-JH8.10E−07
mitochondrion cytochrome c oxidase
subunit 1 (cox1) gene, complete cds
10913646.E20.gz43_519157AE006216Pasteurella multocida PM70 section 1832.30E−05
of 204 of the complete genome
10923646.H04.gz43_518904AF043740Branchiostoma floridae amphioxus Otx2.00E−06
transcription factor (Otx) mRNA,
complete cds
10933646.H09.gz43_518984AP000145Homo sapiens genomic DNA,2.90E−40
chromosome 21q21.2, LL56-APP region,
clone B2291C14-R44F3, segment 10/10,
complete sequence
10943646.H16.gz43_519096U22342Bacteriophage T270 integrase (int) gene,1.00E−07
complete cds
10953646.I01.gz43_518857X54486Human gene for C1-inhibitor6.80E−05
10963646.J03.gz43_518890AB055372Macaca fascicularis brain cDNA,5.40E−190
clone: QflA-12842
10973646.J22.gz43_519194AL133032Homo sapiens mRNA; cDNA2.00E−06
DKFZp586B0317 (from clone
DKFZp586B0317)
10983646.K14.gz43_519067AF239178Paracoccidioides brasiliensis lon4.00E−06
proteinase gene, complete cds; nuclear
gene for mitochondrial product
10993646.L17.gz43_519116Z58907H. sapiens CpG island DNA genomic2.50E−70
Mse1 fragment, clone 116a6, forward
read cpg116a6.ft1a
11003646.O13.gz43_519055AL050391Homo sapiens mRNA; cDNA5.20E−08
DKFZp586A181 (from clone
DKFZp586A181); partial cds
11013646.O16.gz43_519103X00331Drosophila virilis simple DNA sequence5.20E−08
(pDV-161)
11023646.P09.gz43_518992U04527Borrelia burgdorferi 212 DNA gyrase b5.00E−06
subunit (gyrB) and ribonuclease P
protein component (rnpA) genes, partial
cds, DnaA protein (dnaA), DNA
polymerase III beta subunit (dnaN), and
ribosomal protein L34 (rpmH) genes,
complete cds
11033646.P14.gz43_519072AY032863Mus musculus chloride-formate8.00E−06
exchanger mRNA, complete cds
11043646.P17.gz43_519120U19569Human squamous cell carcinoma antigen1.20E−07
(SCCA2) gene, exon 1
11053661.A08.gz43_519483AB017511Hydra magnipapillata mRNA for PLC-1.20E−05
betaH1, complete cds
11063661.D17.gz43_519630J03488Reovirus type 3 L2 gene encoding3.00E−06
guanylyltransferase, complete cds
11073661.D18.gz43_519646AB033024Homo sapiens mRNA for KIAA11981.90E−11
protein, partial cds
11083661.E19.gz43_519663AB014084Homo sapiens genomic DNA,6.00E−05
chromosome 6p21.3, HLA class I region,
Cosmid clone: TY7A5, complete
sequence
11093661.E23.gz43_519727AE001032Archaeoglobus fulgidus section 75 of5.30E−05
172 of the complete genome
11103661.F14.gz43_519584X15063Plasmodium falciparum mRNA for6.80E−05
major merozoite surface antigen gp195
11113661.G16.gz43_519617AF255609Homo sapiens high mobility group2.70E−07
protein HMG1 gene, exons 1 and 2,
partial cds
11123661.G20.gz43_519681AK021558Homo sapiens cDNA FLJ11496 fis,6.40E−09
clone HEMBA1001964
11133661.H11.gz43_519538Z30705Puumala virus (Evo/15Cg/93) gene for N3.90E−07
protein
11143661.H24.gz43_519746X66979X. laevis mRNA XLFLI1.60E−05
11153661.I22.gz43_519715AF029887Caenorhabditis elegans UNC-129 (unc-5.00E−06
129) mRNA, complete cds
11163661.J15.gz43_519604AJ297538Homo sapiens partial RARA gene, intron 24.00E−06
11173661.K22.gz43_519717AK002100Homo sapiens cDNA FLJ11238 fis,1.30E−13
clone PLACE1008532
11183661.L19.gz43_519670AL589643Human DNA sequence from clone2.20E−05
RP11-344C1 on chromosome 6,
complete sequence [Homo sapiens]
11193661.M03.gz43_519415Z57613H. sapiens CpG island DNA genomic1.20E−08
Mse1 fragment, clone 187a12, forward
read cpg187a12.ft1a
11203661.M23.gz43_519735X79547Equus caballus mitochondrial DNA5.80E−05
complete sequence
11213661.P22.gz43_519722AF055668Mus musculus apoptosis-linked gene 4,8.00E−06
deltaC form (Alg-4) mRNA, partial cds
11223662.A13.gz43_519947Z49438S. cerevisiae chromosome X reading3.00E−06
frame ORF YJL163c
11233662.B13.gz43_519948AB045237Xenopus laevis XRPTPb mRNA for7.00E−06
receptor-type protein tyrosine
phosphatase beta.11, complete cds
11243662.C10.gz43_519901BC007905Homo sapiens, Similar to retinal1.20E−09
degeneration B beta, clone MGC: 14375
IMAGE: 4299595, mRNA, complete cds
11253662.C15.gz43_519981M33864Human (cline HGL-3) interstitial1.20E−05
retinoid-binding protein 3 (RBP3) gene,
exon 1
11263662.F13.gz43_519952AB040935Homo sapiens mRNA for KIAA15021.20E−61
protein, partial cds
11273662.H14.gz43_519970AB032757Mus musculus gad65 gene for glutamate8.00E−07
decarboxylase 65, partial cds
11283662.H23.gz43_520114AK013013Mus musculus 10, 11 days embryo2.00E−06
cDNA, RIKEN full-length enriched
library, clone: 2810406L04, full insert
sequence
11293662.H24.gz43_520130D45371Human apM1 mRNA for GS3109 (novel9.60E−10
adipose specific collagen-like factor),
complete cds
11303662.J05.gz43_519828M83554H. sapiens lymphocyte activation antigen1.40E−05
CD30 mRNA, complete cds
11313662.J08.gz43_519876Z11876B. hermsii vmp7 gene encoding Vmp71.11E−04
outer membrane lipoprotein
11323662.J09.gz43_519892AB011101Homo sapiens mRNA for KIAA05296.30E−05
protein, partial cds
11333662.J16.gz43_520004U00484Anabaena PCC7120 protein kinase PknA2.00E−06
(pknA) gene, complete cds
11343662.K03.gz43_519797AL390145Homo sapiens mRNA; cDNA1.40E−05
DKFZp762C115 (from clone
DKFZp762C115)
11353662.L05.gz43_519830U63635Schizosaccharomyces pombe RNA lariat5.80E−10
debranching enzyme (Sp-dbr1) gene,
complete cds
11363662.N24.gz43_520136Z30709L. helveticus genes for prolinase and3.70E−05
putative ABC transporter
11373662.O02.gz43_519785AF084460Gallus gallus potassium channel Shaker6.90E−05
alpha subunit variant cKv1.4(m)
mRNA, complete cds
11383662.P03.gz43_519802AJ011456Schinziella tetragona matK gene7.20E−08
(corresponding location in Tobacco: 963-1244)
11393663.A09.gz43_520267Z69608A. rara SSU rRNA gene (partial)3.30E−07
11403663.C08.gz43_520253Z50756Caenorhabditis elegans cosmid T08D10,7.60E−07
complete sequence
11413663.C19.gz43_520429Z22672H. sapiens cacnl1a3 gene encoding2.80E−07
skeletal muscle dhp-receptor alpha 1
subunit
11423663.E04.gz43_520191U89318Homo sapiens nucleophosmin2.60E−07
phosphoprotein (NPM) gene, intron 9,
partial sequence
11433663.F15.gz43_520368U66073Tritrichomonas foetus putative9.20E−07
superoxide dismutase 1 (SOD1) gene,
complete cds
11443663.F22.gz43_520480U36786Rattus norvegicus putative pheromone7.10E−07
receptor VN7 mRNA, complete cds
11453663.G01.gz43_520145AK024359Homo sapiens cDNA FLJ14297 fis,9.50E−36
clone PLACE1008941
11463663.G08.gz43_520257L19339Molgula oculata zinc finger protein5.20E−07
(manx) mRNA, complete cds
11473663.H20.gz43_520450X61307Staphylococcus aureus spa gene for5.00E−06
protein A
11483663.J06.gz43_520228AE007916Agrobacterium tumefaciens strain C582.02E−04
plasmid AT, section 44 of 50 of the
complete sequence
11493663.J16.gz43_520388U38181Leuconostoc mesenteroides3.90E−07
dextransucrase gene, complete cds
11503663.K02.gz43_520165X68339Mycoplasma-like organism (substrain5.00E−06
ASHY) DNA for 16S rRNA
11513663.K13.gz43_520341AF155221Mus musculus matrix metalloproteinase2.00E−06
19 (Mmp19) mRNA, complete cds
11523663.L18.gz43_520422AB031056Solobacterium moorei gene for 16S1.00E−06
rRNA, isolate: RCA59-74
11533663.L24.gz43_520518D10445Mouse mRNA for protein C, complete6.00E−06
cds
11543663.M24.gz43_520519AE001196Treponema pallidum section 12 of 87 of5.20E−05
the complete genome
11553663.N09.gz43_520280AF081788Homo sapiens putative spliceosome4.00E−20
associated protein mRNA, complete cds
11563663.N10.gz43_520296X62423P. falciparum pol delta gene for DNA4.00E−06
polymerase delta
11573663.N12.gz43_520328AF178079Zygosaccharomyces rouxii ketoreductase5.00E−06
(krd) mRNA, complete cds
11583663.N16.gz43_520392U41060Homo sapiens estrogen regulated LIV-12.00E−06
protein (LIV-1) mRNA, complete cds
11593663.O07.gz43_520249D00442Grapevine fanleaf virus satellite RNA1.50E−08
(RNA3), complete cds
11603663.O09.gz43_520281AK002141Homo sapiens cDNA FLJ11279 fis,5.30E−10
clone PLACE1009444, highly similar to
PHOSPHATIDYLINOSITOL 4-
KINASE ALPHA (EC 2.7.1.67)
11613664.A11.gz43_520683U67525Methanococcus jannaschii section 67 of4.00E−06
150 of the complete genome
11623664.C21.gz43_520845AF064773Staphylococcus aureus extracellular1.30E−07
enterotoxin type G precursor (SEG)
gene, complete cds
11633664.D06.gz43_520606AF178079Zygosaccharomyces rouxii ketoreductase5.00E−06
(krd) mRNA, complete cds
11643664.D12.gz43_520702U10519Human DNA polymerase beta gene,2.00E−07
exon 5
11653664.D17.gz43_520782AK027226Homo sapiens cDNA: FLJ23573 fis,4.90E−07
clone LNG12520
11663664.E18.gz43_520799AF317204Mus musculus C-type lectin superfamily3.20E−05
1 gene, complete cds
11673664.E23.gz43_520879AB050903Mus musculus mRNA for a4 subunit3.00E−06
isoform, complete cds
11683664.E24.gz43_520895Z92793Caenorhabditis elegans cosmid H15M21,1.20E−05
complete sequence
11693664.G12.gz43_520705AF211482Dictyostelium discoideum SdhA (sdhA)2.30E−09
gene, complete cds
11703664.G20.gz43_520833M14450Rat thyrotropin (TSH) beta-subunit gene,4.00E−06
exons 2 and 3
11713664.H15.gz43_520754Y11270E. histolytica INO1 gene2.00E−06
11723664.H22.gz43_520866X97773B. taurus mRNA for mitochondrial1.20E−05
tricarboxylate carrier protein
11733664.J12.gz43_520708M58150Bovine lactoperoxidase (LPO) mRNA,3.20E−05
complete cds
11743664.J23.gz43_520884U67463Methanococcus jannaschii section 5 of3.00E−06
150 of the complete genome
11753664.K16.gz43_520773Z83118Caenorhabditis elegans cosmid M04D5,2.70E−07
complete sequence
11763664.K19.gz43_520821U36927Plasmodium yoelii rhoptry protein gene,3.00E−05
complete cds
11773664.L21.gz43_520854AF057695Haemophilus ducreyi strain 350002.15E−04
putative phosphomannomutase (pmm)
gene, partial cds; large supernatant
protein 1 (lspA1) gene, complete cds;
and putative GMP synthase (guaA) gene,
partial cds
11783664.O22.gz43_520873U43574Hydra vulgaris nucleoporin p62 gene,7.00E−06
complete cds
11793664.P12.gz43_520714AF030883Mus musculus tRNA-His gene, complete9.00E−06
sequence; platelet-activating factor
acetylhydrolase Ib alpha subunit (Pafaha-
psl) pseudogene, complete sequence;
and tRNA-Glu gene, complete sequence
11803664.P18.gz43_520810Z47735H. sapiens NFKB1 gene, exons 11 & 121.32E−04
11813665.A23.gz43_521259X66979X. laevis mRNA XLFLI1.60E−05
11823665.B01.gz43_520908M90058Human serglycin gene, exons 1, 2, and 34.00E−06
11833665.B12.gz43_521084AK020877Mus musculus adult retina cDNA,7.10E−07
RIKEN full-length enriched library,
clone: A930019H03, full insert sequence
11843665.E11.gz43_521071AB024030Arabidopsis thaliana genomic DNA,9.00E−06
chromosome 5, TAC clone: K5A21
11853665.E20.gz43_521215X76584H. sapiens simple DNA sequence region6.80E−08
clone wg1h1
11863665.H20.gz43_521218X95301D. rerio mRNA for HER-5 protein9.50E−07
11873665.K01.gz43_520917X04653Mouse mRNA for Ly-6 alloantigen (Ly-1.30E−05
6E.1)
11883665.M01.gz43_520919AF098352Wiseana copularis haplotype southern5.80E−07
cytochrome oxidase subunit I and
cytochrome oxidase subunit II genes,
partial cds; mitochondrial genes for
mitochondrial products
11893665.M21.gz43_521239AF257480Rana temporaria microsatellite SB803.30E−09
sequence
11903665.M23.gz43_521271Y10623C. pallidivittatus globin gene cluster E1.10E−05
11913665.N24.gz43_521288X95301D. rerio mRNA for HER-5 protein1.00E−06
11923665.O06.gz43_521001AE007033Mycobacterium tuberculosis CDC1551,7.40E−05
section 119 of 280 of the complete
genome
11933665.O14.gz43_521129AB033094Homo sapiens mRNA for KIAA12682.10E−08
protein, partial cds
11943665.O15.gz43_521145AK004557Mus musculus adult male lung cDNA,1.20E−05
RIKEN full-length enriched library,
clone: 1200003C23, full insert sequence
11953665.O19.gz43_521209AY036905Trichoderma atroviride protein GTPase2.10E−08
Tga1 (tga1) gene, complete cds
11963665.O21.gz43_521241U89293Homo sapiens MSH4 (HMSH4) mRNA,1.20E−39
complete cds
11973665.O23.gz43_521273X00048Herpes simplex virus (HSV) type 26.00E−06
transforming region mtr-2 (map
coordinates 0.580-0.625)
11983665.P13.gz43_521114Z48796H. sapiens Ski-W mRNA for helicase1.70E−05
11993666.A07.gz43_521387AK005546Mus musculus adult female placenta1.20E−07
cDNA, RIKEN full-length enriched
library, clone: 1600027G01, full insert
sequence
12003666.A19.gz43_521579AB011101Homo sapiens mRNA for KIAA05295.80E−05
protein, partial cds
12013666.A24.gz43_521659AL050208Homo sapiens mRNA; cDNA2.90E−07
DKFZp586F2323 (from clone
DKFZp586F2323)
12023666.B11.gz43_521452X06932Petunia hsp70 gene3.00E−06
12033666.C18.gz43_521565Z22672H. sapiens cacnl1a3 gene encoding2.80E−07
skeletal muscle dhp-receptor alpha 1
subunit
12043666.D02.gz43_521310AJ297538Homo sapiens partial RARA gene, intron 24.00E−06
12053666.D11.gz43_521454AF057695Haemophilus ducreyi strain 350002.43E−04
putative phosphomannomutase (pmm)
gene, partial cds; large supernatant
protein 1 (lspA1) gene, complete cds;
and putative GMP synthase (guaA) gene,
partial cds
12063666.D15.gz43_521518Z66194H. sapiens CpG island DNA genomic1.70E−66
Mse1 fragment, clone 80b12, forward
read cpg80b12.ft1b
12073666.D16.gz43_521534Z66194H. sapiens CpG island DNA genomic2.10E−37
Mse1 fragment, clone 80b12, forward
read cpg80b12.ft1b
12083666.F22.gz43_521632U97062Staphylococcus aureus NCTC 83251.20E−08
SecA (secA) gene, complete cds
12093666.G12.gz43_521473J03901Maize pyruvate, orthophosphate dikinase1.72E−04
mRNA, complete cds
12103666.I12.gz43_521475AJ225102Pinus lambertiana chloroplast DNA6.40E−10
containing a SSR Black Hills (Oregon)
12113666.L01.gz43_521302M86227Staphylococcus aureus DNA gyrase B5.00E−06
subunit (gyrB) RecF homologue (recF)
and DNA gyrase A subunit (gyrA) gene,
complete cds
12123666.L06.gz43_521382AF224725Trichosurus vulpecula retrovirus TvERV3.30E−08
(type D) gag polyprotein (gag), protease
(pro), and pol polyprotein (pol) genes,
complete cds
12133666.L11.gz43_521462AF147081Homo sapiens gamma-glutamyl3.30E−05
hydrolase gene, exons 1 and 2
12143666.L23.gz43_521654AK020701Mus musculus 6 days neonate skin2.20E−07
cDNA, RIKEN full-length enriched
library, clone: A030009B12, full insert
sequence
12153666.M16.gz43_521543AF158179Drosophila melanogaster strain Canton-S4.40E−07
Chiffon-2 (chiffon) mRNA, alternative
splice form 2, complete cds
12163666.N06.gz43_521384Z48796H. sapiens Ski-W mRNA for helicase1.70E−05
12173667.A15.gz43_524557AF005903Monodelphis domestica GTP-binding7.80E−08
protein homolog mRNA, partial cds
12183754.A08.gz43_532949AF091502Lactobacillus reuteri autoaggregation-1.00E−06
mediating protein (aggH) gene, complete
cds
12193754.A13.gz43_533029U02695Protomelas similis clone PsiI 32 SATA7.60E−07
satellite DNA sequence
12203754.A16.gz43_533077AE006577Streptococcus pyogenes M1 GAS strain9.00E−06
SF370, section 106 of 167 of the
complete genome
12213754.B04.gz43_532886S83995Pst1 fragment [Chlamydia pneumoniae,2.00E−06
Genomic, 474 nt]
12223754.B05.gz43_532902AY008833Staphylococcus aureus tcaR-tcaA-tcaB5.00E−06
operon, complete sequences
12233754.B07.gz43_532934AF270216Staphylococcus epidermidis strain SR19.50E−07
clone step.1054h11 genomic sequence
12243754.B08.gz43_532950AK007308Mus musculus adult male testis cDNA,7.00E−06
RIKEN full-length enriched library,
clone: 1700128E15, full insert sequence
12253754.B10.gz43_532982AE002807Drosophila melanogaster genomic5.40E−05
scaffold 142000013385251, complete
sequence
12263754.C22.gz43_533175D30612Homo sapiens mRNA for repressor4.00E−06
protein, partial cds
12273754.D19.gz43_533128L12043Plasmodium falciparum unidentified3.00E−06
mRNA sequence
12283754.E12.gz43_533017AB062933Macaca fascicularis brain cDNA3.60E−07
clone: QccE-22249, full insert sequence
12293754.E20.gz43_533145AL138746Human DNA sequence from clone RP3-8.30E−10
389B13 on chromosome Xq26.2-27.1,
complete sequence [Homo sapiens]
12303754.F01.gz43_532842AF086820Drosophila melanogaster paired-like8.00E−06
homeodomain protein UNC-4 (unc-4)
mRNA, complete cds
12313754.F08.gz43_532954S66402vascular AT1a angiotensin receptor3.10E−05
{exon 1, promoter} [rats, Sprague-
Dawley, Genomic, 3477 nt]
12323754.F11.gz43_533002X57377Mouse dilute myosin heavy chain gene2.10E−05
for novel heavy chain with unique C-
terminal region
12333754.F15.gz43_533066AJ245620Homo sapiens CTL1 gene2.50E−12
12343754.F20.gz43_533146AE002426Neisseria meningitidis serogroup B strain3.70E−05
MC58 section 68 of 206 of the complete
genome
12353754.G03.gz43_532875AF002166Xenopus laevis Ig mu heavy chain1.20E−07
switch region sequence
12363754.G08.gz43_532955X71020N. tabacum Npg1 gene for6.80E−07
polygalacturonase
12373754.G18.gz43_533115AF126531Homo sapiens putative DNA-directed1.10E−13
RNA polymerase III C11 subunit gene,
complete cds
12383754.H08.gz43_532956L20127Rochalimaea henselae antigen (htrA)4.60E−07
gene, complete cds
12393754.I01.gz43_532845AK022138Homo sapiens cDNA FLJ12076 fis,3.90E−14
clone HEMBB1002442, weakly similar
to LIN-10 PROTEIN
12403754.I03.gz43_532877AF016653Caenorhabditis elegans cosmid C41D7,2.00E−06
complete sequence
12413754.J01.gz43_532846U97408Caenorhabditis elegans cosmid F48A94.00E−06
12423754.J05.gz43_532910Z35484Thermoanaerobacter sp. ATCC536274.00E−06
cgtA gene
12433754.J10.gz43_532990D17094Human HepG2 partial cDNA, clone5.10E−11
hmd5h04m5
12443754.J12.gz43_533022Z56695H. sapiens CpG island DNA genomic1.00E−06
Mse1 fragment, clone 136d4, reverse
read cpg136d4.rt1a
12453754.J24.gz43_533214Y12855Homo sapiens P2X7 gene, exon 12 and2.50E−05
13
12463754.K14.gz43_533055L79913Xenopus laevis rds/peripherin (rds35)5.00E−06
mRNA, complete cds
12473754.K17.gz43_533103AE006251Lactococcus lactis subsp. lactis IL14039.00E−06
section 13 of 218 of the complete
genome
12483754.K20.gz43_533151AB047880Macaca fascicularis brain cDNA,1.00E−06
clone: QnpA-14303
12493754.M08.gz43_532961X58467Human CYP2D7AP pseudogene for4.30E−11
cytochrome P450 2D6
12503754.N16.gz43_533090U33116Saccharomyces cerevisiae high copy1.80E−07
DNA polymerase suppressor alpha
mutation gene (PSP2), complete cds
12513754.N19.gz43_533138AK025312Homo sapiens cDNA: FLJ21659 fis,1.40E−07
clone COL08743
12523754.N22.gz43_533186AF081828Ixodes hexagonus mitochondrial DNA,4.00E−06
complete genome
12533754.O18.gz43_533123Z73229S. cerevisiae chromosome XII reading3.00E−06
frame ORF YLR057w
12543754.O23.gz43_533203AE006900Sulfolobus solfataricus section 259 of1.10E−05
272 of the complete genome
12553754.P13.gz43_533044AF220217Homo sapiens rsec15-like protein1.80E−10
mRNA, partial cds
12563754.P17.gz43_533108AJ250862Bacillus sp. HIL-Y85/54728 mersacidin1.20E−05
biosynthesis gene cluster (mrsK2,
mrsR2, mrsF, mrsG, mrsE, mrsA,
mrsR1, mrsD, mrsM and mrsT genes)
12573756.A02.gz43_533237AF285594Homo sapiens testis protein TEX111.10E−05
(TEX11) mRNA, complete cds
12583756.A11.gz43_533381U43148Human patched homolog (PTC) mRNA,4.00E−06
complete cds
12593756.A13.gz43_533413U56861Nicotiana plumbaginifolia intergenic1.00E−06
region between lhcb1*1 and lhcb1*2
genes
12603756.B03.gz43_533254AF101735Pan troglodytes isolate PTOR3A5P5.70E−08
olfactory receptor pseudogene, complete
sequence
12613756.B04.gz43_533270Z82038C. thermosaccharolyticum etfB, etfA,1.00E−06
hbd, thlA and actA genes
12623756.B15.gz43_533446M96151Mus musculus apolipoprotein B gene1.13E−04
sequence
12633756.B21.gz43_533542Z92793Caenorhabditis elegans cosmid H15M21,1.30E−05
complete sequence
12643756.B22.gz43_533558U43542Nicotiana tabacum diphenol oxidase2.00E−06
mRNA, complete cds
12653756.C06.gz43_533303AB022085Mus musculus Cctz-2 gene for7.00E−05
chaperonin containing TCP-1 zeta-2
subunit, exon 5, 6, 7, 8, 9, 10
12663756.C16.gz43_533463AF143236Homo sapiens apoptosis related protein5.00E−06
APR-2 mRNA, complete cds
12673756.D08.gz43_533336AB049544Porcine enterovirus 10 gene for RNA-7.20E−07
dependent RNA polymerase, partial cds
12683756.D18.gz43_533496X53658E. coli DNA fragment7.60E−08
12693756.D24.gz43_533592X96861H. virescens mRNA for pheromone2.40E−07
binding protein
12703756.E01.gz43_533225AF202892Mus musculus Kif21a (Kif21a) mRNA,4.00E−06
complete cds
12713756.E06.gz43_533305AF139374Homo sapiens DIR1 protein (DIR1)8.00E−06
gene, complete cds
12723756.E12.gz43_533401AF238884Botrytis virus F, complete genome8.00E−06
12733756.E22.gz43_533561U78866Arabidopsis thaliana putative arginine-5.00E−06
aspartate-rich RNA binding protein
(gene1500), (gene1000), and (gene400)
genes, complete cds
12743756.F11.gz43_533386D50091Drosophila ezoana G-3-P dehydrogenase2.00E−06
(alphaGpdh) gene, exon1-8, complete
cds
12753756.F16.gz43_533466AJ233973Gallus gallus microsatellite DNA4.20E−07
GCT028 (CA) repeat
12763756.G07.gz43_533323AE000708Aquifex aeolicus section 40 of 109 of the6.00E−05
complete genome
12773756.G12.gz43_533403M84731Pseudomonas sp. 5-substituted hydantoin1.20E−05
racemase (hyuE) gene, complete cds
12783756.G14.gz43_533435AL116458Botrytis cinerea strain T4 cDNA library6.70E−07
under conditions of nitrogen deprivation
12793756.I03.gz43_533261U67550Methanococcus jannaschii section 92 of2.30E−05
150 of the complete genome
12803756.J05.gz43_533294U11292Human Ki nuclear autoantigen mRNA,7.70E−07
complete cds
12813756.K03.gz43_533263AF073484Homo sapiens MHC class I-related8.00E−06
protein MR1 precursor (MR1) gene,
signal peptide
12823756.K07.gz43_533327M37499Human methylmalonyl CoA mutase2.00E−06
(MUT) gene, exon 2
12833756.K15.gz43_533455AF248820Maoricicada campbelli isolate TB-MC-7.30E−07
016 tRNA-Asp gene, complete sequence;
ATPase subunit 8 gene, complete cds;
and ATPase subunit 6 gene, partial cds;
mitochondrial genes for mitochondrial
products
12843756.K18.gz43_533503M36300S. cerevisiae glutamine amidotransferase2.30E−05
(TRP3) gene, 3′ end
12853756.K20.gz43_533535AY022480Oryza sativa microsatellite MRG48052.00E−10
containing (AGG)X8, genomic sequence
12863756.L02.gz43_533248X03833Human gene for interleukin 1 alpha (IL-12.80E−12
alpha)
12873756.L03.gz43_533264AF244246Dysdera sp. MC cytochrome c oxidase I2.70E−07
(COI) gene, partial cds; mitochondrial
gene for mitochondrial product
12883756.L19.gz43_533520AJ002732Schizosaccharomyces pombe mRNA for2.00E−06
ribosomal protein 114
12893756.M06.gz43_533313AK002951Mus musculus adult male brain cDNA,3.60E−07
RIKEN full-length enriched library,
clone: 0710001E20, full insert sequence
12903756.M07.gz43_533329AF057708Populus balsamifera subsp. trichocarpa2.60E−07
PTD protein (PTD) gene, complete cds
12913756.M20.gz43_533537Z35821S. cerevisiae chromosome II reading2.00E−06
frame ORF YBL060w
12923756.N18.gz43_533506AL591667Human DNA sequence from clone6.10E−05
RP11-389N9 on chromosome 6,
complete sequence [Homo sapiens]
12933756.N21.gz43_533554AK026258Homo sapiens cDNA: FLJ22605 fis,2.00E−06
clone HSI04743
12943756.O03.gz43_533267U61347Leiophyllum buxifolium ribosomal4.20E−07
maturase (matK) gene, chloroplast gene
encoding chloroplast protein, complete
cds
12953756.O07.gz43_533331AF177871Drosophila melanogaster small GTPase5.70E−07
RHO1 (Rho1) gene, alternatively spliced
products and complete cds
12963756.O08.gz43_533347M60705Homo sapiens type I DNA6.00E−06
topoisomerase gene, exons 19 and 20
12973756.P08.gz43_533348M60705Homo sapiens type I DNA1.00E−05
topoisomerase gene, exons 19 and 20
12983759.C01.gz43_533607X71874H. sapiens genes for proteasome-like4.00E−06
subunit (MECL-1), chymotrypsin-like
protease (CTRL-1) and protein serine
kinase (PSK-H1) last exon
12993759.D15.gz43_533832AL356790Human DNA sequence from clone1.10E−07
RP11-238J15 on chromosome 20
Contains ESTs and GSSs. Contains part
of the TOM gene for a putative
mitochondrial outer membrane protein
import receptor similar to yeast pre-
mRNA splicing factors Prp1/Zer1 and
Prp6, complete>
13003759.H08.gz43_533724M31684D. melanogaster cytoskeleton-like2.00E−06
bicaudalD protein (BicD) mRNA,
complete cds
13013759.H15.gz43_533836AB046001Macaca fascicularis brain cDNA,2.60E−07
clone: QccE-12738
13023759.H17.gz43_533868AE000706Aquifex aeolicus section 38 of 109 of the1.30E−05
complete genome
13033759.H23.gz43_533964AK027088Homo sapiens cDNA: FLJ23435 fis,6.20E−34
clone HRC12631
13043759.I05.gz43_533677AF056433Homo sapiens clone FBD3 Cri-du-chat1.70E−07
critical region mRNA
13053759.I19.gz43_533901Z69666Human DNA sequence from cosmid2.06E−04
24F8 from a contig from the tip of the
short arm of chromosome 16, spanning
2 Mb of 16p13.3. Contains ESTs, repeat
polymorphism and CpG island
13063759.K05.gz43_533679L01432Soybean calmodulin (SCaM-3) mRNA,4.10E−08
complete cds
13073759.K17.gz43_533871Z33340M. capricolum DNA for CONTIG4.00E−06
MC456
13083759.L02.gz43_533632U26736Caenorhabditis elegans stomatin-like3.70E−05
protein MEC-2 (mec-2) gene, complete
cds
13093759.L09.gz43_533744M11180Transposon Tn917 (complete),1.50E−07
macrolide-lincosamide-streptogramin-B
(MLS) resistance, complete cds
13103759.L10.gz43_533760AF117022Solaria atropurpurea trnL gene, partial4.40E−07
sequence; chloroplast gene for
chloroplast product
13113759.L15.gz43_533840U22657Mus musculus genomic locus related to1.60E−05
cellular morphology
13123759.L24.gz43_533984AK022990Homo sapiens cDNA FLJ12928 fis,7.60E−10
clone NT2RP2004767
13133759.M19.gz43_533905M96324Lycopersicon esculentum Ca2+-ATPase2.50E−05
gene, complete cds
13143759.N08.gz43_533730AK005546Mus musculus adult female placenta1.30E−07
cDNA, RIKEN full-length enriched
library, clone: 1600027G01, full insert
sequence
13153759.N16.gz43_533858AB014079Homo sapiens genomic DNA,3.80E−12
chromosome 6p21.3, HLA class I region,
Cosmid clone: TY1E11, complete
sequence
13163759.N23.gz43_533970AK018377Mus musculus 16 days embryo lung5.70E−07
cDNA, RIKEN full-length enriched
library, clone: 8430403M08, full insert
sequence
13173759.O16.gz43_533859AE000918Methanobacterium thermoautotrophicum1.40E−05
from bases 1444576 to 1460617 (section
124 of 148) of the complete genome
13183759.P03.gz43_533652L06066Saccharomyces cerevisiae PET1175.90E−07
polypeptide (PET117) gene, complete
cds
13193759.P13.gz43_533812X89414A. thaliana DNA for pyrroline-5-5.00E−06
carboxylase synthetase gene
13203759.P15.gz43_533844X66979X. laevis mRNA XLFLI1.50E−05
13213759.P17.gz43_533876AF039313Moraxella catarrhalis strain LES-12.00E−06
transferrin binding protein B (tbpB)
gene, complete cds
13223762.A09.gz43_534117AE000496Escherichia coli K12 MG1655 section1.63E−04
386 of 400 of the complete genome
13233762.A16.gz43_534229X98371D. subobscura sex-lethal gene7.00E−06
13243762.A19.gz43_534277U95019Human voltage-dependent calcium6.10E−07
channel beta-2c subunit mRNA,
complete cds
13253762.A20.gz43_534293M10014Homo sapiens map 4q28 fibrinogen8.00E−06
(FGG) gene, alternative splice products,
complete cds
13263762.B05.gz43_534054J05614Human proliferating cell nuclear antigen1.40E−05
(PCNA) gene, promoter region
13273762.B15.gz43_534214AJ297559Homo sapiens partial PIK3CB gene for2.50E−05
phosphatidylinositol 3-kinase catalytic
subunit p110beta, exons 15-17
13283762.C20.gz43_534295M58580Rabbit angiotensin-converting enzyme3.10E−05
(ACE) gene, 5′ end
13293762.C23.gz43_534343L27146Human neurofibromatosis 2 (NF2) gene,1.00E−06
exon 16
13303762.D03.gz43_534024U51305Triticum aestivum alpha-gliadin storage1.40E−05
protein pseudogene, complete cds
13313762.D04.gz43_534040AF263274Chionodraco rastrospinosus isolate Cra73.50E−07
alpha tubulin mRNA, complete cds
13323762.D18.gz43_534264M94764Glycine max cv. Dare nodulin 26 gene2.50E−05
fragment
13333762.D19.gz43_534280AE001446Helicobacter pylori, strain J99 section 73.30E−05
of 132 of the complete genome
13343762.D22.gz43_534328M73962Bovine pregnancy-associated4.00E−06
glycoprotein 1 mRNA, complete cds
13353762.E01.gz43_533993X63746S. cerevisiae rpc34 and fun34 genes for4.00E−06
DNA dependant RNA polymerase c (III)
13363762.E10.gz43_534137Z74847S. cerevisiae chromosome XV reading1.00E−05
frame ORF YOL105c
13373762.E15.gz43_534217AF207841Pyricularia grisea AVR-Pita (AVR-Pita)2.20E−09
gene, complete cds
13383762.E23.gz43_534345M58600Human heparin cofactor II (HCF2) gene,3.60E−37
exons 1 through 5
13393762.F08.gz43_534106Z47066Human cosmid Qc14G3 from Xq283.10E−09
contains STSs
13403762.F22.gz43_534330AY034974Arabidopsis thaliana unknown protein4.20E−07
(F24J8.3) mRNA, complete cds
13413762.G18.gz43_534267Z28150S. cerevisiae chromosome XI reading2.00E−06
frame ORF YKL150w
13423762.H12.gz43_534172AF370230Arabidopsis thaliana unknown protein6.60E−08
(T21P5_16/AT3g03420) mRNA,
complete cds
13433762.I07.gz43_534093U19569Human squamous cell carcinoma antigen4.60E−07
(SCCA2) gene, exon 1
13443762.J03.gz43_534030U22421Mus musculus obesity protein (ob) gene,5.30E−07
complete cds
13453762.J18.gz43_534270AB027966Schizosaccharomyces pombe gene for2.30E−08
Hypothetical protein, partial cds,
clone: TB89
13463762.K02.gz43_534015AF273762Homo sapiens 3-hydroxy-3-4.40E−14
methylglutaryl-coenzyme reductase
gene, exon 15
13473762.K20.gz43_534303K01464Rat cardiac alpha-myosin heavy chain3.00E−06
gene, 5′ flank, 1st 3 exons
13483762.L18.gz43_534272Z49438S. cerevisiae chromosome X reading4.00E−06
frame ORF YJL163c
13493762.L20.gz43_534304XM_030040Homo sapiens similar to KIAA08773.00E−06
protein (H. sapiens) (LOC90219),
mRNA
13503762.M04.gz43_534049AF002237Anopheles gambiae clone 227 mRNA4.00E−06
sequence
13513762.M17.gz43_534257M29688S. cerevisiae PMS1 gene encoding DNA1.40E−08
mismatch repair protein, complete cds
13523762.M23.gz43_534353M20006Chicken tumor 10 c-myc DNA, exons 22.90E−09
and 3
1353Clu1014734.con_1AB027966Schizosaccharomyces pombe gene for3.00E−08
Hypothetical protein, partial cds,
clone: TB89
1354Clu1036845.con_1M34429Human PVT-IGLC fusion protein1.37E−03
mRNA, 5′ end
|
Example 21
Members of Protein Families
SEQ ID NOS:134-1352 were used to conduct a profile search as described in the specification above. Several of the polynucleotides of the invention were found to encode polypeptides having characteristics of a polypeptide belonging to a known protein family (and thus represent members of these protein families) and/or comprising a known functional domain. Table 18 (inserted prior to claims) provides: 1) the SEQ ID NO (“SEQ ID”) of the query polynucleotide sequence; 2) the sequence name (“SEQ NAME”) used as an internal identifier of the query-sequence; 3) the name (“PFAM NAME”) of the profile hit; 4) a brief description of the profile hit (“PFAM DESCRIPTION”); 5) the score (“SCORE”) of the profile hit; 6) the starting nucleotide of the profile hit (“START”); and 7) the ending nucleotide of the profile hit (“END”).
TABLE 18
|
|
SEQ
IDSEQ NAMEPFAM NAMEPFAM DESCRIPTIONSCORESTARTEND
|
|
2223547.D19.GZ43_505986DC1DC1 domain30.64411493
2703550.G02.GZ43_506101rvtReverse transcriptase (RNA-47.32321611
dependent DNA polymerase)
4543562.B22.GZ43_5079527tm_17 transmembrane receptor37.16154479
(rhodopsin family)
4543562.B22.GZ43_507952Bowman-Bowman-Birk serine45.92292450
Birk_legprotease inhibitor family
4543562.B22.GZ43_507952Cation_effluxCation efflux family33.32225380
4913565.E16.GZ43_508243AP_endonucleas1AP endonuclease family 138.16406577
5463571.A08.GZ43_508897oxidored_q1NADH-30.04297393
Ubiquinone/plastoquinone
(complex I), various chains
5503571.B13.GZ43_508978EGFEGF-like domain38.88243355
5513571.B22.GZ43_509122EGFEGF-like domain38.88243355
5643571.H10.GZ43_508936WWWW domain54.92487576
7243583.H13.GZ43_510520SreC. elegans Sre G protein-30.36282485
coupled chemoreceptor
7713590.J21.GZ43_512427bZIPbZIP transcription factor33.68166308
7783590.M03.GZ43_512142protamine_P1Protamine P135.88268437
9073608.L14.gz43_514237Transposase_22L1 transposable element62.12491616
9693617.P12.gz43_515361AP_endonucleas1AP endonuclease family 139.8463254
10383632.O06.gz43_51740760s_ribosomal60s Acidic ribosomal protein38.04276444
10383632.O06.gz43_51740760s_ribosomal60s Acidic ribosomal protein36.441398
11283662.H23.gz43_520114Glycoprotein_GPneumovirus attachment43.0421297
glycoprotein G
11283662.H23.gz43_520114Metallothio_5Metallothionein family 547.88231345
11283662.H23.gz43_520114squashSquash family serine34.6222301
protease inhibitor
11283662.H23.gz43_520114SyndecanSyndecan domain35.361308
11453663.G01.gz43_520145KRABKRAB box95.08424484
13503762.M04.gz43_534049protamine_P1Protamine P133.16293468
|
In addition, SEQ ID NOS:1619-1675 were also used to conduct a profile search as described above. Several of the polypeptides of the invention were found to have characteristics of a polypeptide belonging to a known protein family (and thus represent members of these protein families) and/or comprising a known functional domain. Table 19 (inserted prior to claims) provides: 1) the SEQ ID NO (“SEQ ID”) of the query protein sequence; 2) the sequence name (“PROTEIN SEQ NAME”) used as an internal identifier of the query sequence; 3) the name (“PFAM NAME”) of the profile hit; 4) a brief description of the profile hit (“PFAM DESCRIPTION”); 5) the score (“SCORE”) of the profile hit; 6) the starting residue of the profile hit (“START”); and 7) the ending residue of the profile hit (“END”).
TABLE 19
|
|
SEQPFAMPFAM
IDSEQ NAMENAMEDESCRIPTIONSCORESTARTEND
|
|
1619NTP_004511S11.3_4Armadillo_segArmadillo/beta-1.8E−95142184
catenin-like repeat
1619NTP_004511S11.3_4Armadillo_segArmadillo/beta-1.8E−95186226
catenin-like repeat
1619NTP_004511S11.3_4Armadillo_segArmadillo/beta-1.8E−95228269
catenin-like repeat
1619NTP_004511S11.3_4Armadillo_segArmadillo/beta-1.8E−95271311
catenin-like repeat
1619NTP_004511S11.3_4Armadillo_segArmadillo/beta-1.8E−95313353
catenin-like repeat
1619NTP_004511S11.3_4Armadillo_segArmadillo/beta-1.8E−95355395
catenin-like repeat
1619NTP_004511S11.3_4Armadillo_segArmadillo/beta-1.8E−95397437
catenin-like repeat
1619NTP_004511S11.3_4Armadillo_segArmadillo/beta-1.8E−95440480
catenin-like repeat
1619NTP_004511S11.3_4Armadillo_segArmadillo/beta-1.8E−95142184
catenin-like repeat
1619NTP_004511S11.3_4Armadillo_segArmadillo/beta-1.8E−95186226
catenin-like repeat
1619NTP_004511S11.3_4Armadillo_segArmadillo/beta-1.8E−95228269
catenin-like repeat
1619NTP_004511S11.3_4Armadillo_segArmadillo/beta-1.8E−95271311
catenin-like repeat
1619NTP_004511S11.3_4Armadillo_segArmadillo/beta-1.8E−95313353
catenin-like repeat
1619NTP_004511S11.3_4Armadillo_segArmadillo/beta-1.8E−95355395
catenin-like repeat
1619NTP_004511S11.3_4Armadillo_segArmadillo/beta-1.8E−95397437
catenin-like repeat
1619NTP_004511S11.3_4Armadillo_segArmadillo/beta-1.8E−95440480
catenin-like repeat
1619NTP_004511S11.3_4IBBImportin beta binding5.8E−3735124
domain
1619NTP_004511S11.3_4IBBImportin beta binding5.8E−3735124
domain
1630NTP_007592S2.3_10histoneCore histone1.2E−10297
H2A/H2B/H3/H4
1630NTP_007592S2.3_10histoneCore histone1.2E−10297
H2A/H2B/H3/H4
1633NTP_007867S7.3_3GTF2IGTF2I-like repeat7.2E−76106171
1633NTP_007867S7.3_3GTF2IGTF2I-like repeat7.2E−76295370
1633NTP_007867S7.3_3GTF2IGTF2I-like repeat7.2E−76106171
1633NTP_007867S7.3_3GTF2IGTF2I-like repeat7.2E−76295370
1634NTP_007867S8.3_1GTF2IGTF2I-like repeat7.2E−76122187
1634NTP_007867S8.3_1GTF2IGTF2I-like repeat7.2E−76311386
1634NTP_007867S8.3_1GTF2IGTF2I-like repeat7.2E−76122187
1634NTP_007867S8.3_1GTF2IGTF2I-like repeat7.2E−76311386
1640NTP_008858S2.3_2GST_NGlutathione S-4.6E−112195
transferase, N-terminal
domain
1640NTP_008858S2.3_2GST_NGlutathione S-4.6E−112195
transferase, N-terminal
domain
1643NTP_009526S2.3_3CBSCBS domain4.8E−433084
1643NTP_009526S2.3_3CBSCBS domain4.8E−43111165
1643NTP_009526S2.3_3CBSCBS domain4.8E−43186239
1643NTP_009526S2.3_3CBSCBS domain4.8E−43258311
1643NTP_009526S2.3_3CBSCBS domain4.8E−433084
1643NTP_009526S2.3_3CBSCBS domain4.8E−43111165
1643NTP_009526S2.3_3CBSCBS domain4.8E−43186239
1643NTP_009526S2.3_3CBSCBS domain4.8E−43258311
1644NTP_009526S2.3_5CBSCBS domain4.8E−433084
1644NTP_009526S2.3_5CBSCBS domain4.8E−43111165
1644NTP_009526S2.3_5CBSCBS domain4.8E−43186239
1644NTP_009526S2.3_5CBSCBS domain4.8E−43258311
1644NTP_009526S2.3_5CBSCBS domain4.8E−433084
1644NTP_009526S2.3_5CBSCBS domain4.8E−43111165
1644NTP_009526S2.3_5CBSCBS domain4.8E−43186239
1644NTP_009526S2.3_5CBSCBS domain4.8E−43258311
1647NTP_010018S2.3_5DAG_PE-bindPhorbol7.7E−23154203
esters/diacylglycerol
binding domain (C1
domain)
1647NTP_010018S2.3_5DAG_PE-bindPhorbol7.7E−23387426
esters/diacylglycerol
binding domain (C1
domain)
1647NTP_010018S2.3_5DAG_PE-bindPhorbol7.7E−23154203
esters/diacylglycerol
binding domain (C1
domain)
1647NTP_010018S2.3_5DAG_PE-bindPhorbol7.7E−23387426
esters/diacylglycerol
binding domain (C1
domain)
1651NTP_010757S4.3_2T-boxT-box 6E−1149351099
1651NTP_010757S4.3_2T-boxT-box 6E−11411421160
1651NTP_010757S4.3_2T-boxT-box 6E−1149351099
1651NTP_010757S4.3_2T-boxT-box 6E−11411421160
1653NTP_011130S2.3_3GATAGATA zinc finger1.5E−11159198
1653NTP_011130S2.3_3GATAGATA zinc finger1.5E−11159198
1656NTP_011430S6.3_6cadherinCadherin domain7.4E−61174270
1656NTP_011430S6.3_6cadherinCadherin domain7.4E−61284390
1656NTP_011430S6.3_6cadherinCadherin domain7.4E−61405495
1656NTP_011430S6.3_6cadherinCadherin domain7.4E−61174270
1656NTP_011430S6.3_6cadherinCadherin domain7.4E−61284390
1656NTP_011430S6.3_6cadherinCadherin domain7.4E−61405495
1658NTP_017582S2.3_6HMG_boxHMG (high mobility6.8E−093492
group) box
1658NTP_017582S2.3_6HMG_boxHMG (high mobility6.8E−093492
group) box
1675NTP_026331S1.1_1GTF2IGTF2I-like repeat7.2E−76106171
1675NTP_026331S1.1_1GTF2IGTF2I-like repeat7.2E−76295370
1675NTP_026331S1.1_1GTF2IGTF2I-like repeat7.2E−76106171
1675NTP_026331S1.1_1GTF2IGTF2I-like repeat7.2E−76295370
|
Some SEQ ID NOS exhibited multiple profile hits where the query sequence contains overlapping profile regions, and/or where the sequence contains two different functional domains. Each of the profile hits of Tables 18 and 19 is described in more detail below. The acronyms for the profiles (provided in parentheses) are those used to identify the profile in the Pfam, Prosite, and InterPro databases. The Pfam database can be accessed through web sites supported by Genome Sequencing Center at the Washington University School of Medicine or by the European Molecular Biology Laboratories in Heidelberg, Germany. The Prosite database can be accessed at the ExPASy Molecular Biology Server on the internet. The InterPro database can be accessed at a web site supported by the EMBL European Bioinformatics Institute. The public information available on the Pfam, Prosite, and InterPro databases regarding the various profiles, including but not limited to the activities, function, and consensus sequences of various proteins families and protein domains, is incorporated herein by reference.
Epidermal Growth Factor (EGF; Pfam Accession No. PF00008). SEQ ID NOS:550 and 551 represent polynucleotides encoding a member of the EGF family of proteins. The distinguishing characteristic of this family is the presence of a sequence of about thirty to forty amino acid residues found in epidermal growth factor (EGF) which has been shown to be present, in a more or less conserved form, in a large number of other proteins (Davis, New Biol. (1990) 2:410-419; Blomquist et al., Proc. Natl. Acad. Sci. U.S.A. (1984) 81:7363-7367; Barkert et al., Protein Nucl. Acid Enz. (1986) 29:54-86; Doolittle et al., Nature. (1984) 307:558-560; Appella et al., FEBS Lett. (1988) 231:1-4; Campbell and Bork, Curr. Opin. Struct. Biol. (1993) 3:385-392). A common feature of the domain is that the conserved pattern is generally found in the extracellular domain of membrane-bound proteins or in proteins known to be secreted. The EGF domain includes six cysteine residues which have been shown to be involved in disulfide bonds. The main structure is a two-stranded beta-sheet followed by a loop to a C-terminal short two-stranded sheet. Subdomains between the conserved cysteines strongly vary in length. These consensus patterns are used to identify members of this family: C-x-C-x(5)-G-x(2)-C and C-x-C-x(s)-[GP]-[FYW]-x(4,8)-C.
Seven Transmembrane Integral Membrane Proteins—Rhodopsin Family (7tm—1; Pfam Accession No. PF00001). SEQ ID NO:454 corresponds to a sequence encoding a polypeptide that is a member of the seven transmembrane (7tm) receptor rhodopsin family. G-protein coupled receptors of the (7tm) rhodopsin family (also called R7G) are an extensive group of hormones, neurotransmitters, and light receptors which transduce extracellular signals by interaction with guanine nucleotide-binding (G) proteins (Strosberg, Eur. J. Biochem. (1991) 196:1; Kerlavage, Curr. Opin. Struct. Biol. (1991) 1:394; Probst et al., DNA Cell Biol. (1992) 11:1; Savarese et al., Biochem. J. (1992) 283:1. The consensus pattern that contains the conserved triplet and that also spans the major part of the third transmembrane helix is used to detect this widespread family of proteins: [GSTALIVMFYWC]-[GSTANCPDE]-{EDPKRH}-x(2)-[LIVMNQGA]-x(2)-[LIVMFT]-[GSTANC]-[LIVMFYWSTAC]-[DENH]-R-[FYWCSH]-x(2)-[LIVM].
Basic Region Plus Leucine Zipper Transcription Factors (bZIP; Pfam Accession No. PF00170). SEQ ID NO:771 represents a polynucleotide encoding a novel member of the family of basic region plus leucine zipper transcription factors. The bZIP superfamily (Hurst, Protein Prof. (1995) 2:105; and Ellenberger, Curr. Opin. Struct. Biol. (1994) 4:12) of eukaryotic DNA-binding transcription factors encompasses proteins that contain a basic region mediating sequence-specific DNA-binding followed by a leucine zipper required for dimerization. The consensus pattern for this protein family is: [KR]-x(1,3)-[RKSAQ]-N-x(2)-[SAQ](2)-x-[RKTAENQ]-x-R-x-[RK].
Reverse Transcriptase (rvt; Pfam Accession No. PF00078). SEQ ID NO:270 represents a polynucleotide encoding a reverse transcriptase, which occurs in a variety of mobile elements, including retrotransposons, retroviruses, group II introns, bacterial msDNAs, hepadnaviruses, and caulimoviruses (Xiong and Eickbush, EMBO J. (1990) 9:3353-3362). Reverse transcriptases catalyze RNA-template-directed extension of the 3′-end of a DNA strand by one deoxynucleotide at a time and require an RNA or DNA primer.
KRAB box (KRAB; Pfam Accession No. PF01352). SEQ ID NO:1145 represents a polypeptide having a Krueppel-associated box (KRAB). A KRAB box is a domain of around 75 amino acids that is found in the N-terminal part of about one third of eukaryotic Krueppel-type C2H2 zinc finger proteins (ZFPs). It is enriched in charged amino acids and can be divided into subregions A and B, which are predicted to fold into two amphipathic alpha-helices. The KRAB A and B boxes can be separated by variable spacer segments and many KRAB proteins contain only the A box.
The KRAB domain functions as a transcriptional repressor when tethered to the template DNA by a DNA-binding domain. A sequence of 45 amino acids in the KRAB A subdomain has been shown to be necessary and sufficient for transcriptional repression. The B box does not repress by itself but does potentiate the repression exerted by the KRAB A subdomain. Gene silencing requires the binding of the KRAB domain to the RING-B box-coiled coil (RBCC) domain of the KAP-1/TIF1-beta corepressor. As KAP-1 binds to the heterochromatin proteins HP1, it has been proposed that the KRAB-ZFP-bound target gene could be silenced following recruitment to heterochromatin.
KRAB-ZFPs constitute one of the single largest class of transcription factors within the human genome, and appear to play important roles during cell differentiation and development. The KRAB domain is generally encoded by two exons. The regions coded by the two exons are known as KRAB-A and KRAB-B.
Armadillo/beta-catenin-like repeat (Armadillo_seg: Pfam Accession No. PF00514). SEQ ID NO: 1619 represents a polypeptide having sequence similarity with the armadillo/beta-catenin-like repeat (armadillo). The armadillo repeat is an approximately 40 amino acid long tandemly repeated sequence motif first identified in the Drosophila segment polarity gene armadillo. Similar repeats were later found in the mammalian armadillo homolog beta-catenin, the junctional plaque protein plakoglobin, the adenomatous polyposis coli (APC) tumor suppressor protein, and a number of other proteins (Peifer et al., Cell 76(2):786-791 (1994)).
The 3 dimensional fold of an armadillo repeat is known from the crystal structure of beta-catenin (Rojas et al., Cell 95:105-130 (1998)). There, the 12 repeats form a superhelix of alpha-helices, with three helices per unit. The cylindrical structure features a positively charged grove which presumably interacts with the acidic surfaces of the known interaction partners of beta-catenin.
Cadherin domain (cadherin; Pfam Accession No. PF00028). SEQ ID NO: 1656 represents a polypeptide having sequence similarity to a cadherin domain. Cadherins are a family of animal glycoproteins responsible for calcium-dependent cell-cell adhesion (Takeichi, Annu. Rev. Biochem. 59:237-252(1990); Takeichi, Trends Genet. 3:213-217(1987)). Cadherins preferentially interact with themselves in a homophilic manner in connecting cells; thus acting as both receptor and ligand. A wide number of tissue-specific forms of cadherins are known, for example: Epithelial (E-cadherin) (CDH1); Neural (N-cadherin) (CDH2); Placental (P-cadherin) (CDH3); Retinal (R-cadherin) (CDH4); Vascular endothelial (VE-cadherin) (CDH5); Kidney (K-cadherin) (CDH6); Cadherin-8 (CDH8); Cadherin-9 (CDH9); Osteoblast (OB-cadherin) (CDH11); Brain (BR-cadherin) (CDH12); T-cadherin (truncated cadherin) (CDH13); Muscle (M-cadherin) (CDH15); Kidney (Ksp-cadherin) (CDH16); and Liver-intestine (LI-cadherin) (CDH17).
Structurally, cadherins are built of the following domains: a signal sequence, followed by a propeptide of about 130 residues, then an extracellular domain of around 600 residues, then a transmembrane region, and finally a C-terminal cytoplasmic domain of about 150 residues. The extracellular domain can be sub-divided into five parts: there are four repeats of about 110 residues followed by a region that contains four conserved cysteines. The calcium-binding region of cadherins may be located in the extracellular repeats. The signature pattern for the repeated domain is located in the C-terminal extremity, which is its best conserved region. The pattern includes two conserved aspartic acid residues and two asparagines; these residues could be implicated in the binding of calcium. The consensus pattern is: [LIV]-x-[LIV]-x-D-x-N-D-[NH]-x-P.
CBS domain (CBS; Pfam Accession No. PF00571). SEQ ID NOS:1643 and 1644 represent polypeptides having sequence similarity to CBS domains, which are present in all 3 forms of cellular life, including two copies in inosine monophosphate dehydrogenase, of which one is disordered in the crystal structure. A number of disease states are associated with CBS-containing proteins including homocystinuria, Becker's and Thomsen disease.
CBS domains are small intracellular modules of unknown function. They are mostly found in 2 or four copies within a protein. Pairs of CBS domains dimerise to form a stable globular domain (Zhang et al., Biochemistry 38:4691-4700 (1999)). Two CBS domains are found in inosine-monophosphate dehydrogenase from all species, however the CBS domains are not needed for activity. CBS domains are found attached to a wide range of other protein domains suggesting that CBS domains may play a regulatory role. The region containing the CBS domains in Cystathionine-beta synthase is involved in regulation by S-AdoMet (Zhang et al., Biochemistry 38:4691-4700 (1999)). The 3D Structure is found as a sub-domain in TIM barrel of inosine-monophosphate dehydrogenase.
Phorbol esters/diacylglycerol binding domain (C1 domain) (DAG_PE-bind: Pfam Accessin No. PF00130). SEQ ID NO: 1647 represents a polypeptide having sequence similarity to the Phorbol esters/diacylglycerol binding domain (C1 domain). Diacylglycerol (DAG) is an important second messenger. Phorbol esters (PE) are analogues of DAG and potent tumor promoters that cause a variety of physiological changes when administered to both cells and tissues. DAG activates a family of serine/threonine protein kinases, collectively known as protein kinase C (PKC) (Azzi et al., Eur. J. Biochem. 208:547-557 (1992)). Phorbol esters can also directly stimulate PKC.
The N-terminal region of PKC, known as C1, has been shown to bind PE and DAG in a phospholipid and zinc-dependent fashion (Ono et al., Proc. Natl. Acad. Sci. U.S.A. 86:4868-4871 (1989)). The C1 region contains one or two copies (depending on the isozyme of PKC) of a cysteine-rich domain about 50 amino-acid residues long and essential for DAG/PE-binding. The DAG/PE-binding domain binds two zinc ions; the ligands of these metal ions are probably the six cysteines and two histidines that are conserved in the C1 domain. The consensus sequence for the C1 domain is: H-x-[LIVMFYW]-x(8,11)-C-x(2)-C-x(3)-[LIVMFC]-x(5,10)-C-x(2)-C-x(4)-[HD]-x(2)-C-x(5,9)-C [All the C and H are involved in binding Zinc].
GATA zinc finger (GATA; Pfam Accession No. PF00320). SEQ ID NO:1653 represents a polypeptide having sequence similarity to GATA zinc finger. A number of transcription factors, including erythroid-specific transcription factor and nitrogen regulatory proteins, specifically bind the DNA sequence (A/T)GATA(A/G) in the regulatory regions of genes (Yamamoto et al., Genes Dev. 4:1650-1662 (1990)) and are consequently termed GATA-binding transcription factors. The interactions occur via highly-conserved zinc finger domains in which the zinc ion is coordinated by 4 cysteine residues (Evans and Felsenfeld, Cell 58:877-885 (1989); Omichinski et al., Science 261:438-446 (1993)).
NMR studies have shown the core of the zinc finger to comprise 2 irregular anti-parallel beta-sheets and an alpha-helix, followed by a long loop to the C-terminal end of the finger. The N-terminal part, which includes the helix, is similar in structure, but not sequence, to the N-terminal zinc module of the glucocorticoid receptor DNA-binding domain. The helix and the loop connecting the 2 beta-sheets interact with the major groove of the DNA, while the C-terminal tail wraps around into the minor groove. It is this tail that is the essential determinant of specific binding. Interactions between the zinc finger and DNA are mainly hydrophobic, explaining the preponderance of thymines in the binding site; a large number of interactions with the phosphate backbone have also been observed (Omichinski et al., Science 261:438-446 (1993)). Two GATA zinc fingers are found in the GATA transcription factors; however, there are several proteins which only contains a single copy of the domain. The consensus sequence of the domain is: C-x-[DN]-C-x(4,5)-[ST]-x(2)-W-[HR]-[RK]-x(3)-[GN]-x(3,4)-C-N-[AS]-C [The four C's are zinc ligands].
Glutathione S-transferase, N-terminal domain (GST_N: Pfam Accession No. PF02798). SEQ ID NO: 1640 represents a polypeptide having sequence similarity to Glutathione S-transferase, N-terminal domain. In eukaryotes, glutathione S-transferases (GSTs) participate in the detoxification of reactive electrophilic compounds by catalysing their conjugation to glutathione. The GST domain is also found in S-crystallins from squid, and proteins with no known GST activity, such as eukaryotic elongation factors 1-gamma and the HSP26 family of stress-related proteins, which include auxin-regulated proteins in plants and stringent starvation proteins in E. coli. The major lens polypeptide of Cephalopoda is also a GST.
Bacterial GSTs of known function often have a specific, growth-supporting role in biodegradative metabolism: epoxide ring opening and tetrachlorohydroquinone reductive dehalogenation are two examples of the reactions catalysed by these bacterial GSTs. Some regulatory proteins, like the stringent starvation proteins, also belong to the GST family. GST seems to be absent from Archaea in which gamma-glutamylcysteine substitute to glutathione as major thiol.
Glutathione S-transferases form homodimers, but in eukaryotes can also form heterodimers of the A1 and A2 or YC1 and YC2 subunits. The homodimeric enzymes display a conserved structural fold. Each monomer is composed of a distinct N-terminal sub-domain, which adopts the thioredoxin fold, and a C-terminal all-helical sub-domain.
GTF2I-like repeat (GTF2I; Pfam Accession No. PF02946). SEQ ID NOS:1633, 1634, and 1675 represent polypeptides having sequence similarity to proteins having GTF2I-like repeat. This region of sequence similarity is found up to six times in a variety of proteins including GTF2I. It has been suggested that this may be a DNA binding domain (O'Mahoney et al., Mol. Cell. Biol. 18:6641-6652 (1998); Osborne et al., Genomics 57:279-284 (1999)).
Core histone H2A/H2B/H3/H4 (histone; Pfam Accession No. PF00125). SEQ ID NO:1630 represents a polypeptide having sequence similarity to core histone H2A/H2B/H3/H4 family polypeptides. Histone H2A is one of the four histones, along with H2B, H3 and H4, which forms the eukaryotic nucleosome core. Using alignments of histone H2A sequences (Wells and Brown, Nucleic Acids Res. 19:2173-2188(1991); Thatcher and Gorovsky, Nucleic Acids Res. 22:174-179(1994)) a conserved region in the N-terminal part of H2A was used to develop a signature pattern. This region is conserved both in classical S-phase regulated H2A's and in variant histone H2A's which are synthesized throughout the cell cycle. The consensus pattern is: [AC]-G-L-x-F-P-V.
Histone H4, along with H3, plays a central role in nucleosome formation. The sequence of histone H4 has remained almost invariant in more then 2 billion years of evolution (Thatcher and Gorovsky, Nucleic Acids Res. 22:174-179(1994)). The region used as a signature pattern is a pentapeptide found in positions 14 to 18 of all H4 sequences. It contains a lysine residue which is often acetylated (Doenecke and Gallwitz, Mol. Cell. Biochem. 44:113-128(1982)) and a histidine residue which is implicated in DNA-binding (Ebralidse et al., Nature 331:365-367(1988)). The consensus pattern is: G-A-K-R-H.
Histone H3 is a highly conserved protein of 135 amino acid residues (Wells and Brown, Nucleic Acids Res. 19:2173-2188(1991); Thatcher and Gorovsky, Nucleic Acids Res. 22:174-179(1994)). Two signature patterns have been developed, the first one corresponds to a perfectly conserved heptapeptide in the N-terminal part of H3, while the second one is derived from a conserved region in the central section of H3. The consensus patterns are: K-A-P-R-K-Q-L and P-F-x-[RA]-L-[VA]-[KRQ]-[DEG]-[IV].
The signature pattern of histone H2B corresponds to a conserved region in the C-terminal part of the protein. The consensus pattern is: [KR]-E-[LIVM]-[EQ]-T-x(2)-[KR]-x-[LIVM](2)-x-[PAG]-[DE]-L-x-[KR]-H-A-[LIVM]-[STA]-E-G
HMG (high mobility group) box (HMG_box: Pfam Accession No. PF00505). SEQ ID NO:1658 corresponds to a polypeptide having sequence similarity to high mobility group proteins, a family of relatively low molecular weight non-histone components in chromatin. HMG1 (also called HMG-T in fish) and HMG2 (Bustin et al., Biochim. Biophys. Acta 1049: 231-243(1990)) are two highly related proteins that bind single-stranded DNA preferentially and unwind double-stranded DNA. HMG1/2 have about 200 amino acid residues with a highly acidic C-terminal section which is composed of an uninterrupted stretch of from 20 to 30 aspartic and glutamic acid residues; the rest of the protein sequence is very basic. In addition to the HMG1 and HMG2 proteins, HMG-domains occur in single or multiple copies in the following protein classes; the SOX family of transcription factors; SRY sex determining region Y protein and related proteins; LEF1 lymphoid enhancer binding factor 1; SSRP recombination signal recognition protein; MTF1 mitochondrial transcription factor 1; UBF1/2 nucleolar transcription factors; Abf2 yeast ARS-binding factor; and yeast transcription factors Ixr1, Rox1, Nhp6a, Nhp6b and Spp41.
Importin beta binding domain (IBB: Pfam Accession No. PF01749). SEQ ID NO: 1619 represents a polypeptide having sequence similarity to importin beta binding domain family polypeptides. This family consists of the importin alpha (karyopherin alpha), importin beta (karyopherin beta) binding domain. The domain mediates formation of the importin alpha beta complex; required for classical NLS import of proteins into the nucleus, through the nuclear pore complex and across the nuclear envelope. Also in the alignment is the NLS of importin alpha which overlaps with the IBB domain (Moroianu et al., Proc. Natl. Acad. Sci. U.S.A. 93:6572-6576(1996)).
T-box domain (T-box: Pfam Accession No. PF00907). SEQ ID NOS:1651 represents a polypeptide having sequence similarity to proteins having a T-box domain. The T-box gene family is an ancient group of putative transcription factors that appear to play a critical role in the development of all animal species. These genes were uncovered on the basis of similarity to the DNA binding domain (Papaioannou and Silver, Bioessays 20:9-19 (1998)) of murine Brachyury (T) gene product, which similarity is the defining feature of the family. The Brachyury gene is named for its phenotype, which was identified 70 years ago as a mutant mouse strain with a short blunted tail. The gene, and its paralogues, have become a well-studied model for the family, and hence much of what is known about the T-box family is derived from the murine Brachyury gene.
Consistent with its nuclear location, Brachyury protein has a sequence-specific DNA-binding activity and can act as a transcriptional regulator (Wattler et al., Genomics 48:24-33(1998)). Homozygous mutants for the gene undergo extensive developmental anomalies, thus rendering the mutation lethal (Kavka and Green, Biochim. Biophys. Acta 1333(2) (1997)). The postulated role of Brachyury is as a transcription factor, regulating the specification and differentiation of posterior mesoderm during gastrulation in a dose-dependent manner (Papaioannou and Silver, Bioessays 20:9-19 (1998)).
Common features shared by T-box family members are, DNA-binding and transcriptional regulatory activity, a role in development and conserved expression patterns. Most of the known genes in all species are expressed in mesoderm or mesoderm precursors (Papaioannou, Trends Genet. 13:212-213(1997)). Members of the T-box family contain a domain of about 170 to 190 amino acids known as the T-box domain (Papaioannou, Trends Genet. 13: 212-213(1997); Bollag et al., Nat. Genet. 7: 383-389(1994); Agulnik et al., Genetics 144:249-254(1996)) and which probably binds DNA. As signature patterns for the T-domain, we selected two conserved regions. The first region corresponds to the N-terminal of the domain and the second one to the central part. The consensus sequences are: L-W-x(2)-[FC]-x(3,4)-[NT]-E-M-[LIV](2)-T-x(2)-G-[RG]-[KRQ] and [LIVMFYW]-H-[PADH]-[DENQ]-[GS]-x(3)-G-x(2)-W-M-x(3)-[IVA]-x-F.
60s Acidic ribosomal protein (60s_ribosomal; Pfam Accession No. PF00428). SEQ ID NO: 1038 represents a polynucleotide encoding a member of the 60s acidic ribosomal protein family. The 60S acidic ribosomal protein plays an important role in the elongation step of protein synthesis. This family includes archaebacterial L12, eukaryotic P0, P1 and P2 (Remacha et al., Biochem. Cell Biol. 73:959-968(1995)).
Some of the proteins in this family are allergens. A nomenclature system has been established for antigens (allergens) that cause IgE-mediated atopic allergies in humans (WHO/IUIS Allergen Nomenclature Subcommittee King T. P., Hoffmann D., Loewenstein H., Marsh D. G., Platts-Mills T. A. E., Thomas W. Bull. World Health Organ. 72:797-806(1994)). This nomenclature system is defined by a designation that is composed of the first three letters of the genus; a space; the first letter of the species name; a space and an arabic number. In the event that two species names have identical designations, they are discriminated from one another by adding one or more letters (as necessary) to each species designation. The allergens in this family include allergens with the following designations: Alt a 6, Alt a 12, Cla h 3, Cla h 4, and Cla h 12.
AP endonuclease family 1 (AP_endonucleas1; Pfam Accession No. PF01260). SEQ ID NOS:491 and 969 correspond to a polynucleotide encoding a member of the family of polypeptides designated AP endonuclease family 1. DNA damaging agents such as the antitumor drugs bleomycin and neocarzinostatin or those that generate oxygen radicals produce a variety of lesions in DNA. Amongst these is base-loss which forms apurinic/apyrimidinic (AP) sites or strand breaks with atypical 3′-termini. DNA repair at the AP sites is initiated by specific endonuclease cleavage of the phosphodiester backbone. Such endonucleases are also generally capable of removing blocking groups from the 3′-terminus of DNA strand breaks.
AP endonucleases can be classified into two families on the basis of sequence similarity. This family contains members of AP endonuclease family 1. Except for Rrp1 and arp, these enzymes are proteins of about 300 amino-acid residues. Rrp1 and arp both contain additional and unrelated sequences in their N-terminal section (about 400 residues for Rrp1 and 270 for arp). The proteins contain glutamate which has been shown (Mol et al., Nature 374: 381-386(1995)), in the Escherichia coli enzyme to bind a divalent metal ion such as magnesium or manganese. The consensus sequences for this family of polypeptides are: [APF]-D-[LIVMF](2)-x-[LIVM]-Q-E-x-K [E binds a divalent metal ion]; D-[ST]-[FY]-R-[KH]-x(7,8)-[FYW]-[ST]-[FYW](2); and N-x-G-x-R-[LIVM]-D-[LIVMFYH]-x-[LV]-x-S
Bowman-Birk serine protease inhibitor family (Bowman-Birk_leg; Pfam Accession No. 00228). SEQ ID NO: 454 represents a polynucleotide encoding a polypeptide having sequence similarity to a member of the Bowman-Birk serine protease inhibitor family. The Bowman-Birk inhibitor family (Laskowski and Kato, Annu. Rev. Biochem. 49:593-626(1980)) is one of the numerous families of serine proteinase inhibitors and has a duplicated structure and generally possesses two distinct inhibitory sites.
These inhibitors are found in the seeds of all leguminous plants as well as in cereal grains. In cereals they exist in two forms, one of which is a duplication of the basic structure (Tashiro et al., J. Biochem. 102:297-306(1987)). The signature pattern for sequences belonging to this family of inhibitors is in the central part of the domain and includes four cysteines. The consensus pattern is: C-x(5,6)-[DENQKRHSTA]-C-[PASTDH]-[PASTDK]-[ASTDV]-C-[NDEKS]-[DEKRHSTA]-C [The four C's are involved in disulfide bonds]. Note that this pattern can be found twice in some duplicated cereal inhibitors.
Cation efflux family (Cation_efflux: Pfam Accession No. PF01545). SEQ ID NO: 454 encodes a polypeptide having sequence similarity to members of the cation efflux family of proteins. Members of this family are integral membrane proteins, that are found to increase tolerance to divalent metal ions such as cadmium, zinc, and cobalt. These proteins are thought to be efflux pumps that remove these ions from cells (Xiong and Jayaswal, J. Bacteriol. 180: 4024-4029(1998); Kunito et al, Biosci. Biotechnol. Biochem. 60: 699-704(1996)).
DC1 domain (DC1; Pfam Accession No. PF03107). SEQ ID NO: 222 corresponds to a polypeptide having sequence similarity to a DC1 domain. This short domain is rich in cysteines and histidines. The pattern of conservation is similar to that found in DAG_PE-bind (Pfam Accession No. PF00130), therefore this domain has been termed DC1 for divergent C1 domain. Like the DAG_PE-bind domain, this domain probably also binds to two zinc ions. The function of proteins with this domain is uncertain, however this domain may bind to molecules such as diacylglycerol. This family are found in plant proteins.
Pneumovirus attachment glycoprotein G (Glycoprotein_G; Pfam Accession No. PF00802). SEQ ID NO:1128 represents a polypeptide having sequence similarity to members of the Pneumovirus attachment glycoprotein G protein family. This family includes attachment proteins from respiratory synctial virus. Glycoprotein G has not been shown to have any neuramimidase or hemagglutinin activity. The amino terminus is thought to be cytoplasmic, and the carboxyl terminus extracellular. The extracellular region contains four completely conserved cysteine residues.
NADH-Ubiquinone/plastoquinone (complex I), various chains (oxidored_q 1; Pfam Accession No. PF00361). SEQ ID NO:546 represents a polypeptide having sequence similarity to NADH-Ubiquinone/plastoquinone (complex I), various chains protein family. This family is part of the NADH:ubiquinone oxidoreductase (complex I) which catalyses the transfer of two electrons from NADH to ubiquinone in a reaction that is associated with proton translocation across the membrane (Walker, Q. Rev. Biophys. 25: 253-324(1992)). Sub-families within this protein family include NADH-ubiquinone oxidoreductase chain 5; NADH-ubiquinone oxidoreductase chain 2; NADH-ubiquinone oxidoreductase chain 4; and Multicomponent K+:H+antiporter.
Protamine P1 (protamine_P1; Pfam Accession No. PF00260). SEQ ID NOS:778 and 1450 represent polypeptides having sequence similarity to Protamine P1 protein family. Protamines are small, highly basic proteins, that substitute for histones in sperm chromatin during the haploid phase of spermatogenesis. They pack sperm DNA into a highly condensed, stable and inactive complex. There are two different types of mammalian protamine, called P1 and P2. P1 has been found in all species studied, while P2 is sometimes absent. There also seems to be a single type of avian protamine whose sequence is closely related to that of mammalian P1 (Oliva et al., J. Biol. Chem. 264:17627-17630(1989)). A conserved region at the N-terminal extremity of the sequence is used as a signature pattern for this family of proteins. The consensus pattern is: [AV]-R-[NFY]-R-x(2,3)-[ST]-x-S-x-S.
Squash family serine protease inhibitor (squash; Pfam Accession No. PF00299). SEQ ID NO:1128 represents a polypeptide having sequence similarity to Squash family serine protease inhibitor proteins. The squash inhibitors form one of a number of serine protease inhibitor families. The proteins, found in the seeds of cucurbitaceae plants (squash, cucumber, balsam pear, etc.), are approximately 30 residues in length, and contain 6 Cys residues, which form 3 disulfide bonds (Bode et al., FEBS Lett. 242: 285-292(1989)). The inhibitors function by being taken up by a serine protease (such as trypsin), which cleaves the peptide bond between Arg/Lys and Ile residues in the N-terminal portion of the protein (Bode et al., FEBS Lett. 242: 285-292(1989); Krishnamoorthi et al., Biochemistry 31: 898-904(1992)). Structural studies have shown that the inhibitor has an ellipsoidal shape, and is largely composed of beta-turns (Bode et al., FEBS Lett. 242: 285-292(1989)). The fold and Cys connectivity of the proteins resembles that of potato carboxypeptidase A inhibitor (Krishnamoorthi et al., Biochemistry 31: 898-904(1992)). The pattern used to detect this family of proteins spans the major part of the sequence and includes five of the six cysteines involved in disulfide bonds. The consensus pattern is: C-P-x(5)-C-x(2)-[DN]-x-D-C-x(3)-C-x-C [The five C's are involved in disulfide bonds]
Metallothionein family 5 (Metallothio—5: Pfam Accession No. PF02067). SEQ ID NO:1128 represents a polypeptide having sequence similarity to metallothionein family 5 proteins. Metallothioneins (MT) are small proteins that bind heavy metals, such as zinc, copper, cadmium, and nickel. They have a high content of cysteine residues that bind the metal ions through clusters of thiolate bonds (Kagi, Meth. Enzymol. 205: 613-626(1991); Kagi and Kojima, Experientia Suppl. 52: 25-61(1987); Kagi and Schaffer, Biochemistry 27: 8509-8515(1988)).
Due to limitations in the original classification system of MTs, which did not allow clear differentiation of patterns of structural similarities, either between or within classes, all class I and class II MTs (the proteinaceous sequences) have now been grouped into families of phylogenetically-related and thus alignable sequences. Diptera (Drosophila, family 5) MTs are 40-43 residue proteins that contain 10 conserved cysteines arranged in five Cys-X-Cys groups. In particular, the consensus pattern C-G-x(2)-C-x-C-x(2)-Q-x(5)-C-x-C-x(2)-D-C-x-C has been found to be diagnostic of family 5 MTs. The protein is found primarily in the alimentary canal, and its induction is stimulated by ingestion of cadmium or copper (Lastowski et al., J. Biol. Chem. 260: 1527-1530(1985)). Mercury, silver and zinc induce the protein to a lesser extent.
Caenorhabditis. elegans Sre G protein-coupled chemoreceptor (Sre; Pfam Accession No. PF03125). SEQ ID NO:724 represents a polypeptide having sequence similarity to C. elegans Sre G protein-coupled chemoreceptor family proteins. C. elegans Sre proteins are candidate chemosensory receptors. There are four main recognized groups of such receptors: Odr-10, Sra, Sro, and Srg. Sre (this family), Sra Sra and Srb Srb comprise the Sra group. All of the above receptors are thought to be G protein-coupled seven transmembrane domain proteins (Troemel, Bioessays 21:1011-1020 (1999); Troemel et al., Cell 83:207-218 (1995)).
Syndecan domain (Syndecan; Pfam Accession No. PF01034). SEQ ID NO:1128 corresponds to a polypeptide having a syndecan domain. Syndecans (Bernfield et al., Annu. Rev. Cell Biol. 8:365-393(1992); David, FASEB J. 7:1023-1030(1993)) are a family of transmembrane heparan sulfate proteoglycans which are implicated in the binding of extracellular matrix components and growth factors. Syndecans bind a variety of molecules via their heparan sulfate chains and can act as receptors or as co-receptors. Structurally, these proteins consist of four separate domains: a) a signal sequence; b) an extracellular domain (ectodomain) of variable length containing the sites of attachment of the heparan sulfate glycosaminoglycan side chains and whose sequence is not evolutionarily conserved in the various forms of syndecans; c) a transmembrane region; and d) a highly conserved cytoplasmic domain of about 30 to 35 residues which could interact with cytoskeletal proteins.
The signature pattern for syndecans starts with the last residue of the transmembrane region and includes the first 10 residues of the cytoplasmic domain. This region, which contains four basic residues, may act as a stop transfer site. The consensus pattern is: [FY]-R-[IM]-[KR]-K(2)-D-E-G-S-Y.
L1 transposable element (Transposase—22; Pfam Accession No. PF02994). SEQ ID NO:907 represents a polypeptide having an L1 transposable element. Many human L1 elements are capable of retrotransposition and some of these have been shown to exhibit reverse transcriptase (RT) activity (Sassaman et al., Nat Genet 16(1):37-43(1997)) although the function of many are, as yet, unknown. There are estimated to be 30-60 active L1 elements reside in the average diploid genome.
WW domain (WW; Pfam Accession No. PF00397). SEQ ID NO:564 represents a polypeptide having WW domain. The WW domain (also known as rsp5 or WWP) is a short conserved region in a number of unrelated proteins, among them dystrophin, responsible for Duchenne muscular dystrophy. This short domain may be repeated up to four times in some proteins (Bork and Sudol, Trends Biochem. Sci. 19: 531-533(1994); Andre and Springael, Biochem. Biophys. Res. Commun. 205: 1201-1205(1994); Hofmann and Bucher, FEBS Lett. 358: 153-157(1995); Sudol et al., FEBS Lett. 369: 67-71(1995)). The WW domain binds to proteins with particular proline-motifs, [AP]-P-P-[AP]-Y, and having four conserved aromatic positions that are generally Trp (Chen and Sudol, Proc. Natl. Acad. Sci. U.S.A. 92: 7819-7823(1995)). The name WW or WWP derives from the presence of these Trp as well as that of a conserved Pro. The WW domain is frequently associated with other domains typical for proteins in signal transduction processes.
A large variety of proteins containing the WW domain are known. These include; dystrophin, a multidomain cytoskeletal protein; utrophin, a dystrophin-like protein of unknown function; vertebrate YAP protein, substrate of an unknown serine kinase; mouse NEDD-4, involved in the embryonic development and differentiation of the central nervous system; yeast RSP5, similar to NEDD-4 in its molecular organization; rat FE65, a transcription-factor activator expressed preferentially in liver; tobacco DB10 protein and others. The consensus pattern is: W-x(9,11)-[VFY]-[FYW]-x(6,7)-[GSTNE]-[GSTQCR]-[FYW]-x(2)-P.
Example 22
Detection of Differential Expression Using Arrays and Source of Patient Tissue Samples
mRNA isolated from samples of cancerous and normal breast and colon tissue obtained from patients were analyzed to identify genes differentially expressed in cancerous and normal cells. Normal and cancerous tissues were collected from patients using laser capture microdissection (LCM) techniques, which techniques are well known in the art (see, e.g., Ohyama et al. (2000) Biotechniques 29:530-6; Curran et al. (2000) Mol. Pathol. 53:64-8; Suarez-Quian et al. (1999) Biotechniques 26:328-35; Simone et al. (1998) Trends Genet 14:272-6; Conia et al. (1997) J. Clin. Lab. Anal. 11:28-38; Emmert-Buck et al. (1996) Science 274:998-1001).
Table 20 (inserted prior to claims) provides information about each patient from which colon tissue samples were isolated, including: the Patient ID (“PT ID”) and Path ReportID (“Path ID”), which are numbers assigned to the patient and the pathology reports for identification purposes; the group (“Grp”) to which the patients have been assigned; the anatomical location of the tumor (“Anatom Loc”); the primary tumor size (“Size”); the primary tumor grade (“Grade”); the identification of the histopathological grade (“Histo Grade”); a description of local sites to which the tumor had invaded (“Local Invasion”); the presence of lymph node metastases (“Lymph Met”); the incidence of lymph node metastases (provided as a number of lymph nodes positive for metastasis over the number of lymph-nodes examined) (“Lymph Met Incid”); the regional lymphnode grade (“Reg Lymph Grade”); the identification or detection of metastases to sites distant to the tumor and their location (“Dist Met & Loc”); the grade of distant metastasis (“Dist Met Grade”); and general comments about the patient or the tumor (“Comments”). Histophatology of all primary tumors indicated the tumor was adenocarcinmoa except for Patient ID Nos. 130 (for which no information was provided), 392 (in which greater than 50% of the cells were mucinous carcinoma), and 784 (adenosquamous carcinoma). Extranodal extensions were described in three patients, Patient ID Nos. 784, 789, and 791. Lymphovascular invasion was described in Patient ID Nos. 128, 278, 517, 534, 784, 786, 789, 791, 890, and 892. Crohn's-like infiltrates were described in seven patients, Patient ID Nos. 52, 264, 268, 392, 393, 784, and 791. Table 21 (below) provides information about each patient from which the breast tissue samples were isolated, including: 1) the “Pat Num”, a number assigned to the patient for identification purposes; 2) the “Histology”, which indicates whether the tumor was characterized as an intraductal carcinoma (IDC) or ductal carcinoma in situ (DCIS); 3) the incidence of lymph node metastases (LMF), represented as the number of lymph nodes positive to metastases out of the total number examined in the patient; 4) the “Tumor Size”; 5) “TNM Stage”, which provides the tumor grade (T#), where the number indicates the grade and “p” indicates that the tumor grade is a pathological classification; regional lymph node metastasis (N#), where “0” indicates no lymph node metastases were found, “1” indicates lymph node metastases were found, and “X” means information not available and; the identification or detection of metastases to sites distant to the tumor and their location (M#), with “X” indicating that no distant mesatses were reported; and the stage of the tumor (“Stage Grouping”). “nr” indicates “no reported”.
TABLE 20
|
|
LymphRegDistDist
PathAnatomHistoLymphMetLymphMet &Met
Pt IDIDGrpLocSizeGradeGradeLocal InvasionMetIncidGradeLocGradeComment
|
|
1521IIIAscending4.0T3G2Extending intoPos3/8 N1NegMXinvasive
colonsubserosal adiposeadenocarcinoma,
tissuemoderately
differentiated;
focal perineural
invasion is seen
5271IICecum9.0T3G3Invasion throughNeg0/12N0NegM0Hyperplastic
muscularispolyp in
propria, subserosalappendix.
involvement;
ileocec. valve
involvement
121140IISigmoid6T4G2Invasion ofNeg0/34N0NegM0Perineural
muscularis propriainvasion; donut
into serosa,anastomosis
involvingNeg. One
submucosa oftubulovillous
urinary bladderand one tubular
adenoma with
no high grade
dysplasia.
125144IICecum6T3G2Invasion throughNeg0/19N0NegM0patient history
the muscularisof metastatic
propria intomelanoma
suserosal adipose
tissue. Ileocecal
junction.
128147IIITransverse5.0T3G2Invasion ofPos1/5 N1NegM0
colonmuscularis propria
into percolonic fat
130149Splenic5.5T3through wall andPos10/24 N2NegM1
flexureinto surrounding
adipose tissue
133152IIRectum5.0T3G2Invasion throughNeg0/9 N0NegM0Small separate
muscularis propriatubular
into non-adenoma (0.4 cm)
peritonealized
pericolic tissue;
gross
configuration is
annular.
141160IVCecum5.5T3G2Invasion ofPos7/21N2Pos -M1Perineural
muscularis propriaLiverinvasion
into pericolonicidentified
adipose tissue, butadjacent to
not through serosa.metastatic
Arising fromadenocarcinoma.
tubular adenoma.
156175IIIHepatic3.8T3G2Invasion throughPos2/13N1NegM0Separate
flexuremuscularis propriatubolovillous
intoand tubular
subserosa/pericolicadenomas
adipose, no serosal
involvement.
Gross
configuration
annular.
228247IIIRectum5.8T3G2 toInvasion throughPos1/8 N1NegMXHyperplastic
G3muscularis propriapolyps
to involve
subserosal,
perirectoal
adipose, and
serosa
264283IIAscending5.5T3G2Invasion throughNeg0/10N0NegM0Tubulovillous
colonmuscularis propriaadenoma with
into subserosalhigh grade
adipose tissue.dysplasia
266285IIITransverse9T3G2Invades throughNeg0/15N1Pos -MX
colonmuscularis propriaMesen-
to involveteric
pericolonicdeposit
adipose, extends to
serosa.
268287ICecum6.5T2G2Invades fullNeg0/12N0NegM0
thickness of
muscularis
propria, but
mesenteric adipose
free of malignancy
278297IIIRectum4T3G2Invasion intoPos7/10N2NegM0Descending
perirectal adiposecolon polyps,
tissue.no HGD or
carcinoma
identified.
296315IIICecum5.5T3G2Invasion throughPos2/12N1NegM0Tubulovillous
muscularis propriaadenoma (2.0 cm)
and invadeswith no
pericolic adiposehigh grade
tissue. Ileocecaldysplasia. Neg.
junction.liver biopsy.
339358IIRecto-6T3G2Extends intoNeg0/6 N0NegM01 hyperplastic
sigmoidperirectal fat butpolyp identified
does not reach
serosa
341360IIAscending2 cmT3G2Invasion throughNeg0/4 N0NegMX
coloninvasivemuscularis propria
to involve
pericolonic fat.
Arising from
villous adenoma.
356375IISigmoid6.5T3G2Through colonNeg0/4 N0NegM0
wall into
subserosal adipose
tissue. No serosal
spread seen.
360412IIIAscending4.3T3G2Invasion thruPos1/5 N1NegM0Two mucosal
colonmuscularis propriapolyps
to pericolonic fat
392444IVAscending2T3G2Invasion throughPos1/6 N1Pos -M1Tumor arising
colonmuscularis propriaLiverat prior
into subserosalileocolic
adipose tissue, notsurgical
serosa.anastomosis.
393445IICecum6.0T3G2Cecum, invadesNeg0/21N0NegM0
through
muscularis propria
to involve
subserosal adipose
tissue but not
serosa.
413465IVCecum4.8T3G2Invasive throughNeg0/7 N0Pos -M1rediagnosis of
muscularis toLiveroophorectomy
involve periserosalpath to
fat; abuttingmetastatic
ileocecal junction.colon cancer.
505383IV7.5T3G2Invasion throughPos2/17N1Pos -M1Anatomical
muscularis propriaLiverlocation of
involving pericolicprimary not
adipose, serosalnotated in
surface uninvolvedreport.
Evidence of
chronic colitis.
517395IVSigmoid3T3G2penetratesPos6/6 N2NegM0No mention of
muscularisdistant met in
propria, involvesreport
pericolonic fat.
|
TABLE 21
|
|
|
Breast cancer patient data.
|
Pat
Tumor
|
Num
Histology
LMF
Size
TNM Stage
Stage Grouping
|
|
280
IDC, DCIS +
nr
2 cm
T2NXMX
probable Stage II
|
D2
|
284
IDC, DCIS
0/16
2 cm
T2pN0MX
Stage II
|
285
IDC, DCIS
nr
4.5 cm
T2NXMX
probable Stage II
|
291
IDC, DCIS
0/24
4.5 cm
T2pN0MX
Stage II
|
302
IDC, DCIS
nr
2.2 cm
T2NXMX
probable Stage II
|
375
IDC, DCIS
nr
1.5 cm
T1NXMX
probable Stage I
|
408
IDC
0/23
3.0 cm
T2pN0MX
Stage II
|
416
IDC
0/6
3.3 cm
T2pN0MX
Stage II
|
421
IDC, DCIS
nr
3.5 cm
T2NXMX
probable Stage II
|
459
IDC
2/5
4.9 cm
T2pN1MX
Stage II
|
465
IDC
0/10
6.5 cm
T3pN0MX
Stage II
|
470
IDC, DCIS
0/6
2.5 cm
T2pN0MX
Stage II
|
472
IDC, DCIS
6/45
5.0+ cm
T3pN1MX
Stage III
|
474
IDC
0/18
6.0 cm
T3pN0MX
Stage II
|
476
IDC
0/16
3.4 cm
T2pN0MX
Stage II
|
605
IDC, DCIS
1/25
5.0 cm
T2pN1MX
Stage II
|
649
IDC, DCIS
1/29
4.5 cm
T2pN1MX
Stage II
|
|
Identification of Differentially Expressed Genes
cDNA probes were prepared from total RNA isolated from the patient cells described above. Since LCM provides for the isolation of specific cell types to provide a substantially homogenous cell sample, this provided for a similarly pure RNA sample.
Total RNA was first reverse transcribed into cDNA using a primer containing a T7 RNA polymerase promoter, followed by second strand DNA synthesis. cDNA was the transcribed in vitro to produce antisense RNA using the T7 promoter-mediated expression (see, e.g., Luo et al. (1999) Nature Med 5:117-122), and the antisense RNA was then converted into cDNA. The second set of cDNAs were again transcribed in vitro, using the T7 promoter, to provide antisense RNA. Optionally, the RNA was again converted into cDNA, allowing for up to a third round of T7-mediated amplification to produce more antisense RNA. Thus the procedure provided for two or three rounds of in vitro transcription to produce the final RNA used for fluorescent labeling.
Fluorescent probes were generated by first adding control RNA to the antisense RNA mix, and producing fluorescently labeled cDNA from the RNA starting material. Fluorescently labeled cDNAs prepared from the tumor RNA sample were compared to fluorescently labeled cDNAs prepared from normal cell RNA sample. For example, the cDNA probes from the normal cells were labeled with Cy3 fluorescent dye (green) and the cDNA probes prepared from the tumor cells were labeled with Cy5 fluorescent dye (red), and vice versa.
Each array used had an identical spatial layout and control spot set. Each microarray was divided into two areas, each area having an array with, on each half, twelve groupings of 32×12 spots, for a total of about 9,216 spots on each array. The two areas are spotted identically which provide for at least two duplicates of each clone per array.
Polynucleotides for use on the arrays were obtained from both publicly available sources and from cDNA libraries generated from selected cell lines and patient tissues. PCR products of from about 0.5 kb to 2.0 kb amplified from these sources were spotted onto the array using a Molecular Dynamics Gen III spotter according to the manufacturer's recommendations. The first row of each of the 24 regions on the array had about 32 control spots, including 4 negative control spots and 8 test polynucleotides. The test polynucleotides were spiked into each sample before the labeling reaction with a range of concentrations from 2-600 pg/slide and ratios of 1:1. For each array design, two slides were hybridized with the test samples reverse-labeled in the labeling reaction. This provided for about four duplicate measurements for each clone, two of one color and two of the other, for each sample.
The differential expression assay was performed by mixing equal amounts of probes from tumor cells and normal cells of the same patient (“matched”) or from tumor cells and normal cells of different patients (“unmatched”) (i.e., the tumor cells are from one patient and the normal cells are from a different patient). The arrays were prehybridized by incubation for about 2 hrs at 60° C. in 5×SSC/0.2% SDS/1 mM EDTA, and then washed three times in water and twice in isopropanol. Following prehybridization of the array, the probe mixture was then hybridized to the array under conditions of high stringency (overnight at 42° C. in 50% formamide, 5×SSC, and 0.2% SDS. After hybridization, the array was washed at 55° C. three times as follows: 1) first wash in 1×SSC/0.2% SDS; 2) second wash in 0.1×SSC/0.2% SDS; and 3) third wash in 0.1×SSC.
The arrays were then scanned for green and red fluorescence using a Molecular Dynamics Generation III dual color laser-scanner/detector. The images were processed using BioDiscovery Autogene software, and the data from each scan set normalized to provide for a ratio of expression relative to normal. Data from the microarray experiments was analyzed according to the algorithms described in U.S. application Ser. No. 60/252,358, filed Nov. 20, 2000, by E. J. Moler, M. A. Boyle, and F. M. Randazzo, and entitled “Precision and accuracy in cDNA microarray data,” which application is specifically incorporated herein by reference.
The experiment was repeated, this time labeling the two probes with the opposite color in order to perform the assay in both “color directions.” Each experiment was sometimes repeated with two more slides (one in each color direction). The level fluorescence for each sequence on the array expressed as a ratio of the geometric mean of 8 replicate spots/genes from the four arrays or 4 replicate spots/gene from 2 arrays or some other permutation. The data were normalized using the spiked positive controls present in each duplicated area, and the precision of this normalization was included in the final determination of the significance of each differential. The fluorescent intensity of each spot was also compared to the negative controls in each duplicated area to determine which spots have detected significant expression levels in each sample.
A statistical analysis of the fluorescent intensities was applied to each set of duplicate spots to assess the precision and significance of each differential measurement, resulting in a p-value testing the null hypothesis that there is no differential in the expression level between the tumor and normal samples of each patient in matched samples or between tumor and normal samples of tissue from different patients in unmatched samples. During initial analysis of the microarrays, the hypothesis was accepted if p>10−3, and the differential ratio was set to 1.000 for those spots. All other spots have a significant difference in expression between the tumor and normal sample. If the tumor sample has detectable expression and the normal does not, the ratio is truncated at 1000 since the value for expression in the normal sample would be zero, and the ratio would not be a mathematically useful value (e.g., infinity). If the normal sample has detectable expression and the tumor does not, the ratio is truncated to 0.001, since the value for expression in the tumor sample would be zero and the ratio would not be a mathematically useful value. These latter two situations are referred to herein as “on/off.” Database tables were populated using a 95% confidence level (p>0.05).
Table 22 (inserted prior to claims) provides the results for gene products expressed by at least 2-fold or greater in cancerous prostate, colon, or breast tissue samples relative to normal tissue samples in at least 20% of the patients tested. Table 22 includes: 1) the SEQ ID NO (“SEQ ID”) assigned to each sequence for use in the present specification; 2) the sequence name (“SEQ NAME”) used as an internal identifier of the sequence; 3) the name assigned to the clone from which the sequence was isolated (“CLONE ID”); 4) the percentage of patients tested in which expression levels (e.g., as message level) of the gene was at least 2-fold greater in cancerous breast tissue than in matched normal tissue (“BREAST PATIENTS >=2×”); 5) the breast number ratios, indicating the number of patients upon which the provided ratio using matched breast tissue was based (“BREAST NUM RATIOS”); 6) the percentage of patients tested in which expression levels (e.g., as message level) of the gene was at least 2-fold greater in cancerous colon tissue than in matched normal tissue (“COLON PATIENTS >=2×”); 7) the colon number ratios, indicating the number of patients upon which the provided ratio using matched colon tissue was based (“COLON NUM RATIOS”); 8) the percentage of patients tested in which expression levels (e.g., as message level) of the gene was at least 2-fold greater in cancerous colon tissue than in unmatched normal tissue (“COLON UM>=2×”); 9) the unmatched colon number ratios, indicating the number of patients upon which the provided ratio using unmatched colon tissue was based (“COLON UM NUM RATIOS”).
TABLE 22
|
|
COLON
BREASTBREASTCOLONCOLONUM
SEQPATIENTSNUMPATIENTSNUMCOLONNUM
IDSEQ NAMECLONE ID>=2xRATIOS>=2xRATIOSUM >=2xRATIOS
|
|
1893544.G06.GZ43_505397M00084443A:E10508
4203559.B18.GZ43_507504M00084700A:C1041.0256413933.3333327
7543590.D19.GZ43_512389M00085031B:E0337.58
8163596.P03.GZ43_512529M00085171D:F05704060.7142928
8393599.K02.GZ43_512892M00085222D:D07704060.7142928
11923665.O06.gz43_521001M00086277B:E0657.5405012
12833756.K15.gz43_533455M00085835B:E1135.29411763435.7142928
12893756.M06.gz43_533313M00085815C:E1147.058823517
13203759.P15.gz43_533844M00085100B:C1263.414634141
1549NT_007592S2.3_105010
1560NT_009296S1.3_142.928
1560NT_009296S1.3_146.23933.327
1560NT_009296S1.3_148.73944.427
1577NT_017582S2.3_661.53955.627
1577NT_017582S2.3_661.53955.627
|
Table 25 (inserted prior to claims) provides the results for other gene products expressed by at least 2-fold or greater in cancerous prostate, colon, or breast tissue sample, which may be metastasized cancer samples, relative to normal tissue samples in at least 20% of the patients tested. For each set of data (i.e., the percentage of patients in which a particular sequence is up-regulated in a cancer tissue) the number of patients (Colon Cancer Patients; Colon Unmatched Met Patients and Colon Match Met Patients) is shown. If a sample is matched, it is matched to a sample from the same patient, if a sample is unmatched, the results obtained from that sample are compared to a pooled sample of an appropriate tissue type from the patients. If a sample is not from a metastasized tissue, it is from a primary tumor.
TABLE 25
|
|
BreastColonProstate
CancerCancerCancer
Tumor/BreastTumor/ColonTumor/
SEQNormalCancerNormalCancerNormal
IDSeq NameSpotID>=2xPatients>=2xPatients>=2x
|
1383541.A16.GZ43_505167586482326.09
1513538.K12.GZ43_504729567752326.00
1813544.A17.GZ43_50556756939
2053544.L13.GZ43_5055146010031.96
2623550.D16.GZ43_5063224210844.7476
3243553.J14.GZ43_5066806023347.371937.11
3313553.K03.GZ43_5065055577326.092321.051922.45
3743556.C15.GZ43_5070736010031.96
3923556.J14.GZ43_5070646023347.371937.11
4023556.M02.GZ43_5068754210844.7476
4083556.N06.GZ43_5069405844020.41
4673562.I02.GZ43_5076395807521.43
5993574.F10.GZ43_5093185807521.43
6033574.G11.GZ43_5093355678221.7423
6053574.I02.GZ43_5091936010031.96
7543590.D19.GZ43_5123896010031.96
7683590.J01.GZ43_5121076010031.96
7773590.L10.GZ43_5122536010031.96
7923596.E08.GZ43_5125986010031.96
8043596.K14.GZ43_5127006010031.96
8143596.O10.GZ43_5126406010031.96
8183596.P07.GZ43_5125936010031.96
8413599.K23.GZ43_5132285742934.69
8413599.K23.GZ43_5132286010031.96
8463599.M24.GZ43_5132463506524.0075
8513599.O06.GZ43_5129606010031.96
8543602.A09.GZ43_5133786010031.96
8733602.K06.GZ43_5133406010031.96
8833605.I19.gz43_5139305675320.41
9233611.I04.gz43_5144586010031.96
9313611.L22.gz43_5147496010031.96
9533614.P11.gz43_5149615677526.0923
9553617.B16.gz43_515411136834.2935
9553617.B16.gz43_5154112587350.0076
9553617.B16.gz43_5154112665859.2176
9583617.H16.gz43_5154175990425.77
9593617.I01.gz43_5151785700823.47
9663617.N14.gz43_5153915765121.43
9683617.P11.gz43_5153455675320.41
9693617.P12.gz43_5153616010031.96
9713620.B03.gz43_5158105765121.43
9793620.G17.gz43_5160396010031.96
9993623.N23.gz43_5165262707828.9576
10053626.G01.gz43_5165513395839.132324.51
10053626.G01.gz43_5165513511339.132323.53
10053626.G01.gz43_5165515892130.432322.45
10093626.M15.gz43_5167815982926.092336.8419
10303632.G01.gz43_5173192593339.22
10323632.K20.gz43_5176276010031.96
10343632.M13.gz43_5175176010031.96
10353632.M19.gz43_5176135742934.69
10353632.M19.gz43_5176136010031.96
10423635.A13.gz43_5178896010031.96
10633638.L10.gz43_5182366010031.96
10743643.I24.gz43_5188415990425.77
11173661.K22.gz43_5197175675320.41
11763664.K19.gz43_5208212593339.22
11793664.P12.gz43_5207145700823.47
11793664.P12.gz43_5207145779721.43
12143666.L23.gz43_5216545311426.47
12243754.B08.gz43_5329506010031.96
12573756.A02.gz43_5332376010031.96
12663756.C16.gz43_5334636010031.96
12723756.E12.gz43_5334015765121.43
12783756.G14.gz43_5334356010031.96
13063759.K05.gz43_5336795990425.77
13253762.A20.gz43_5342936023347.371937.11
13483762.L18.gz43_5342726010031.96
1354Clu8293.con_16010031.96
1372Clu403488.con_16010031.96
1409Clu609914.con_124511
1411Clu621702.con_13506524.0075
1427Clu733840.con_124511
1435Clu777670.con_16010031.96
1441Clu854573.con_15742934.69
1441Clu854573.con_16010031.96
1459Clu1053799.con_15807521.43
1465Clu1054813.con_16023347.371937.11
1471Clu1055326.con_12593339.22
1492Clu1088930.con_12707828.9576
1522Clu1224379.con_14210844.7476
1541Clu1228277.con_16010031.96
1546Clu1259069.con_22584451.96
1546Clu1259069.con_22899649.02
1549Clu1292262.con_15675320.41
1554Clu1292436.con_15990425.77
1573NTN_007592S2.3_105310025.3375
1584NTN_009296S1.3_1136834.2935
1584NTN_009296S1.3_12587350.0076
1584NTN_009296S1.3_12665859.2176
1585NTN_009296S3.3_25675320.41
1600NTN_011512S51.3_33508622.6775
1600NTN_011512S51.3_33682421.3375
1601NTN_017582S2.3_62634569.7476
1615NTN_025842S13.2_12707828.9576
|
Colon
ColonColonColonColonMatchedColon
ProstateUnmatchedUnmatchedMatchedMatchedMet/Match
SEQCancerMet/NormalMetMet/NormalMetTumorMet
IDPatients>=2xPatients>=2xPatients>=2xPatients
|
138
151
18122.2218
20597
26263.643352.7836
3249741.1817
33198
37497
3929741.1817
40263.643352.7836
40898
46798
59998
603
60597
75497
76897
77797
79297
80497
81497
81897
84198
84197
84621.2133
85197
85497
87397
88398
92397
93197
953
95556.673028.577
95557.583355.5636
95566.673344.4436
95897
95998
96698
96898
96997
97198
97997
99915.1533
100510218.1833
100510215.1533
100598
100947.0617
10301020.0033
103297
103497
103598
103597
104297
106397
107497
111798
11761020.0033
117998
117998
12141020.0033
122497
125797
126697
127298
127897
130697
13259741.1817
134897
135497
137297
140924.243326.0923
141121.2133
142724.243326.0923
143597
144198
144197
145998
14659741.1817
14711020.0033
149215.1533
152263.643352.7836
154197
15461020.0033
15461020.0033
154998
155497
15736.063328.5735
158456.673028.577
158457.583355.5636
158466.673344.4436
158598
16009.093325.0036
160012.123333.3336
160178.793366.6736
161515.1533
|
These data provide evidence that the genes represented by the polynucleotides having the indicated sequences are differentially expressed in breast, prostate, cancer as compared to normal non-cancerous breast tissue and are differentially expressed in colon cancer as compared to normal non-cancerous colon tissue
The above methods can be performed to identify genes differentially expressed in cancerous and normal cells of any type of tissue, such as prostate, lung, colon, breast, and the like.
Example 23
Antisense Regulation of Gene Expression
The expression of the differentially expressed genes represented by the polynucleotides in the cancerous cells can be further analyzed using antisense knockout technology to confirm the role and function of the gene product in tumorigenesis, e.g., in promoting a metastatic phenotype.
Methods for analysis using antisense technology are well known in the art. For example, a number of different oligonucleotides complementary to the mRNA generated by the differentially expressed genes identified herein can be designed as antisense oligonucleotides, and tested for their ability to suppress expression of the genes. Sets of antisense oligomers specific to each candidate target are designed using the sequences of the polynucleotides corresponding to a differentially expressed gene and the software program HYBsimulator Version 4 (available for Windows 95/Windows NT or for Power Macintosh, RNAture, Inc. 1003 Health Sciences Road, West, Irvine, Calif. 92612 USA). Factors considered when designing antisense oligonucleotides include: 1) the The expression of the differentially expressed genes represented by the polynucleotides in the cancerous cells can be analyzed using antisense knockout technology to confirm the role and function of the gene product in tumorigenesis, e.g., in promoting a metastatic phenotype.
A number of different oligonucleotides complementary to the mRNA generated by the differentially expressed genes identified herein can be designed as potential antisense oligonucleotides, and tested for their ability to suppress expression of the genes. Sets of antisense oligomers specific to each candidate target are designed using the sequences of the polynucleotides corresponding to a differentially expressed gene and the software program HYBsimulator Version 4 (available for Windows 95/Windows NT or for Power Macintosh, RNAture, Inc. 1003 Health Sciences Road, West, Irvine, Calif. 92612 USA). Factors that are considered when designing antisense oligonucleotides include: 1) the secondary structure of oligonucleotides; 2) the secondary structure of the target gene; 3) the specificity with no or minimum cross-hybridization to other expressed genes; 4) stability; 5) length and 6) terminal GC content. The antisense oligonucleotide is designed so that it will hybridize to its target sequence under conditions of high stringency at physiological temperatures (e.g., an optimal temperature for the cells in culture to provide for hybridization in the cell, e.g., about 37° C.), but with minimal formation of homodimers.
Using the sets of oligomers and the HYBsimulator program, three to ten antisense oligonucleotides and their reverse controls are designed and synthesized for each candidate mRNA transcript, which transcript is obtained from the gene corresponding to the target polynucleotide sequence of interest. Once synthesized and quantitated, the oligomers are screened for efficiency of a transcript knock-out in a panel of cancer cell lines. The efficiency of the knock-out is determined by analyzing mRNA levels using lightcycler quantification. The oligomers that resulted in the highest level of transcript knock-out, wherein the level was at least about 50%, preferably about 80-90%, up to 95% or more up to undetectable message, are selected for use in a cell-based proliferation assay, an anchorage independent growth assay, and an apoptosis assay.
The ability of each designed antisense oligonucleotide to inhibit gene expression is tested through transfection into LNCaP, PC3, 22Rv1, MDA-PCA-2b, or DU145 prostate carcinoma cells. For each transfection mixture, a carrier molecule (such as a lipid, lipid derivative, lipid-like molecule, cholesterol, cholesterol derivative, or cholesterol-like molecule) is prepared to a working concentration of 0.5 mM in water, sonicated to yield a uniform solution, and filtered through a 0.45 μm PVDF membrane. The antisense or control oligonucleotide is then prepared to a working concentration of 100 μM in sterile Millipore water. The oligonucleotide is further diluted in OptiMEM™ (Gibco/BRL), in a microfuge tube, to 2 μM, or approximately 20 μg oligo/ml of OptiMEM™. In a separate microfuge tube, the carrier molecule, typically in the amount of about 1.5-2 nmol carrier/μg antisense oligonucleotide, is diluted into the same volume of OptiMEM™ used to dilute the oligonucleotide. The diluted antisense oligonucleotide is immediately added to the diluted carrier and mixed by pipetting up and down. Oligonucleotide is added to the cells to a final concentration of 30 nM.
The level of target mRNA that corresponds to a target gene of interest in the transfected cells is quantitated in the cancer cell lines using the Roche LightCycler™ real-time PCR machine. Values for the target mRNA are normalized versus an internal control (e.g., beta-actin). For each 20 μl reaction, extracted RNA (generally 0.2-1 μg total) is placed into a sterile 0.5 or 1.5 ml microcentrifuge tube, and water is added to a total volume of 12.5 μl. To each tube is added 7.5 μl of a buffer/enzyme mixture, prepared by mixing (in the order listed) 2.5 μl H2O, 2.0 μl 10× reaction buffer, 10 μl oligo dT (20 pmol), 1.0 μl dNTP mix (10 mM each), 0.5 μl RNAsin® (20 u) (Ambion, Inc., Hialeah, Fla.), and 0.5 μl MMLV reverse transcriptase (50 u) (Ambion, Inc.). The contents are mixed by pipetting up and down, and the reaction mixture is incubated at 42° C. for 1 hour. The contents of each tube are centrifuged prior to amplification.
An amplification mixture is prepared by mixing in the following order: 1× PCR buffer 11, 3 mM MgCl2, 140 μM each dNTP, 0.175 pmol each oligo, 1:50,000 dil of SYBR® Green, 0.25 mg/ml BSA, 1 unit Taq polymerase, and H20 to 20 μl. (PCR buffer II is available in 10× concentration from Perkin-Elmer, Norwalk, Conn.). In 1× concentration it contains 10 mM Tris pH 8.3 and 50 mM KCl. SYBR® Green (Molecular Probes, Eugene, Oreg.) is a dye which fluoresces when bound to double stranded DNA. As double stranded PCR product is produced during amplification, the fluorescence from SYBR® Green increases. To each 20 μl aliquot of amplification mixture, 2 μl of template RT is added, and amplification is carried out according to standard protocols. The results are expressed as the percent decrease in expression of the corresponding gene product relative to non-transfected cells, vehicle-only transfected (mock-transfected) cells, or cells transfected with reverse control oligonucleotides.
Example 24
Effect of Expression on Proliferation
The effect of gene expression on the inhibition of cell proliferation can be assessed in metastatic breast cancer cell lines (MDA-MB-231 (“231”)); SW620 colon colorectal carcinoma cells; SKOV3 cells (a human ovarian carcinoma cell line); or LNCaP, PC3, 22Rv1, MDA-PCA-2b, or DU145 prostate cancer cells.
Cells are plated to approximately 60-80% confluency in 96-well dishes. Antisense or reverse control oligonucleotide is diluted to 2 μM in OptiMEM™. The oligonucleotide-OptiMEM™ can then be added to a delivery vehicle, which delivery vehicle can be selected so as to be optimized for the particular cell type to be used in the assay. The oligo/delivery vehicle mixture is then further diluted into medium with serum on the cells. The final concentration of oligonucleotide for all experiments can be about 300 nM.
Antisense oligonucleotides are prepared as described above (see Example 3). Cells are transfected overnight at 37° C. and the transfection mixture is replaced with fresh medium the next morning. Transfection is carried out as described above in Example 23.
Those antisense oligonucleotides that result in inhibition of proliferation of SW620 cells indicate that the corresponding gene plays a role in production or maintenance of the cancerous phenotype in cancerous colon cells. Those antisense oligonucleotides that inhibit proliferation in SKOV3 cells represent genes that play a role in production or maintenance of the cancerous phenotype in cancerous breast cells. Those antisense oligonucleotides that result in inhibition of proliferation of MDA-MB-231 cells indicate that the corresponding gene plays a role in production or maintenance of the cancerous phenotype in cancerous ovarian cells. Those antisense oligonucleotides that inhibit proliferation in LNCaP, PC3, 22Rv1, MDA-PCA-2b, or DU145 cells represent genes that play a role in production or maintenance of the cancerous phenotype in cancerous prostate cells.
Using the following antisense oligonucleotides: TTGGTTCCCAAGACAAGCCGTGAC (SEQ ID NO:1676); TCTCAACGCTACCAGGCACTCCTTG (SEQ ID NO:1677); GCACAGCCCAAAGTCAAAGGCATTA (SEQ ID NO:1678); CAGGCACTCCTTGGTCAAATGTGGG (SEQ ID NO:1679); GGACAGGGAAAGGAGAGGCTAGTCA (SEQ ID NO:1680) and TGCATTCTCTCCCACATCTCAACGC SEQ ID NO:1681, corresponding to a glutothione transferase omega identified by SEQ ID NOS: 1510 and 1674 (Chiron Candidate Id 21), were used to inhibit proliferation of SW620 colon colorectal carcinoma cells. These antisense molecules reduced glutothione transferase omega RNA expression by approximately 90%.
Example 25
Effect of Gene Expression on Cell Migration
The effect of gene expression on the inhibition of cell migration can be assessed in LNCaP, PC3, 22Rv1, MDA-PCA-2b, or DU145 prostate cancer cells using static endothelial cell binding assays, non-static endothelial cell binding assays, and transmigration assays.
For the static endothelial cell binding assay, antisense oligonucleotides are prepared as described above (see Example 23). Two days prior to use, prostate cancer cells (CaP) are plated and transfected with antisense oligonucleotide as described above (see above). On the day before use, the medium is replaced with fresh medium, and on the day of use, the medium is replaced with fresh medium containing 2 μM CellTracker green CMFDA (Molecular Probes, Inc.) and cells are incubated for 30 min. Following incubation, CaP medium is replaced with fresh medium (no CMFDA) and cells are incubated for an additional 30-60 min. CaP cells are detached using CMF PBS/2.5 mM EDTA or trypsin, spun and resuspended in DMEM/1% BSA/10 mM HEPES pH 7.0. Finally, CaP cells are counted and resuspended at a concentration of 1×106 cells/ml.
Endothelial cells (EC) are plated onto 96-well plates at 40-50% confluence 3 days prior to use. On the day of use, EC are washed 1× with PBS and 50λ DMDM/1% BSA/10 mM HEPES pH 7 is added to each well. To each well is then added 50K (50λ) CaP cells in DMEM/1% BSA/10 mM HEPES pH 7. The plates are incubated for an additional 30 min and washed 5× with PBS containing Ca++ and Mg++. After the final wash, 100 μL PBS is added to each well and fluorescence is read on a fluorescent plate reader (Ab492/Em 516 nm).
For the non-static endothelial cell binding assay, CaP are prepared as described above. EC are plated onto 24-well plates at 30-40% confluence 3 days prior to use. On the day of use, a subset of EC are treated with cytokine for 6 hours then washed 2× with PBS. To each well is then added 150-200K CaP cells in DMEM/1% BSA/10 mM HEPES pH 7. Plates are placed on a rotating shaker (70 RPM) for 30 min and then washed 3× with PBS containing Ca++ and Mg++. After the final wash, 500 μL PBS is added to each well and fluorescence is read on a fluorescent plate reader (Ab492/Em 516 nm).
For the transmigration assay, CaP are prepared as described above with the following changes. On the day of use, CaP medium is replaced with fresh medium containing 5 μM CellTracker green CMFDA (Molecular Probes, Inc.) and cells are incubated for 30 min. Following incubation, CaP medium is replaced with fresh medium (no CMFDA) and cells are incubated for an additional 30-60 min. CaP cells are detached using CMF PBS/2.5 mM EDTA or trypsin, spun and resuspended in EGM-2-MV medium. Finally, CaP cells are counted and resuspended at a concentration of 1×106 cells/ml.
EC are plated onto FluorBlok transwells (BD Biosciences) at 30-40% confluence 5-7 days before use. Medium is replaced with fresh medium 3 days before use and on the day of use. To each transwell is then added 50K labeled CaP. 30 min prior to the first fluorescence reading, 10 μg of FITC-dextran (10K MW) is added to the EC plated filter. Fluorescence is then read at multiple time points on a fluorescent plate reader (Ab492/Em 516 nm).
Those antisense oligonucleotides that result in inhibition of binding of LNCaP, PC3, 22Rv1, MDA-PCA-2b, or DU145 prostate cancer cells to endothelial cells indicate that the corresponding gene plays a role in the production or maintenance of the cancerous phenotype in cancerous prostate cells. Those antisense oligonucleotides that result in inhibition of endothelial cell transmigration by LNCaP, PC3, 22Rv1, MDA-PCA-2b, or DU145 prostate cancer cells indicate that the corresponding gene plays a role in the production or maintenance of the cancerous phenotype in cancerous prostate cells.
Example 26
Effect of Gene Expression on Colony Formation
The effect of gene expression upon colony formation of SW620 cells, SKOV3 cells, MD-MBA-231 cells, LNCaP cells, PC3 cells, 22Rv1 cells, MDA-PCA-2b cells, and DU145 cells can be tested in a soft agar assay. Soft agar assays are conducted by first establishing a bottom layer of 2 ml of 0.6% agar in media plated fresh within a few hours of layering on the cells. The cell layer is formed on the bottom layer by removing cells transfected as described above from plates using 0.05% trypsin and washing twice in media. The cells are counted in a Coulter counter, and resuspended to 106 per ml in media. 10 μl aliquots are placed with media in 96-well plates (to check counting with WST1), or diluted further for the soft agar assay. 2000 cells are plated in 800 μl 0.4% agar in duplicate wells above 0.6% agar bottom layer. After the cell layer agar solidifies, 2 ml of media is dribbled on top and antisense or reverse control oligo (produced as described in above) is added without delivery vehicles. Fresh media and oligos are added every 3-4 days. Colonies form in 10 days to 3 weeks. Fields of colonies are counted by eye. Wst-1 metabolism values can be used to compensate for small differences in starting cell number. Larger fields can be scanned for visual record of differences.
Those antisense oligonucleotides that result in inhibition of colony formation of SW620 cells indicate that the corresponding gene plays a role in production or maintenance of the cancerous phenotype in cancerous colon cells. Those antisense oligonucleotides that inhibit colony formation in SKOV3 cells represent genes that play a role in production or maintenance of the cancerous phenotype in cancerous breast cells. Those antisense oligonucleotides that result in inhibition of colony formation of MDA-MB-231 cells indicate that the corresponding gene plays a role in production or maintenance of the cancerous phenotype in cancerous ovarian cells. Those antisense oligonucleotides that inhibit colony formation in LNCaP, PC3, 22Rv1, MDA-PCA-2b, or DU145 cells represent genes that play a role in production or maintenance of the cancerous phenotype in cancerous prostate cells.
Example 27
Induction of Cell Death Upon Depletion of Polypeptides by Depletion of mRNA (“Antisense Knockout”)
In order to assess the effect of depletion of a target message upon cell death, LNCaP, PC3, 22Rv1, MDA-PCA-2b, or DU145 cells, or other cells derived from a cancer of interest, can be transfected for proliferation assays. For cytotoxic effect in the presence of cisplatin (cis), the same protocol is followed but cells are left in the presence of 2 μM drug. Each day, cytotoxicity is monitored by measuring the amount of LDH enzyme released in the medium due to membrane damage. The activity of LDH is measured using the Cytotoxicity Detection Kit from Roche Molecular Biochemicals. The data is provided as a ratio of LDH released in the medium vs. the total LDH present in the well at the same time point and treatment (rLDH/tLDH). A positive control using antisense and reverse control oligonucleotides for BCL2 (a known anti-apoptotic gene) is included; loss of message for BCL2 leads to an increase in cell death compared with treatment with the control oligonucleotide (background cytotoxicity due to transfection).
Example 28
Functional Analysis of Gene Products Differentially Expressed in Cancer
The gene products of sequences of a gene differentially expressed in cancerous cells can be further analyzed to confirm the role and function of the gene product in tumorigenesis, e.g., in promoting or inhibiting development of a metastatic phenotype. For example, the function of gene products corresponding to genes identified herein can be assessed by blocking function of the gene products in the cell. For example, where the gene product is secreted or associated with a cell surface membrane, blocking antibodies can be generated and added to cells to examine the effect upon the cell phenotype in the context of, for example, the transformation of the cell to a cancerous, particularly a metastatic, phenotype. In order to generate antibodies, a clone corresponding to a selected gene product is selected, and a sequence that represents a partial or complete coding sequence is obtained. The resulting clone is expressed, the polypeptide produced isolated, and antibodies generated. The antibodies are then combined with cells and the effect upon tumorigenesis assessed.
Where the gene product of the differentially expressed genes identified herein exhibits sequence homology to a protein of known function (e.g., to a specific kinase or protease) and/or to a protein family of known function (e.g., contains a domain or other consensus sequence present in a protease family or in a kinase family), then the role of the gene product in tumorigenesis, as well as the activity of the gene product, can be examined using small molecules that inhibit or enhance function of the corresponding protein or protein family.
Additional functional assays include, but are not necessarily limited to, those that analyze the effect of expression of the corresponding gene upon cell cycle and cell migration. Methods for performing such assays are well known in the art.
Example 29
Deposit Information
Deposits of the biological materials in the tables referenced below were made with either the Agricultural Research Service Culture Collection (NRRL), 1815 North University Street, Peoria, Ill. 61604, or with the American Type Culture Collection (ATCC), 10801 University Blvd., Manasas, Va. 20110-2209, under the provisions of the Budapest Treaty, on or before the filing date of the present application. The accession number indicated is assigned after successful viability testing, and the requisite fees were paid. Access to said cultures will be available during pendency of the patent application to one determined by the Commissioner to be entitled to such under 37 C.F.R. § 1.14 and 35 U.S.C. §122. All restriction on availability of said cultures to the public will be irrevocably removed upon the granting of a patent based upon the application. Moreover, the designated deposits will be maintained for a period of thirty (30) years from the date of deposit, or for five (5) years after the last request for the deposit; or for the enforceable life of the U.S. patent, whichever is longer. Should a culture become nonviable or be inadvertently destroyed, or, in the case of plasmid-containing strains, lose its plasmid, it will be replaced with a viable culture(s) of the same taxonomic description.
These deposits are provided merely as a convenience to those of skill in the art, and are not an admission that a deposit is required. A license may be required to make, use, or sell the deposited materials, and no such license is hereby granted. The deposit below was received by the ATCC on or before the filing date of the present application.
TABLE 23
|
|
Cell Lines Deposited with ATCC
ATCC AccessionCMCC Accession
Cell LineDeposit DateNo.No.
|
KM12L4-AMar. 19, 1998CRL-1249611606
Km12CMay 15, 1998CRL-1253311611
MDA-MB-May 15, 1998CRL-1253210583
231
MCF-7Oct. 9, 1998CRL-1258410377
|
In addition, pools of selected clones, as well as libraries containing specific clones, were assigned an “ES” number and a “CMCC” number (both internal references) and deposited with the NRRL. Table 24 provides the NRRL Accession Nos. of the clones deposited as librarires named ES219-ES225 (CMCC5471-CMCC5477, respectively) on Nov. 1, 2001, and of the clones deposited as a library named ES226 (CMCC5478) on Nov. 7, 2001.
TABLE 24
|
|
LibraryCMCCNRRL
IDNumberCloneIdNumber
|
|
ES2195471M00084879B:E01B-30523
ES2195471M00083819B:E10B-30523
ES2195471M00084942C:B10B-30523
ES2195471M00084704C:B09B-30523
ES2195471M00084887C:C07B-30523
ES2195471M00084976B:A08B-30523
ES2195471M00085011B:A01B-30523
ES2195471M00084961A:C07B-30523
ES2195471M00084960D:D02B-30523
ES2195471M00084973A:B06B-30523
ES2195471M00084928D:F06B-30523
ES2195471M00084968C:D10B-30523
ES2195471M00084973A:B06B-30523
ES2195471M00084966A:A08B-30523
ES2195471M00084919C:B04B-30523
ES2195471M00085003C:D03B-30523
ES2195471M00084968A:D01B-30523
ES2195471M00084969D:C11B-30523
ES2195471M00084899D:B01B-30523
ES2195471M00084893C:A12B-30523
ES2195471M00084890D:F09B-30523
ES2195471M00084904A:D03B-30523
ES2195471M00085029A:C02B-30523
ES2195471M00084963D:D07B-30523
ES2195471M00085147C:A04B-30523
ES2195471M00085144B:C12B-30523
ES2195471M00085124B:G05B-30523
ES2195471M00085702B:G11B-30523
ES2195471M00085203A:E06B-30523
ES2195471M00085242A:C06B-30523
ES2195471M00084980D:H08B-30523
ES2195471M00085187B:C11B-30523
ES2195471M00085021C:F06B-30523
ES2195471M00085182B:E04B-30523
ES2195471M00084930D:B08B-30523
ES2195471M00084941B:E07B-30523
ES2195471M00084424D:G07B-30523
ES2195471M00084938B:F12B-30523
ES2195471M00084853D:G03B-30523
ES2195471M00084878B:B12B-30523
ES2195471M00084889B:C02B-30523
ES2195471M00084885D:A12B-30523
ES2195471M00084845A:E02B-30523
ES2195471M00084972B:H03B-30523
ES2195471M00084908A:F03B-30523
ES2195471M00084975A:G05B-30523
ES2195471M00084941C:H04B-30523
ES2195471M00084997D:H09B-30523
ES2195471M00084491A:E08B-30523
ES2195471M00083815C:H08B-30523
ES2195471M00084501A:D06B-30523
ES2195471M00084558D:G08B-30523
ES2195471M00084510C:F02B-30523
ES2195471M00084521C:H11B-30523
ES2195471M00084446A:A05B-30523
ES2195471M00084458A:G06B-30523
ES2195471M00084377D:E08B-30523
ES2195471M00084382A:D06B-30523
ES2195471M00083816B:D08B-30523
ES2195471M00084449B:C09B-30523
ES2195471M00084431C:B02B-30523
ES2195471M00084463A:B07B-30523
ES2195471M00084487D:F04B-30523
ES2195471M00083800C:E07B-30523
ES2195471M00084468C:E07B-30523
ES2195471M00084638A:E10B-30523
ES2195471M00084439B:A08B-30523
ES2195471M00084479D:E10B-30523
ES2195471M00084455D:B03B-30523
ES2195471M00084368D:C02B-30523
ES2195471M00084642D:E08B-30523
ES2195471M00084373A:F08B-30523
ES2195471M00084364C:B06B-30523
ES2195471M00084521B:E11B-30523
ES2195471M00084385B:D03B-30523
ES2195471M00084443C:H06B-30523
ES2195471M00083803C:F03B-30523
ES2195471M00084421C:B11B-30523
ES2195471M00084434B:E06B-30523
ES2195471M00083820B:C03B-30523
ES2195471M00084246B:H03B-30523
ES2195471M00084484C:B11B-30523
ES2195471M00084410C:F10B-30523
ES2195471M00083801B:H03B-30523
ES2195471M00084980C:B07B-30523
ES2195471M00084499C:C11B-30523
ES2195471M00084526C:G09B-30523
ES2195471M00084406C:A01B-30523
ES2195471M00084380D:B07B-30523
ES2195471M00084383B:A11B-30523
ES2195471M00083834C:E02B-30523
ES2195471M00083839A:H03B-30523
ES2195471M00084505C:H08B-30523
ES2195471M00084511D:A02B-30523
ES2195471M00084494C:C01B-30523
ES2195471M00084451D:F06B-30523
ES2195471M00084604A:D02B-30523
ES2195471M00084771D:G03B-30523
ES2195471M00084817A:H11B-30523
ES2195471M00084827D:D04B-30523
ES2195471M00084843D:C06B-30523
ES2195471M00084750C:B08B-30523
ES2195471M00084757A:D01B-30523
ES2195471M00084771D:A01B-30523
ES2195471M00084730B:A09B-30523
ES2195471M00084826B:E11B-30523
ES2195471M00084595C:C07B-30523
ES2195471M00084724A:C02B-30523
ES2195471M00084833A:G07B-30523
ES2195471M00084600D:B10B-30523
ES2195471M00084634C:H02B-30523
ES2195471M00084614D:A08B-30523
ES2195471M00084620B:F05B-30523
ES2195471M00084607A:B03B-30523
ES2195471M00084633A:B12B-30523
ES2195471M00084597A:F06B-30523
ES2195471M00084575A:A11B-30523
ES2195471M00084547B:B10B-30523
ES2195471M00084525A:E08B-30523
ES2195471M00084578B:E12B-30523
ES2195471M00084669A:A05B-30523
ES2195471M00084419C:A09B-30523
ES2195471M00084769C:H03B-30523
ES2195471M00085007A:B03B-30523
ES2195471M00084865D:B04B-30523
ES2195471M00084743D:G01B-30523
ES2195471M00084770B:G12B-30523
ES2195471M00084584B:A02B-30523
ES2195471M00084647C:E12B-30523
ES2195471M00084766D:F12B-30523
ES2195471M00084648D:F05B-30523
ES2195471M00084843A:D06B-30523
ES2195471M00084709C:B02B-30523
ES2195471M00084834B:G02B-30523
ES2195471M00084718D:C04B-30523
ES2195471M00084702B:C12B-30523
ES2195471M00084645D:G02B-30523
ES2195471M00084849B:F11B-30523
ES2195471M00084859C:H05B-30523
ES2195471M00084850D:H02B-30523
ES2195471M00084857B:A09B-30523
ES2195471M00084867A:C11B-30523
ES2195471M00084823A:H01B-30523
ES2195471M00084756B:H01B-30523
ES2195471M00084700D:E09B-30523
ES2195471M00085010C:H01B-30523
ES2195471M00085060B:C05B-30523
ES2195471M00085012C:A08B-30523
ES2195471M00085047D:F03B-30523
ES2195471M00085049B:E03B-30523
ES2195471M00085051C:A01B-30523
ES2195471M00085050A:E11B-30523
ES2195471M00085676C:C04B-30523
ES2195471M00085121A:D10B-30523
ES2195471M00085166D:C10B-30523
ES2195471M00084992D:B02B-30523
ES2195471M00085148B:H01B-30523
ES2195471M00085123B:C04B-30523
ES2195471M00085173B:A08B-30523
ES2195471M00085172C:F06B-30523
ES2195471M00084937D:B04B-30523
ES2195471M00085026D:A01B-30523
ES2195471M00084994D:F11B-30523
ES2195471M00085190C:D10B-30523
ES2195471M00085194D:F04B-30523
ES2195471M00085222D:D07B-30523
ES2195471M00085223A:G01B-30523
ES2195471M00084740C:B08B-30523
ES2195471M00085056A:G12B-30523
ES2195471M00084671A:C12B-30523
ES2195471M00084571C:D05B-30523
ES2195471M00084587B:H07B-30523
ES2195471M00084582C:H03B-30523
ES2195471M00084618C:A03B-30523
ES2195471M00084687A:A03B-30523
ES2195471M00085038A:C06B-30523
ES2195471M00084722A:H12B-30523
ES2195471M00084676B:E02B-30523
ES2195471M00084615D:H12B-30523
ES2195471M00084659C:G05B-30523
ES2195471M00084536B:A03B-30523
ES2195471M00084929C:B02B-30523
ES2195471M00084652D:G11B-30523
ES2195471M00084611B:A11B-30523
ES2195471M00084530D:G07B-30523
ES2195471M00084527C:H07B-30523
ES2195471M00084545C:C05B-30523
ES2195471M00084535D:C12B-30523
ES2195471M00084684C:D02B-30523
ES2195471M00084679D:G12B-30523
ES2195471M00084734A:E04B-30523
ES2195471M00084696D:H04B-30523
ES2205472M00084724D:F04B-30524
ES2205472M00084559B:F10B-30524
ES2205472M00084707D:H03B-30524
ES2205472M00084525D:H01B-30524
ES2205472M00084578C:G09B-30524
ES2205472M00084710B:G07B-30524
ES2205472M00084537B:C05B-30524
ES2205472M00084560A:G08B-30524
ES2205472M00085129B:C02B-30524
ES2205472M00084620D:E05B-30524
ES2205472M00084576A:E12B-30524
ES2205472M00084720A:A01B-30524
ES2205472M00084654A:E04B-30524
ES2205472M00084596D:E10B-30524
ES2205472M00084646A:D02B-30524
ES2205472M00084572D:F07B-30524
ES2205472M00084620A:E08B-30524
ES2205472M00084553B:F04B-30524
ES2205472M00084614D:B07B-30524
ES2205472M00084604D:D08B-30524
ES2205472M00084722D:A03B-30524
ES2205472M00084958C:B03B-30524
ES2205472M00084523C:A05B-30524
ES2205472M00085166C:A08B-30524
ES2205472M00084467A:D06B-30524
ES2205472M00084890C:A06B-30524
ES2205472M00084609C:F10B-30524
ES2205472M00084413C:A11B-30524
ES2205472M00084834A:A03B-30524
ES2205472M00085172A:G05B-30524
ES2205472M00085146B:C01B-30524
ES2205472M00085038A:B10B-30524
ES2205472M00084246A:D03B-30524
ES2205472M00084967B:D09B-30524
ES2205472M00085035D:E04B-30524
ES2205472M00084736B:H03B-30524
ES2205472M00085025A:D11B-30524
ES2205472M00084900C:A04B-30524
ES2205472M00085127C:C03B-30524
ES2205472M00084424A:G07B-30524
ES2205472M00085131D:A06B-30524
ES2205472M00084987B:H12B-30524
ES2205472M00084967C:D10B-30524
ES2205472M00084420A:G02B-30524
ES2205472M00084452B:F07B-30524
ES2205472M00084705C:D01B-30524
ES2205472M00085156A:G04B-30524
ES2205472M00084447D:F03B-30524
ES2205472M00084495B:C11B-30524
ES2205472M00084745A:A08B-30524
ES2205472M00084458B:G05B-30524
ES2205472M00084449C:C01B-30524
ES2205472M00084867B:A03B-30524
ES2205472M00084680A:F08B-30524
ES2205472M00084585B:D06B-30524
ES2205472M00084835D:H03B-30524
ES2205472M00084685C:B12B-30524
ES2205472M00084500C:D01B-30524
ES2205472M00084469A:C09B-30524
ES2205472M00084381C:A05B-30524
ES2205472M00084477C:C07B-30524
ES2205472M00084647C:A05B-30524
ES2205472M00084687C:F12B-30524
ES2205472M00084756C:H01B-30524
ES2205472M00084565D:F08B-30524
ES2205472M00084560B:F12B-30524
ES2205472M00084640D:A08B-30524
ES2205472M00084443A:E10B-30524
ES2205472M00084521A:E11B-30524
ES2205472M00085019C:D05B-30524
ES2205472M00084587C:A07B-30524
ES2205472M00084616A:G03B-30524
ES2205472M00084732B:A04B-30524
ES2205472M00084666A:C04B-30524
ES2205472M00084633A:H05B-30524
ES2205472M00084510C:F05B-30524
ES2205472M00084648B:F06B-30524
ES2205472M00084700D:H04B-30524
ES2205472M00084506C:A05B-30524
ES2205472M00084475C:G11B-30524
ES2205472M00084673B:H11B-30524
ES2205472M00084595D:D08B-30524
ES2205472M00084636C:A06B-30524
ES2205472M00084612C:B01B-30524
ES2205472M00084644A:H05B-30524
ES2205472M00084602D:B09B-30524
ES2205472M00084584B:G07B-30524
ES2205472M00084678C:C11B-30524
ES2205472M00084546C:C06B-30524
ES2205472M00084755A:D02B-30524
ES2205472M00084536D:F07B-30524
ES2205472M00084699A:G05B-30524
ES2205472M00084438D:H04B-30524
ES2205472M00084766B:F02B-30524
ES2205472M00084703B:D09B-30524
ES2205472M00084856B:D03B-30524
ES2205472M00084857A:G05B-30524
ES2205472M00084868B:D01B-30524
ES2205472M00084823D:E05B-30524
ES2205472M00084485C:B04B-30524
ES2205472M00084910D:E07B-30524
ES2205472M00084996B:D08B-30524
ES2205472M00084487D:F07B-30524
ES2205472M00084824C:C10B-30524
ES2205472M00084949B:B12B-30524
ES2205472M00084746B:B04B-30524
ES2205472M00084944D:E05B-30524
ES2205472M00084851C:F10B-30524
ES2205472M00084849A:H08B-30524
ES2205472M00084843A:G01B-30524
ES2205472M00084921C:E04B-30524
ES2205472M00084742A:F07B-30524
ES2205472M00083799D:F10B-30524
ES2205472M00084760D:D09B-30524
ES2205472M00084845C:H05B-30524
ES2205472M00084927A:C01B-30524
ES2205472M00084935B:E10B-30524
ES2205472M00084974D:F11B-30524
ES2205472M00084935C:E07B-30524
ES2205472M00084503D:G10B-30524
ES2205472M00084907C:C01B-30524
ES2205472M00084893C:B01B-30524
ES2205472M00083803B:F11B-30524
ES2205472M00084945A:D10B-30524
ES2205472M00084765B:A10B-30524
ES2205472M00084455D:G03B-30524
ES2205472M00084874D:E03B-30524
ES2205472M00084889C:A04B-30524
ES2205472M00084846B:H07B-30524
ES2205472M00084967B:B10B-30524
ES2205472M00084838A:F12B-30524
ES2205472M00084885A:C01B-30524
ES2205472M00084823A:H06B-30524
ES2205472M00084958B:E10B-30524
ES2205472M00084399B:E05B-30524
ES2205472M00084880B:D03B-30524
ES2205472M00084877D:G07B-30524
ES2205472M00084406B:C03B-30524
ES2205472M00084856B:A12B-30524
ES2205472M00084888D:A11B-30524
ES2205472M00083831D:H11B-30524
ES2205472M00084481D:C06B-30524
ES2205472M00083834B:F09B-30524
ES2205472M00084707D:B08B-30524
ES2205472M00084976C:C12B-30524
ES2205472M00085201C:C12B-30524
ES2205472M00084379D:A05B-30524
ES2205472M00084392C:G06B-30524
ES2205472M00084492B:F03B-30524
ES2205472M00085697B:G05B-30524
ES2205472M00085683B:B10B-30524
ES2205472M00084988B:B08B-30524
ES2205472M00084969C:H11B-30524
ES2205472M00084988C:G03B-30524
ES2205472M00085123A:E07B-30524
ES2205472M00084988C:A04B-30524
ES2205472M00084363A:C02B-30524
ES2205472M00084975A:G05B-30524
ES2205472M00084431C:G08B-30524
ES2205472M00084972B:H03B-30524
ES2205472M00084376A:E06B-30524
ES2205472M00084859C:D09B-30524
ES2205472M00084957B:H07B-30524
ES2205472M00085053D:D04B-30524
ES2205472M00084425A:A01B-30524
ES2205472M00084367D:E06B-30524
ES2205472M00084938B:A11B-30524
ES2205472M00085051A:G03B-30524
ES2205472M00083817B:G09B-30524
ES2205472M00085229B:C10B-30524
ES2205472M00085178D:F01B-30524
ES2205472M00084980D:D02B-30524
ES2205472M00085228B:C10B-30524
ES2205472M00085243A:D07B-30524
ES2205472M00085031B:E03B-30524
ES2205472M00085164C:G05B-30524
ES2205472M00085031C:D05B-30524
ES2205472M00084251D:C05B-30524
ES2205472M00085027A:C02B-30524
ES2205472M00084248D:H09B-30524
ES2205472M00085209C:F11B-30524
ES2205472M00084368D:D03B-30524
ES2205472M00083818A:E09B-30524
ES2205472M00084980C:E06B-30524
ES2205472M00084248B:C06B-30524
ES2205472M00085244C:D03B-30524
ES2205472M00084987A:D09B-30524
ES2205472M00084994A:H04B-30524
ES2205472M00084970D:E08B-30524
ES2205472M00085038D:D10B-30524
ES2205472M00085035B:C12B-30524
ES2205472M00085184D:B08B-30524
ES2215473M00084666C:A06B-30525
ES2215473M00084657C:E01B-30525
ES2215473M00084540B:B08B-30525
ES2215473M00084415C:C05B-30525
ES2215473M00084812A:C02B-30525
ES2215473M00084396B:B03B-30525
ES2215473M00084844B:H08B-30525
ES2215473M00084877D:H09B-30525
ES2215473M00084925C:G01B-30525
ES2215473M00084970A:C11B-30525
ES2215473M00084961C:F01B-30525
ES2215473M00084391B:D06B-30525
ES2215473M00084694D:F04B-30525
ES2215473M00084698B:D02B-30525
ES2215473M00084388A:G03B-30525
ES2215473M00084973A:C01B-30525
ES2215473M00084423C:G11B-30525
ES2215473M00084497D:D03B-30525
ES2215473M00084889D:G06B-30525
ES2215473M00084959B:C07B-30525
ES2215473M00084432B:C05B-30525
ES2215473M00084489A:D12B-30525
ES2215473M00084748A:D09B-30525
ES2215473M00084962C:F10B-30525
ES2215473M00084767B:D10B-30525
ES2215473M00084711B:A05B-30525
ES2215473M00084743A:E03B-30525
ES2215473M00084466B:E01B-30525
ES2215473M00084450C:A09B-30525
ES2215473M00084492C:B05B-30525
ES2215473M00084487B:A06B-30525
ES2215473M00084480B:A05B-30525
ES2215473M00084764D:G08B-30525
ES2215473M00084743D:H04B-30525
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Retrieval of Individual Clones from Deposit of Pooled Clones. Where the biological deposit is composed of a pool of cDNA clones or a library of cDNA clones, the deposit was prepared by first transfecting each of the clones into separate bacterial cells. The clones in the pool or library were then deposited as a pool of equal mixtures in the composite deposit. Particular clones can be obtained from the composite deposit using methods well known in the art. For example, a bacterial cell containing a particular clone can be identified by isolating single colonies, and identifying colonies containing the specific clone through standard colony hybridization techniques, using an oligonucleotide probe or probes designed to specifically hybridize to a sequence of the clone insert (e.g., a probe based upon unmasked sequence of the encoded polynucleotide having the indicated SEQ ID NO). The probe should be designed to have a Tm of approximately 80° C. (assuming 2° C. for each A or T and 4° C. for each G or C). Positive colonies can then be picked, grown in culture, and the recombinant clone isolated. Alternatively, probes designed in this manner can be used to PCR to isolate a nucleic acid molecule from the pooled clones according to methods well known in the art, e.g., by purifying the cDNA from the deposited culture pool, and using the probes in PCR reactions to produce an amplified product having the corresponding desired polynucleotide sequence.
Those skilled in the art will recognize, or be able to ascertain, using not more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such specific embodiments and equivalents are intended to be encompassed by the following claims.
All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
Patent Application
20060234246
