P53-induced apoptosis

Abstract

The most well-documented biochemical property of p53 is its ability to transcriptionally activate genes. Many of the genes which are activated by p53 expression prior to the onset of apoptosis are predicted to encode proteins which could generate or respond to oxidative stress, including one that is implicated in apoptosis within plant meristems. p53 may result in apoptosis through a three-step process: (I) the transcriptional induction of specific redox-related genes; (ii) the formation of reactive oxygen species (ROS); and (iii) the oxidative degradation of mitochondrial components, rapidly leading to cell death. Transcription of other genes is decreased by p53. Examination of the level of transcription of p53-induced or repressed genes can be used to determine p53 status, to diagnose cancer, and to evaluate cytotoxicity or carcinogenicity of a test agent.

Description

TECHNICAL FIELD OF THE INVENTION

This invention is related to genes and proteins involved in cell cycle control and tumorigenesis. These genes can be used diagnostically and therapeutically because of their role in cancers.

BACKGROUND OF THE INVENTION

The inactivation of the p53 gene in a large fraction of human cancers has inspired an intense search for the encoded protein's physiologic and biologic properties. Expression of p53 induces either a stable growth arrest or programmed cell death (apoptosis). In human colorectal cancers (CRC), the growth arrest is dependent on the transcriptional induction of p21WAF1/CIP1 (1), but the biochemical mechanisms underlying the development of p53-dependent apoptosis are largely unknown (2). Thus, there is a continuing need in the art for discovering new genes which are regulated by p53 and genes which are related to cell cycle control and tumorigenesis.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide methods of diagnosing cancer or determining p53 status in a sample suspected of being neoplastic.

It is another object of the present invention to provide an isolated and purified nucleic acid molecule which is identified by a SAGE tag.

It is an object of the present invention to provide an isolated nucleotide probe comprising at least 12 nucleotides of a rat nucleic acid molecule identified by a SAGE tag.

Another object of the invention is to provide methods and kits for evaluating cytotoxicity or carcinogenicity of an agent.

It is still another object of the invention to provide a DNA construct useful for screening drugs as anti-neoplastic agents.

It is even another object of the invention to provide a preparation of antibodies.

These and other objects of the invention are provided by one or more of the embodiments described below. One embodiment of the invention provides a method of diagnosing cancer or determining p53 status in a sample suspected of being neoplastic. The level of transcription of an RNA transcript in a first sample of a first tissue is compared to the level of transcription of the transcript in a second sample of a second tissue. The first tissue is suspected of being neoplastic and the second tissue is a normal human tissue. The first and second tissue are of the same tissue type. The transcript is identified by a tag selected from the group consisting of SEQ ID NOS:10, 15-22, 26, 27, and 30. The first sample is characterized as neoplastic or as having a mutant p53 when transcription is found to be the same or lower in the first sample than in the second sample.

Another embodiment of the invention provides a method of diagnosing cancer or determining p53 status in a sample suspected of being neoplastic. The level of transcription of an RNA transcript in a first sample of a first tissue is compared to the level of transcription of the transcript in a second sample of a second tissue. The first tissue is suspected of being neoplastic, and the second tissue is a normal human tissue. The first and second tissue are of the same tissue type. The transcript is identified by a tag selected from the group consisting of SEQ ID NOS:37-67. The first sample is categorized as neoplastic or as having a mutant p53 when transcription is found to be the same or higher in the first sample than in the second sample.

Yet another embodiment of the invention provides an isolated and purified nucleic acid molecule which comprises a SAGE tag selected from the group consisting of SEQ ID NOS:15, 16, 17, 19, 21, 22, and 30.

Even another embodiment of the invention provides an isolated nucleotide probe comprising at least 12 contiguous nucleotides of a human nucleic acid molecule. The human nucleic acid molecule comprises a SAGE tag selected from the group consisting of SEQ ID NOS:15, 16, 17, 19, 21, 22, and 30.

A further embodiment of the invention provides a kit for evaluating toxicity or carcinogenicity of an agent. The kit comprises at least 2 probes. The probes comprise at least 12 contiguous nucleotides of a human nucleic acid molecule. The human nucleic acid molecule comprises a SAGE tag selected from the group consisting of SEQ ID NOS:15, 16, 17, 19, 21, 22, and 30.

Another embodiment of the invention provides a kit for evaluating cytotoxicity or carcinogenicity. The kit comprises at least 2 probes. The probes comprise a SAGE tag selected from the group consisting of SEQ ID NOS:15, 16, 17, 19, 21, 22, and 30.

Even another embodiment of the invention provides a method for evaluating cytotoxicity or carcinogenicity of an agent. A test agent is contacted with a human cell. The level of transcription of a transcript in the human cell after contacting with the agent is determined. An agent which increases the level of a transcript identified by a SAGE tag selected from the group consisting of SEQ ID NOS: 10, 15-22, 26, 27, and 30, or an agent which decreases the level of a transcript identified by a SAGE tag selected from the group consisting of SEQ ID NOS:37-67 is a potential cytotoxin or carcinogen.

Another embodiment of the invention provides a method to determine the neoplastic status or p53 status of a cell. ROS levels in a first sample of a first tissue are compared to ROS levels in a second sample of a second tissue. The first tissue is or is suspected of being neoplastic, and the second tissue is a normal human tissue. Elevated levels of ROS in the first sample indicate expression of p53, and low levels of ROS in the first sample indicate lack of expression of p53. Lack of expression of p53 is an indicator of neoplasia.

Still another embodiment of the invention provides a DNA construct for screening drugs as anti-neoplastic agents. The DNA construct comprises a reporter gene under the control of a PIG-3 promoter. The reporter gene is 3′ and covalently linked to the PIG-3 promoter. The PIG-3 promoter comprises the sequence CAGCTTGCCCACCCATGCTC (SEQ ID NO:1).

A further embodiment of the invention provides a method of diagnosing cancer or determining p53 status in a sample suspected of being neoplastic. Cells of a test sample are treated with a DNA-damaging agent. The level of transcription of an RNA transcript in cells of the sample is compared to the level of transcription of the transcript in cells of the sample which are not subject to said treating. The transcript is identified by a tag selected from the group consisting of SEQ ID NOS:10, 15-22, 26, 27, and 30. The sample is characterized as neoplastic or as having a mutant p53 when transcription is found to be the same or lower in the treated cells than in the untreated cells.

Another embodiment of the invention provides a method of diagnosing cancer or determining p53 status in a sample suspected of being neoplastic. Cells of a test sample are treated with a DNA-damaging agent. The level of transcription of an RNA transcript in the cells is compared to the level of transcription of the transcript in cells of the sample which are not subject to said treating. The transcript is identified by a tag selected from the group consisting SEQ ID NOS:37-67. The sample is categorized as neoplastic or as having a mutant p53 when transcription is found to be the same or higher in the treated cells than in the untreated cells.

Even another embodiment of the invention provides a preparation of antibodies which specifically bind to a PIG protein having an amino acid sequence selected from the group consisting of SEQ ID NOS:81, 83, 84, 86, 87, and 88.

Still another embodiment of the invention provides a method of diagnosing cancer or determining p53 status in a sample suspected of being neoplastic. The level of a PIG protein having an amino acid sequence selected from the group consisting of SEQ ID NOS:79-88 and the amino acid sequence encoded by SEQ ID NO:72 in a first sample of a first tissue is compared to the level of the PIG protein in a second sample of a second tissue. The first tissue is suspected of being neoplastic, and the second tissue is a normal human tissue. The first and second tissue are of the same tissue type. The first sample is categorized as neoplastic or as having a mutant p53 when the level of the PIG protein is found to be the same or lower in the first sample than in the second sample.

Yet another embodiment of the invention provides a method of diagnosing cancer or determining p53 status in a sample suspected of being neoplastic. The level of a protein of Table 2 in a first sample of a first tissue is compared to the level of the protein of Table 2 in a second sample of a second tissue. The first tissue is suspected of being neoplastic, and the second tissue is a normal human tissue. The first and second tissue are of the same tissue type. The first sample is categorized as neoplastic or as having a mutant p53 when the level of the protein of Table 2 is found to be the same or higher in the first sample than in the second sample.

Even another embodiment of the invention provides a kit for evaluating toxicity or carcinogenicity of an agent. The kit comprises at least 2 antibodies which specifically bind to a PIG protein having an amino acid sequence selected from the group consisting of SEQ ID NOS:81, 83, 84, 86, 87, and 88.

Still another embodiment of the invention provides a method for evaluating cytotoxicity or carcinogenicity of an agent. A test agent is contacted with a human cell. The level of a PIG protein having an amino acid sequence selected from the group consisting of SEQ ID NOS:79-88 and the amino acid sequence encoded by SEQ ID NO:72 or of a protein of Table 2 in the human cell is determined after contacting with the agent. An agent which increases the level of the PIG protein or an agent which decreases the level of the protein of Table 2 is identified as a potential cytotoxin or carcinogen.

A further embodiment of the invention provides a method of diagnosing cancer or determining p53 status in a sample suspected of being neoplastic. Cells of a test sample are treated with a DNA-damaging agent. The level of a PIG protein having an amino acid sequence selected from the group consisting of SEQ ID NOS:79-88 and the amino acid sequence encoded by SEQ ID NO:72 in cells of the sample is compared to the level of the PIG protein in cells of the sample which are not subject to said treating. The sample is categorized as neoplastic or as having a mutant p53 when the level of the PIG protein is found to be the same or lower in the treated cells than in the untreated cells.

Even another embodiment of the invention provides a method of diagnosing cancer or determining p53 status in a sample suspected of being neoplastic. Cells of a test sample are treated with a DNA-damaging agent. The level of a protein of Table 2 in cells of the sample is compared to the level of the protein of Table 2 in cells of the sample which are not subject to said treating. The sample is categorized as neoplastic or as having a mutant p53 when the level of the protein of Table 2 is found to be the same or higher in the treated cells than in the untreated cells.

These and other embodiments of the invention provide the art with tools for assessing p53 status in cells, which can provide diagnostic and prognostic information useful in the evaluation of patients and the management of cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 A. Summary of SAGE data. For each of 7,202 different transcripts identified, the ratio of their abundances in two libraries is plotted. The y-axis indicates the number of tags expressed at the ratio indicated on the x-axis. Bars representing tags exhibiting less than 5-fold differences in expression are shown in green, and those induced or repressed more than 8-fold are shown in blue and red, respectively.

FIG. 1 B. Northern blot analysis after Ad-p53 infection. Representative Northern blots are shown for several transcripts identified by SAGE to be expressed at higher levels in p53-expressing cells at the indicated times post infection. Uninfected cells (column marked “0”) and cells infected with Ad-lacZ for 48 hrs (column marked “B”) were included for comparison. EF1 is a control transcript expressed at relatively equal levels in cells 16 hours after infection with Ad-p53 and Ad-acZ. The SAGE tag abundances (16 hours after infection) are included at the right.

FIG. 2 A. Schematic of PIG3 gene, illustrating intron-exon structure and promoter region. Numbers refer to nucleotides relative to the 5′ end of the cDNA. The fragments used for the luciferase constructs had their distal ends at the Eag I site within exon 1 and their 5′ ends at either the Apa LI or Nsi I sites (FULL and DEL, respectively). The 53-binding site located at nucleotides 328-308 is indicated, with the upper case letters corresponding to the highly conserved residues that were altered in one of the oligonucleotides used for immunoprecipitation.

FIG. 2B. p 53-induction of the PIG3 promoter. Fragments encompassing 5.6 or 0.7 kb (FULL and DEL, respectively) of the PIG3 gene promoter were cloned upstream of a luciferase reporter and transfected into the indicated cell types in the presence of wt and mutant p53 expression vectors. The levels of luciferase activity were determined in cell lysates 24 hours after transfection.

FIG. 2 C. In vitro binding assay with end-labeled fragments containing wild type (WT) and mutant (M) p53 binding sites. A fragment containing thirteen copies of a p53 binding site from the WAF1 promoter region 3026 was used as a control (C). The “input” lanes contained 0.5% of the amount of fragment used in the binding assays.

FIG. 3 . Sequences of selected genes identified through SAGE. In each case, the indicated gene is compared to the homologue from the non-human species that revealed a clue to its possible function. The amino acid sequences were aligned using Macaw Version 2.0.3, and the most significant similarities are indicated by shading. With the exception of PIG6, the cloned human sequences appeared to be full length with respect to the coding region. Accession numbers are provided in Table 1.

FIG. 3A , human PIG3 (SEQ ID NO:87) and rat micGST (SEQ ID NO:88).

FIG. 3B , human PIG3 (SEQ ID NO:81) and Vigna TED2 (SEQ ID NO:89).

FIG. 3C , human PIG8 (SEQ ID NO:90) and C. elegans f37c12 (SEQ ID NO:91). FIG. 3D , human PIG6 (SEQ ID NO:92) and Drosophila PUT1 (SEQ ID NO:93).

FIG. 4 . Oxidative stress and mitochondrial damage in p53-mediated apoptosis.

FIG. 4 A. DLD-1 cells were infected with Ad-p53 or control (Ad-lacZ) viruses and harvested after 27, 35, or 42 hours. Cells were incubated with CM-DCF-DA, a probe of ROS, or NAO, a probe of the mitochondrial membrane cardiolipin, and analyzed by flow cytometry. The mean fluorescence of the control cells is indicated by vertical lines in each box. The pro-oxidant drug menadione was used as a positive control to induce oxidative stress. An increase in ROS and a decrease in cardiolipin concentration could be clearly observed by cytometry at 27 hours and increased as the p53-expressing cells entered apoptosis.

FIG. 4 B. Time course of apoptosis-related events following p53 expression. Cells were infected with Ad-p53 at 0 hours and PIG3 expression (&Circlesolid;) was quantitated by densitometry of Northern blots. ROS production (∘) was assessed with lucigenin; glutathione depletion exhibited a similar time course (not shown). Cardiolipin concentration (Δ) was assessed with nonyl-acridine orange staining. Caspase activation (▴) was assessed by cleavage of PARP, and chromatin condensation/fragmentation (⋄) was assessed by staining with DAPI.

DETAILED DESCRIPTION

The most well-documented biochemical property of p53 is its ability to transcriptionally activate genes. Of 7,202 transcripts induced by p53 expression prior to the onset of apoptosis, only 14 (0.19%) are found at markedly higher levels in p53-expressing cells than in control cells. The genes encoding these transcripts are termed PIGS (p53-induced genes). Many of these genes are predicted to encode proteins which could generate or respond to oxidative stress, including one that is implicated in apoptosis within plant meristems. Thus, p53 may result in apoptosis through a three-step process: (i) the transcriptional induction of specific redox-related genes; (ii) the formation of reactive oxygen species (ROS); and (iii) the oxidative degradation of mitochondrial components, rapidly leading to in cell death.

Using the SAGE tags disclosed in Tables 1 and 2, transcripts can be evaluated for enhanced or reduced expression, respectively. A SAGE tag is a short sequence tag, preferably 10 or 11 base pairs, which is generated from defined positions within each mRNA molecule. Expression patterns are deduced from the abundance of individual tags. The altered expression can provide an indication of the status of the p53 genes in the cells, which themselves reflect the neoplastic status of cells. While the presence of wild-type p53 is not determinative of normalcy, the presence of mutant p53 is an indication of neoplasia.

The tags which are shown in Table 1 identify transcripts which are enhanced by p53; the tags of Table 2 identify transcripts which are decreased by p53. Wild-type p53 is required for these modulations. Thus failure to so-modulate is an indication of mutant p53 in the cell. Similarly, DNA-damaging agents which cause apoptosis do so via wild-type p53. In the absence of wild-type p53 these agents cannot induce transcription of the Table 1 tag-identified transcripts nor can they decrease transcription of the Table 2 tag-identified transcripts. Thus, analysis of the status of these transcripts can provide an indication of the presence or absence of wild-type p53.

Cells can be compared from suspect tissues to normal tissues. Similarly, a suspect or test tissue sample can be treated with a DNA-damaging agent and the response of the cells in the tissue assessed. The response assessed is the induction or reduction in the transcripts identified by the tags. Tags “identify” transcripts by hybridization to them. This hybridization can be determined using any method of measuring transcription, including but not limited to Northern blots, quantitative RT PCR, etc. Conditions for optimizing hybridization signals and minimizing background are known in the art and can be selected by the skilled artisan. Preferably an assay is done with at least two, five, or ten of the transcripts which are known to be modulated by p53. More preferably one or more of the tags used is selected from SEQ ID NOS:15-17, 19, 21, 22, or 30. Suitable DNA-damaging agents include adriamycin, mitomycin, alkylating agents, and γ- and UV-radiation.

Isolated and purified nucleic acid molecules which include a SAGE tag particularly SEQ ID NOS:15-17, 19, 21, 22, or 30, are also provided. These can be made using the SAGE tags to isolate a full length RNA, which is then reverse transcribed using reverse transcriptase to form cDNA. Alternatively the SAGE tags can be used to identify clones from cDNA libraries using hybridization. The SAGE tags can also be used as primers to generate PCR products which contain the SAGE tags. Any such method known in the art can be used. Isolated and purified nucleic acid molecules are free of other nucleic acid molecules with which they are found in cells. Preferably they are also free of the genes to which they are adjacent in the chromosome.

Nucleotide probes are typically less than full length genes and can be labeled so that they can be used in hybridization experiments. Such probes are typically at least 12 contiguous nucleotides in length. Probes of the invention can comprise a SAGE tag of Tables 1 and 2, particularly SEQ ID NOS:15-17, 19, 21, 22, or 30, or can comprise a different portion of a transcript or cDNA molecule identified by such SAGE tags.

Kits can be formulated for evaluating toxicity or carcinogenicity of test agents. The kits comprise at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 probes which are complementary to the transcripts identified by the SAGE tags of Table 1 and 2. Just as DNA-damaging agents induce apoptosis via p53, which can be measured by measuring the induction or repression of expression of specific transcripts, so can other as yet unknown agents. Such agents which cause DNA damage are likely to be toxic or carcinogenic. Thus, human cells can be contacted with a test agent, and the levels of one or more transcripts identified by a SAGE tag in Table 1 or 2 can be measured. If the agent causes the modulation which is caused by the introduction of wild-type p53 or the modulation which is caused by DNA-damaging agents in wild-type p53-containing cells, then the agent is a suspected carcinogen or toxic agent.

Reactive Oxygen Species PROS) production can also be used as an indicator of p53 status and hence neoplasia. Levels of ROS can be determined and compared between cells of a tissue which is suspected of being neoplastic and normal cells. Elevated levels of ROS indicate expression of p53, and low levels indicate lack of p53 expression. These levels can be measured after contacting the cells with an agent which induces DNA damage. Alternatively a test sample can be tested before and after treatment with DNA damaging agents. The ability to induce ROS indicates a wild-type p53. Any method for measuring ROS can be used, including but not limited to carboxymethyl dichlorofluorescein diacetate and flow cytometry, nonylacridine orange as a probe for cardiolipin, lucigenin chemiluminescence, and intracellular glutathione.

DNA constructs which contain a reporter gene under the transcriptional control of a PIG promoter can be used to test agents for the ability to induce apoptosis. Such agents have potential use as anti-neoplastic agents. One such construct contains the PIG-3 promoter which contains the p53-binding site CAGCTTGCCCACCCATGCTC (SEQ ID NO:1). Other PIG promoters can be used similarly.

PIG-specific antibodies can be used in assays to determine the status of the p53 gene in cells similar to those described above employing SAGE tags. Proteins or polypeptides encoded by PIGs 1-7 and 9-12 (PIG proteins) can be purified by any method known in the art or produced by recombinant DNA methods or by synthetic chemical methods and used as immunogens, to obtain a preparation of antibodies which specifically bind to a PIG protein, preferably to PIG 3, 6, 7, 10, 11, or 12. The antibodies can be used to detect wild-type PIG proteins in human tissue and fractions thereof.

Preparations of polyclonal or monoclonal PIG antibodies can be made using standard methods known in the art. The antibodies specifically bind to epitopes present in PIG proteins. Preferably, the PIG epitopes are not present in other human proteins. Typically, at least 6, 8, 10, or 12 contiguous amino acids are required to form an epitope. However, epitopes which involve non-contiguous amino acids may require more, e.g., at least 15, 25, or 50 amino acids. Antibodies which specifically bind to PIG proteins provide a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in Western blots or other immunochemical assays. Preferably, antibodies which specifically bind PIG proteins do not detect other proteins in immunochemical assays and can immunoprecipitate PIG proteins from solution.

Antibodies which specifically bind to PIG proteins, particularly to PIG 3, 6, 7, 10, 11, or 12, can be purified by methods well known in the art. Preferably, the antibodies are affinity purified, by passing antiserum over a column to which a PIG protein or polypeptide is bound. The bound antibodies can then be eluted from the column, for example, using a buffer with a high salt concentration.

As disclosed above, wild-type p53 is required to modulate the level of transcripts identified in Tables 1 and 2, and the presence of mutant p53 is an indication of neoplasia. For example, wild-type p53 increases transcription of genes shown in Table 1 and decreases transcription of genes shown in Table 2. The status of the p53 gene in a tissue suspected of being neoplastic can be determined by comparing the levels of one or more of the products of genes whose transcription is modulated by wild-type p53 in the suspect tissue with the level of a PIG protein in a tissue which is normal.

Such comparisons can be made by any methods known in the art. Preferably, antibodies which specifically bind to the protein products of the modulated genes are used, for example in radioimmunoassays or immunocytochemical methods, as is known in the art. Antibodies which specifically bind to the proteins of Table 2 can be used to measure the levels of the proteins of Table 2. Antibodies which specifically bind to PIGs 1-7 and 9-12, particularly those which specifically bind to PIG 3, 6, 7, 10, 11, and 12, can be used to measure the levels of PIG proteins.

The same or a lower level of a PIG protein in the suspect tissue indicates the presence of mutant p53. Similarly, the same or a higher level of a protein of Table 2 in the suspect tissue indicates the presence of mutant p53. The levels of two, 3, 4, 5, 6, 7, 8, 9, or 10 or more proteins can be compared. Detection of binding of PIG-specific antibodies to PIG proteins, or of antibodies which specifically bind to the proteins of Table 2, can also be used to determine if a suspect tissue contains a wild-type or mutant p53 gene after treatment with DNA-damaging agents.

Antibodies of the invention which specifically bind to PIG 3, 6, 7, 10, 11, or 12 can be provided in kits, for evaluating cytotoxicity or carcinogenicity of test agents, as described above. A kit can contain one, 2, 3, 4, 5, or 6 of the antibodies of the invention.

The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention. The following methods were used in the examples reported below.

Methods

Cells and RNA. All cell lines used in this study were obtained from the American Type Culture Collection and were cultured in McCoy's medium supplemented with 10% fetal bovine serum (FBS). Cells were infected with recombinant adenoviruses containing either the p53 gene or the β-galactosidase gene (26) at a multiplicity of infection of 10-100. RNA was purified from cells at various times after infection using the MessageMaker Kit (Gibco/BRL). Northern blot analysis was performed as described (26).

SAGE. SAGE was performed as previously described (3, 27). Briefly, polyadenylated RNA was converted to double-stranded cDNA with a BRL synthesis kit using the manufacturer's protocol with the inclusion of primer biotin-5′-T18-3′. The cDNA was cleaved with NlaIll, and the 3′-terminal cDNA fragments were bound to streptavidin-coated magnetic beads (Dynal). After ligation of oligonucleotides containing recognition sites for BsmFI, the linkered-cDNA was released from the beads by digestion with BsmFI. The released tags were ligated to one another, concatemerized, and cloned into the Sph I site of pZero 21.0 (Invitrogen). Colonies were screened with PCR using M13 forward and M13 reverse primers. PCR products containing inserts of greater than 300 bp (>20 tags) were sequenced with the TaqFS DyePrimer kit and analyzed using a 377 ABI automated sequencer Perkin Elmer).

Statistical analysis. 53,022 and 51,853 tags were identified from DLD-1 cells infected with Ad-p53 and Ad-lacZ, respectively. The two libraries were compared using the SAGE program group (3). Corrections for tags containing linker sequences and other potential artifacts were made as described previously (27). Of 104,875 total tags identified, 3,181 were excluded from analysis on this basis. Monte Carlo simulations revealed that the computational analyses had a >99% probability of detecting a transcript expressed at an abundance of 0.00005 in either RNA sample.

cDNA clones. Cellular mRNA from Ad-p53-infected cells was used to prepare cDNA as described for the SAGE libraries, except that the 3′ primer contained an additional M13 forward sequence between the olio-DT tract and the biotinylated 5′ residue. To determine the sequence of the transcript from which an individual tag was derived, this cDNA was used as a template for PCR, employing an M13 forward primer and a primer containing the tag sequence. In other cases, mRNA from Ad-p53-infected cells was used to construct a cDNA library in the ZAP Express vector (Stratagene) and the library was screened by hybridization with oligonucleotides corresponding to tags, as described (3). Of 14 tags identified by SAGE as differentially expressed in p53-expressing cells, 8 corresponding genes could be identified simply by searching public databases, particularly those including expressed sequence tags. In 5 cases, one of the two strategies described above was used to obtain the corresponding PIG. In one of the 14 cases PIG13), no cDNA clone could be recovered corresponding to the tag sequence.

Analysis of PIG3 genomic structure. An arrayed BAC library (Research Genetics) was screened by PCR using the following primers derived from the 5′ end of the PIG3 gene: 5′-GGC-CAG-GAG-TAA-GTA-ACT-3′ (SEQ ID NO:2) and 5′-GCC-CTG-GTC-TGC-CGC-GGA-3′ (SEQ ID NO:3). Eco RI fragments encompassing the PIG3 coding sequences were subcloned into pBR322 and partially sequenced to determine the intron-exon borders. A 6.1 kb Apa LI fragment whose 3′ end was at a Eag I site 308 bp downstream of the transcription start site was then cloned into a promoterless luciferase reporter vector (FIG. 2 A). This fragment was completely sequenced by primer walking. Subclones were then generated by restriction endonuclease digestion. Luciferase activity was determined after co-transfection with expression vectors encoding wt or H175R mutant p53. For in vitro p53 binding experiments, oligonucleotides containing two copies of the predicted p53-binding site ( FIG. 2A ) were subcloned into a modified pBR322 vector, excised as a ˜260 bp restriction fragment, and end-labeled. Immunochemical assays were performed as described previously (28).

Flow cytometry and other assays. Cells were collected with the aid of trypsin and incubated with CM-H2DCF-DA or NAO (Molecular Probes, Eugene, Oreg.) at concentrations of 10 and 0.4 μM, respectively, for 20 minutes at 37° C. prior to analysis by flow cytometry (14,15). To determine the fraction of apoptotic cells after various treatments, cells were stained with the DNA-binding dye H33258 and evaluated by fluorescence microscopy or flow cytometry as described (1). Superoxide production was assessed with lucigenin (29). In brief, 4-5×10 6 cells were collected with rubber policeman and resuspended in 1 ml of Earle's Balanced Salt Solution (Gibco BRL 14015-069, Life Technologies). Dark-adapted lucigenin (bis-N-methulacridinium nitrate, Sigma M8010) was added to the samples to a final concentration of 20 μM. Light emission was detected using a Berthold LB 9505C luminometer for 60 minutes at 37° C. Glutathione concentrations were measured using an assay kit purchased from Oxford Biomedical Res. Inc. according to the manufacturer's instructions. Caspase activation was assessed by cleavage of PARP (polyADP-ribose polymerase). Lysates from cells infected with Ad-p53 were Western blotted with an anti-PARP antibody, and the cleavage fragments were quantitated by densitometry (4).

EXAMPLE 1

To evaluate the patterns of gene expression following p53 expression, we employed SAGE, a technique which allows the quantitative evaluation of cellular mRNA populations (3). In brief, the method revolves around short sequence “tags” (11 bp), generated from defined positions within each mRNA molecule. Expression patterns are deduced from the abundance of individual tags. To induce apoptosis, the colorectal cancer line DLD-1, containing an inactive endogenous p53 gene, was infected with a replication defective adenovirus encoding p53 (Ad-p53). As previously shown, DLD-1 cells are among the ˜50% of CRC lines that undergo apoptosis in response to p53 (4). RNA was purified from cells 16 hours after infection, at least 8 hours before the onset of morphological signs of apoptosis.

A total of 101,694 tags were analyzed, approximately half from cells infected with Ad-p53 and half from cells infected with a control virus (Ad-lacZ) encoding β-galactosidase. These tags corresponded to 7,202 different transcripts. Comparison of the two SAGE libraries indicated a remarkable similarity in expression profiles (FIG. 1 A). Of the 7,202 transcripts detected, only 14 (0.19%) were expressed at levels more than 10-fold greater in p53-expressing than in control cells; conversely, only 20 transcripts were expressed at levels less than 10-fold lower in the p53-expressing cells.

As previous data indicated p53-mediated transcriptional activation as the likely basis of p53 action (5), we concentrated on the 14 tags appearing at higher levels in the p53-expressing cells. The mRNA transcripts corresponding to 13 of these tags were successfully identified (Table 1). In each case, the induction was confirmed by Northern blot analysis (examples in FIG. 1 B). Only two of these genes (called PIGs, for p53-induced genes) had been implicated as targets of p53-transcriptional activation (1, 2, 5, 6) and seven had not previously been described at all. Other genes previously implicated in p53-mediated responses were induced to lower levels (e.g., MDM2, thrombospondin) or not at all (e.g., bax and cyclin G1) in the human CRC cells studied here (4).

EXAMPLE 2

PIGs were induced at relatively short times after p53 expression, at least 12 hours prior to any morphological or biochemical signs of apoptosis FIG. 1 B). This time course suggested that PIGs were directly induced by the transcriptional activation properties of p53. To formally test this conjecture in a representative case, we evaluated the genomic structure and sequence of PIG3. By screening a bacterial artificial chromosome (SAC) library, a genomic clone was identified that contained all PIG3 coding sequences. The gene was localized to chromosome 2p (see Methods), and the intron-exon structure and sequence of the promoter region were determined.

A 6.1 kb ApaLI fragment of genomic DNA containing the presumptive promoter was then cloned upstream of a luciferase reporter gene (FIG. 2 A). The resulting construct was transfected into three different human cell lines together with wild type (wt) or mutant p53.

As shown in FIG. 2B , wt p53 induced substantial activity through the PIG3 promoter in all three lines. Mutant p53 had no transcriptional activation capacity. Analysis of a truncated promoter showed that the p53-responsive elements lay within a fragment containing only 862 bp of sequence upstream of the PIG3 transcription start site (FIG. 2 A). Determination of the sequence of this 6.1 kb Apa L1 fragment revealed a single 20 bp sequence predicted to bind p53, located at 308 nt upstream of the transcription start site p53. A DNA fragment containing two copies of this sequence, but not a derivative of this fragment altered at critical residues, was found to bind strongly to p53 in vitro FIG. 2 C).

EXAMPLE 3

As a further test of the p53-dependency of PIG3 induction, we determined whether PIG3 could be induced by endogenous p53 rather than through the exogenous Ad-p53 source. Six CRC cancer cell lines were each treated with adriamycin, a DNA-damaging and apoptotic-inducing agent known to increase endogenous p53 levels. PIG3, like p21, was found to be strongly induced in the three lines with wild-type p53 genes, but not in the three lines with mutant p53 genes.

EXAMPLE 4

The sequences of the PIGs provided important clues to their potential functions (Table 1). In particular, several were predicted to encode proteins with activities related to the redox status of cells. PIG12 is a novel member of the microsomal glutathione transferase family of genes (FIG. 3 A). PIG8 is the human homologue of a mouse gene (Ei24) whose expression is induced in a p53-dependent manner by etoposide, a quinone known to generate reactive oxygen species (ROS) (6) (FIG. 3 C). PIG6 is a homolog of proline oxidoreductase (FIG. 3 D), a mitochondrial enzyme that catalyzes the first step in the conversion of proline to glutamate (7). Glutamate is one of the three amino acids required for formation of glutathione, a major regulator of cellular redox status. The p21 gene, which can also be considered a PIG, can be induced by ROS, independently of p53 (8). PIG4 encodes a serum amyloid protein that can be induced by oxidative stress (9). PIG1 belongs to the galectin family, members of which can stimulate superoxide production (10). PIG7 has been shown to be induced by TNF-β, a known inducer of oxidative stress. PIG3 is a novel gene that is highly related to TED2, a plant NADPH oxidoreductase (11) (FIG. 3 B). Interestingly, TED2 is one of the few genes implicated in the apoptotic process necessary for the formation of plant meristems (11). The closest relative of PIG3 in mammals is an NADPH-quinone oxidoreductase which has been shown to be a potent generator of ROS (12).

Previous studies have shown that ROS are powerful inducers of apoptosis (13). The SAGE-based characterization of p53-induced genes suggested that p53 might induce apoptosis by stimulating the production of ROS. To test this hypothesis, the production of ROS was measured in p53-expressing cells using carboxy-methyl dichlorofluorescein (DCF-diacetate (CM-DCF-DA) and flow cytometry (14). This analysis showed that ROS were induced following Ad-p53 infection and that ROS continued to increase as apoptosis progressed (FIG. 4 A). The magnitude of the increase in ROS, as assessed by DCF fluorescence, was similar in p53-expressing cells to that observed in cells treated with the powerful oxidant menadione (FIG. 4 A). No change in DCF fluorescence was observed following infection with a control adenovirus (FIG. 4 A).

As an assay for the functional consequences of ROS production, we examined the cellular content of cardiolipin, a major component of the mitochondrial membrane which is especially sensitive to cellular oxidation (15). Using nonyl-acridine orange (NAO) as a probe, cardiolipin was found to decrease soon after p53-induced ROS was detected (FIG. 4 A), demonstrating significant injury to a major mitochondrial component.

EXAMPLE 5

To determine the specificity of PIG expression for the p53-dependent apoptotic process, we performed experiments with other inducers of ROS or apoptosis. We found that PIGs were not expressed simply as a result of ROS production, as none were induced following treatment with menadione and only p21 was induced by hydrogen peroxide in DLD-1 cells. Similarly, the specificity of PIG induction for p53-dependent apoptosis was confirmed by the demonstration that other inducers of apoptosis (indomethacin or ceramide) did not result in the expression of any PIG, despite extensive cell death.

EXAMPLE 6

To clarify the relationship between p53 expression, PIG activation, ROS production, and apoptosis, we carried out more detailed time course experiments. PIG induction began within six hours after Ad-53 infection (FIG. 1 B and FIG. 4 B), while intracellular ROS production, as assessed with lucigenin chemiluminescence, could first be observed at 18 hours (FIG. 4 B). This ROS production led to oxidative stress, as evidenced by a 48+/−12% decrease in intracellular glutathione concentration at 21 hours. Mitochondrial lipid degradation (NAO) was not observed until three to six hours after the onset of a measurable ROS increase and was accompanied by morphologic (chromatin condensation and fragmentation) and biochemical (caspase-mediated degradation of PARP) signs of apoptosis (FIG. 4 B). These observations are consistent with previous studies showing that mitochondrial damage is rapidly followed by classic signs of programmed cell death (13).

The time courses illustrated in FIG. 4B suggest a cascade wherein p53 transcriptionally induces redox-controlling genes resulting in the production of ROS, in turn leading to oxidative damage to mitochondria and apoptosis. To determine whether these steps were causally associated, we inhibited each step with specific pharmacologic agents and determined the effect of this inhibition on other components of the pathway.

First, cells were treated with the transcriptional inhibitor 5,6-dichlorobenimidizole riboside (DRB) at 8 hours following Ad-p53 infection (16). Though p53 expression was already near maximal at this time, DRB was found to block apoptosis at 24 hours by 83+/−3% as well as to inhibit the expression of PIGs. The translational inhibitor cycloheximide, when given up to 8 hours following Ad-p53 infection, was found to similarly block apoptosis (by 79% at 24 hours). Thus both transcription and translation were required for p53-induced apoptosis in CRC cells, as observed in some other systems (2, 5) and as expected for classic programmed cell death (2, 5).

Second, p53-expressing cells were treated with pyrrolidine dithiocarbamate (PDTC), an anti-oxidant which has been shown to block ROS-associated apoptosis (17). PDTC was indeed able to block the apoptosis elicited by p53. However, PDTC inhibits many enzymes, and its specificity is questionable (17). We therefore treated cells with diphenyleneiodonium chloride (DPI), a specific inhibitor of flavin-dependent oxidoreductases which has been used to block production of ROS in a variety of systems (18). Cells were treated with DPI 12 hours after Ad-p53 infection, when PIG production was already underway. PIG3 expression, apoptosis, and ROS production were measured 12 hours later. DPI (25 μM) did not inhibit PIG3 production but did inhibit ROS production by 71-85% and inhibited apoptosis by 73-77% in three independent experiments (FIG. 4 D).

Finally, we treated cells with bongkrekic acid (BA), a specific inhibitor of mitochondrial ATP translocase which can block the mitochondrial permeability transition pore opening thought to be required for ROS-dependent forms of apoptosis (13). When cells were treated 12 hours after Ad-p53 infection, BA was found to inhibit neither PIG3 expression nor ROS production, but inhibited subsequent apoptosis by 86-93% (FIG. 4 D). BA was non-toxic at the dose used (100 μM). While BA inhibited the p53-apoptotic process dependent on ROS production, it had no effect on the p53-mediated growth arrest dependent on p21 as assessed by flow cytometry.

The gene expression profile, time courses, and pharmacologic inhibition studies reported above strongly support a three step model underlying p53's induction of apoptosis. We propose that p53 transcriptionally activates a specific subset of genes, including oxidoreductases, long before any morphological or biochemical evidence of cell death (Table 1 and FIG. 4 B). The proteins encoded by these genes then collectively increase the content of ROS, which in turn damage mitochondria. Leakage of calcium and proteinaceous components from damaged mitochondria then stimulate the caspases that are ubiquitously activated during the apoptotic process. (19-22).

Data from several experimental systems are consistent with this model. For example, apoptosis induced by irradiation, which is dependent on p53 in certain cell types, has been suggested to proceed through a process involving ROS and mitochondrial damage (23). Additionally, an SV40 large T antigen mutant, which binds p53 only at the permissive temperature, was shown to induce apoptosis at the non-permissive temperature through a ROS-related mechanism (24). More recently, it was shown that p53-induced apoptosis in smooth muscle cells is ROS-dependent (25). Though the basis for ROS production and the involvement of mitochondria were not investigated in these previous studies, they suggest that the events we observed in CRC cells are unlikely to be cell-type or species specific and may often underlie p53-associated apoptotic processes. The fact that one of the PIGs is highly related to Ted2, an oxidoreductase implicated in plant cell apoptosis (11), and that apoptosis in plants may also proceed through a ROS-directed pathway (11), adds further interest to this model.

Though observations by us and others are consistent with this model, they raise several unanswered questions. For example, we do not yet know which of the PIGS, are primarily responsible for the induction of ROS. We suspect that their combination, rather than any single one, is necessary for ROS generation. This conjecture is supported by preliminary experiments which demonstrate that PIG3 alone does not induce apoptosis when overexpressed. Though we have concentrated on the most highly induced PIGs, the SAGE analysis revealed at least 26 other genes which were induced by p53 to significant but lower levels than p21 and PIG1-PIG13. Some of these genes may play a role in redox regulation.

It is also not known why some cells enter into apoptosis following p53 expression while others undergo a prolonged growth arrest (4). The possibility that PIGs are only induced in the former has been excluded by examination of PIG expression in such lines; most PIGs were induced by p53 in each of ten CRC lines tested, regardless of whether the cells underwent apoptosis or growth arrest. A more likely possibility is that different cells have different capacities to cope with generators of oxidative stress and that cells with a low capacity succumb to apoptosis. This possibility is supported by numerous studies which show that the response to ROS varies significantly with cell type and growth conditions (13). Hopefully, the experiments and genes reported here will open a new window into the p53 apoptotic process that will facilitate inquiry into these issues.

References

1. Waldman, T., Kinzler, K. W. & Vogelstein, B. p21 is necessary for the p53-mediated G1 arrest in human cancer cells. Cancer Res. 55, 5187-5190 (1995).

2. Oren, M. Relationship of p53 to the control of apoptotic cell death. Semin. Cancer Biol. 5, 221-227 (1994).

3. Velculescu, V. E., Zhang, L., Vogelstein, B. & Kinzler, K. W. Serial Analysis Of Gene Expression. Science 270, 484-487 (1995).

4. Polyak, K., Waldman, T., He, T.-C., Kinzler, K. W. & Vogelstein, B. Genetic determinants of p53 induced apoptosis and growth arrest. Genes & Dev. 10, 1945-1952 (1996).

5. Levine, A. J. p53, the cellular gatekeeper for growth and division. Cell 88, 323-331 (1997).

6. Lehar, S. M., et al. Identification and cloning of Ei24, a gene induced by p53 in etoposide-treated cells. Oncogene 12, 1181-1187 (1996).

7. Hayward, D. C., et al. The sluggish-A gene of Drosophila melanogaster is expressed in the nervous system and encodes proline oxidase, a mitochondrial enzyme involved in glutamate biosynthesis. Proc. Natl. Acad. Sci. U.S.A. 90, 2979-2983 (1993).

8. Russo, T., et al. A p53-independent pathway for activation of WAF1/CIP1 expression following oxidative stress. J. Biol. Chem. 270, 29386-29391 (1995).

9. Rienhoff, H. Y., Jr., Huang, J. H., Li, X. X. & Liao, W. S. Molecular and cellular biology of serum amyloid A. Mol. Biol. Med. 7, 287-298 (1990).

10. Yamaoka, A., Kuwabara, I., Frigeri, I. G. & Liu, F. T. A human lectin, galectin-3 (epsilon bp/Mac-2) stimulates superoxide production by neutrophils. J. Immunol. 154, 3479-3487 (1995).

11. Greenberg, J. T. Programmed cell death: A way of life for plants. Proc. Natl. Acad. Sci. U.S.A. 93, 12094-12097 (1996).

12. Rao, P. V., Krishna, C. M. & Zigler, J. S., Jr. Identification and characterization of the enzymatic activity of zeta-crystallin from guinea PIG lens. A novel NADPH:quinone oxidoreductase. J. Biol. Chem. 267, 96-102 (1992).

13. Kroemer, G., Zamzami, N. & Susin, S. A. Mitochondrial control of apoptosis. Immun. Today 18, 45-51 (1997).

14. Zamzami, N., et al. Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. 3. Exp. Med. 181, 1661-1672 (1995).

15. Petit, P. X., et al. Alterations in mitochondrial structure and function are early events of dexamethasone-induced thymocyte apoptosis. J. Cell. Biol. 130, 157-167 (1995).

16. Tamm, I. & Sehgal, P. B. Halobenzimidazole ribosides and RNA synthesis of cells and viruses. Adv. Virus Res. 22, 187-258 (1978).

17. Orrenius, S., Nobel, C. S. I., van den Dobbelsteen, D. J., Burkitt, M. J. & Slater, A. F. G. Dithiocarbamates and the redox regulation of cell death. Biochem. Soc. Transact. 24, 1032-1038 (1996).

18. Holland, P. C., Clark, M. G., Bloxham, D. P. &Lardy, H. A. Mechanism of action of the hypoglycemic agent diphenyleneiodonium. J. Biol. Chem. 248, 6050-6056 (1973).

19. Korsmeyer, S. J. Regulators of cell death. Trends Gen. 11, 101-105.

20. Susin, S. A., et al. Bcl-2 inhibits the mitochondrial release of an apoptogenic protease. J. Exp. Med. 184, 1331-1341 (1996).

21. Yang, J., et al. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275, 1129-1132 (1997).

22. KMuck, R. M., Bossy-Wetzel, E., Green, D. R. & Newmeyer, D. D. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 275, 1132-1136 (1997).

23. Borek, C. Radiation and chemically induced transformation: free radicals, antioxidants and cancer. Br. J. Cancer Suppl. 8, 74-86 (1987).

24. Vayssiere, J. L., Petit, P. X., Risler, Y. & Mignotte, B. Commitment to apoptosis is associated with changes in mitochondrial biogenesis and activity in cell lines conditionally immortalized with simian virus 40. Proc. Natl. Acad. Sci. U. S. A. 91, 11752-11756 (1994).

25. Johnson, T. M., Yu, Z.-X., Ferrans, V. J., Lowenstein, R. A. & Finkel, T. Reactive oxygen species are downstream mediators of p53-dependent apoptosis. Proc. Natl. Acad. Sci. U. S. A. 93, 11848-11852 (1996).

26. El-Deiry, W. S., Tokino, T., Velculescu, V. E., Levy, D. B., Parsons, R., Trent, J. M., Lin, D., Mercer, W. E., Kinzler, K. W. & Vogelstein, B. WAF1, a potential mediator of p53 tumor supression. Cell 75, 817-825 (1993).

27. Velculescu, V. E., et al. Characterization of the yeast transcriptome. Cell 88 (1997).

28. El-Deiry, W. S., Kern, S. E., Pietenpol, J. A., Kinzer, K. W. & Vogelstein, B. Definition of a consensus binding site for p53. Nature Gen. 1, 45-49 (1992).

29. Faulkner, K. & Fridovich, I. Lurinol and lucigenin as detectors for 0 2 ,-.Free Rad. Biol.&Med. 15, 447-451 (1993).

TABLE 1 1
SEQ
ID
NO:	SAGE TAG	ACCESSION	DESCRIPTION
4	CCCGCCTCTT	D38112	mitochondrial 16S rRNA
4	CCCGCCTCTT	T10098	seq816 human cDNA clone
			b4HB3MA-COT8-HAP-Ft
4	CCCGCCTCTT	T10208	seq907 human cDNA clone
			b4HB3MA-COT8-HAP-Ft
4	CCCGCCTCTT	T26521	AB291H2F human cDNA clone LLAB291H2
			3′.
4	CCCGCCTCTT	W27281	28g3 human retina cDNA randomly primed
			sublibrary
4	CCCGCCTCTT	T17062	NIB250 human cDNA 3′end similar to human
			mitochondrial mRNA
5	AATCTGCGCC	M13755	human interferon-induced 17-kDa/15-kDa
			protein mRNA*
5	AATCTGCGCC	M21786	human interferon-induced 15-Kd protein
			(ISG) gene*
6	GTGACCACGG	K03432	18S rRNA
7	TTTCCTCTCA	X57348	human mRNA (clone 9112).
8	TGCCTGCACC	X61683	human gene for cystatin C exon 3
8	TGCCTGCACC	X05607	human mRNA for cysteine proteinase inhibitor
			precursor
9	TCACCCACAC	R01174	ye77b03.s1 human cDNA clone 123725 3′
9	TCACCCACAC	N95827	zb66e05.s1 human cDNA clone 308576 3′
10	TAAACCTGCT	U06643	PIG1, human keratinocyte lectin 14 (HKL-14)
			mRNA*
10	TAAACCTGCT	L07769	PIG1, human galectin-7 mRNA, complete
			CDS.*
11	CCCAAGCTAG	X54079	human mRNA for heat shock protein HSP27
12	AGCCCGCCGC	AF001294	human IPL (IPL) mRNA
12	AGCCCGCCGC	N29541	yw89f12.s1 human cDNA clone 259439 3′
13	GACATCAAGT	Y00503	human mRNA for keratin 19.
13	GACATCAAGT	J03607	human 40-kDa keratin intermediate filament
			precursor
14	TGTCCTGGTT	U03106	human wild-type p53 activated fragment-1
			(WAF1)*
14	TGTCCTGGTT	U09579	human melanoma differentiation associated
			(mda-6)*
14	TGTCCTGGTT	L26165	human DNA synthesis inhibitor mRNA,
			complete CDS.*
14	TGTCCTGGTT	L25610	human cyclin-dependent kinase inhibitor
			mRNA*
15	AGCTCACTCC	AF010314	PIG10, homologous to none*
16	AGGCTGTCCA	AF010315	PIG11, homologous to none*
17	TGAGTCCCTG	AF010316	PIG12, microsomal GST homolog*
18	CCCTCCTCCG	F19653	PIG2, human EST sequence (011-X4-27)
			from skeletal muscle*
18	CCCTCCTCCG	Z49878	PIG2, Guanidinoacetate N-methyltransferase*
19	GAGGCCAACA	AF010309	PIG3, quinone oxidoreductase homologue
19	GAGGCCAACA	H42923	yo10e11.s1 human cDNA clone 177548 3′.
19	GAGGCCAACA	W07320	za94c09.r1 Soares fetal lung NbHL19W
			human
20	TGGGGCCGCA	U33271	PIG5, human normal keratinocyte mRNA,
			clone B4, partial*
21	TCCTTGGACC	AF010311	PIG36, homologous to Drosophila PUT1,
			partial*
22	CTGGGCCTGA	AF010312	PIG7*
23	AGCTGGTTTCC	AF010313	PIG8, human homolog of mouse EI24*
24	GAGGTGCCGG	J00277	human (genomic clones lambda-[SK2-T2,
		J00206	HS578T]; cDNA clones RS-[3, 4, 6])
		J00276	c-Ha-ras1 proto-oncogene, complete coding
		K00954	sequence
24	GAGGTGCCGG	W25059	zb67e08.r1 Soares fetal lung NbHL19W
			human
25	ACAACGTCCA	T16546	NIB1466 human cDNA 3′end
25	ACAACGTCCA	D85815	human DNA for rhoHP1
26	GTGCGGAGGA	X56653	PIG4, human SAA2 alpha gene, exon 3 and
			exon 4*
26	GTGCGGAGGA	X51439	PIG4, human mRNA for serum amyloid A
			(SAA) protein partial*
26	GTGCGGAGGA	X51441	PIG4, human mRNA for serum amyloid A
			(SAA) protein partial*
26	GTGCGGAGGA	X51442	PIG4, human mRNA for serum amyloid A
			(SAA) protein partial*
26	GTGCGGAGGA	X51445	PIG4, human mRNA for serum amyloid A
			(SAA) protein partial*
26	GTGCGGAGGA	M23698	PIG4, human serum amyloid A1 (SAA1)
			mRNA, complete*
26	GTGCGGAGGA	M23699	PIG4, human serum amyloid A2-alpha
			(SAA2) mRNA*
26	GTGCGGAGGA	M26152	PIG4, human serum amyloid A (SAA)
			mRNA, complete*
26	GTGCGGAGGA	M10906	PIG4, human serum amyloid A (SAA)
			mRNA*
26	GTGCGGAGGA	H45773	PIG4, yp23c09.r1 human cDNA clone
			188272 5′ simil*
26	GTGCGGAGGA	T28677	PIG4, E5T51616 human cDNA 5′ end similar
			to serum*
27	CGTCCCGGAG	U33822	PIG9, human tax1-binding protein TXBP181
			mRNA, complete*
27	CGTCCCGGAG	D52048	PIG9, human fetal brain cDNA 5′-end
			GEN-064D09*
28	GTGCTCATTC	AB000584	human mRNA for TGF-beta superfamily
			protein
29	GCTGACTCAG	M99425	human thrombospondin mRNA, 3′ end.
30	AGATGCTGCA		PIG13
31	CTCAGACAGT	AA046881	EST homologous to 40S ribosomal protein
32	TCCGGCCGCG		NO MATCH
33	AGCCACTGCA		Alu repeat
34	GCTTTTAAGG	L06498	human ribosomal protein S20 (RPS20) mRNA
35	GGGCCAATAA	D29121	human keratinocyte cDNA, clone 142
35	GGGCCAATAA	AA178918	human cDNA clone 612020
36	AAGGGCTCTT	M20560	human lipocortin-III mRNA
36	AAGGGCTCTT	M63310	human 1,2-cyclic-inositol-phosphate
			phosphodiesterase (ANX3) mRNA
1 Gene assignments were based on the following list of GenBank sequences (GenBank Release 94). In each case, tentative assignments were based on the identification of a 10 bp SAGE tag adjacent to a N1aIII site. The final assignment was further refined by using an 11 bp SAGE tag and elimination of non 3′ end N1aIII sites and genomic sequences. In some cases,
# the assignment was confirmed by Northern blot analysis as indicated by the asterisk following the description. In other cases, a single assignment could not be made, and more than one gene is listed.

TABLE 2 2
SEQ
ID		ACCESSION
NO:	SAGE TAG	NUMBER	DESCRIPTION
37	GTAAGTGTAC	J01415	12S rRNA
38	TGTACCTGTA	K00558	human alpha-tubulin mRNA
39	AACGACCTCG	V00599	human mRNA fragment encoding beta-
			tubulin
40	AGTTTGTTAG	M33011	human carcinoma-associated antigen
			GA733-2 mRNA
41	GACTCGCCCA	M98326	human P1-Cdc46 mRNA
42	GGGCCAATAA	D29121	human keratinocyte cDNA, clone 142
42	GGGCCAATAA	AA178918	human cDNA clone 612020
43	GGGTTTTTAT	L28809	human dbpB-like protein mRNA
44	AGAAATACCA	AA455253	human cDNA clone 814816 3′
45	TACCATCAAT	J02642	human glyceraldehyde 3-phosphate
			dehydrogenase mRNA
46	GGATTGTCTG	M34081	human small nuclear ribonucleoprotein
			particle SmB mRNA
47	TACTAGTCCT	X15183	human mRNA for 90-kDa heat-shock
			protein
48	AATATTGAGA	U62962	human Int-6 mRNA, complete CDs
49	GAGGGAGTTT	U14968	human ribosomal protein L27a mRNA
50	AAGGGCGCGG	M20560	human lipocortin-III mRNA
50	AAGGGCGCGG	M63310	human 1,2-cyclic-inositol-phosphate
			phosphodiesterase (ANX3) mRNA
51	TTCACAAAGG	X61970	human mRNA for macropain subunit zeta
52	CTGCACTTAC	D28480	human mRNA for hMCM2
53	GATCCCAACT	V00594	human mRNA for metallothionein from
			cadmium-treated cells
54	GGGAAGCAGA	X77770	mitochondrial mRNA
55	GCTTTCTCAC	K00365	human mitochondrial Ser-tRNA
56	TTCATTATAA	M26708	human prothymosin alpha mRNA
57	TAAGGAGCTG	X77770	human RPS26 mRNA
58	TGAGGGAATA	M10036	human triosephosphate isomerase mRNA
59	GGGATGGCAG	M98326	human transfer valyl-tRNA synthetase
			mRNA
60	TCTTCTCTG		NO MATCH
61	GCACCTTATT		NO MATCH
62	ACTTTAAACT		NO MATCH
63	CCATTCCACT		NO MATCH
64	TCAAATGCAT	M16342	human small nuclear ribonucleoprotein
			(hnRNP) C protein mRNA
65	GAAAAATGGT	X61156	human mRNA for laminin-binding protein
66	ACTAACACCC	U18810	human PACAP type-e/VIP type-2 receptor
			mRNA
67	TTGGGGTTTC	M12937	human ferritin heavy subunit mRNA
2 Gene assignments were based on the following list of GenBank sequences (GenBank Release 94). In each case, tentative assignments were based on the identification of a 10 bp SAGE tag adjacent to a N1aIII site. The final assignment was further refined by using an 11 bp SAGE tag and elimination of non 3′ end N1aIII sites and genomic sequences.

93 1 20 DNA Homo sapiens 1cagcttgccc acccatgctc 20 2 18 DNA Homo sapiens 2ggccaggagt aagtaact 18 3 18 DNA Homo sapiens 3gccctggtct gccgcgga 18 4 10 DNA Homo sapiens 4cccgcctctt 10 5 10 DNA Homo sapiens 5aatctgcgcc 10 6 10 DNA Homo sapiens 6gtgaccacgg 10 7 10 DNA Homo sapiens 7tttcctctca 10 8 10 DNA Homo sapiens 8tgcctgcacc 10 9 10 DNA Homo sapiens 9tcacccacac 10 10 10 DNA Homo sapiens 10taaacctgct 10 11 10 DNA Homo sapiens 11cccaagctag 10 12 10 DNA Homo sapiens 12agcccgccgc 10 13 10 DNA Homo sapiens 13gacatcaagt 10 14 10 DNA Homo sapiens 14tgtcctggtt 10 15 10 DNA Homo sapiens 15agctcactcc 10 16 10 DNA Homo sapiens 16aggctgtcca 10 17 10 DNA Homo sapiens 17tgagtccctg 10 18 10 DNA Homo sapiens 18ccctcctccg 10 19 10 DNA Homo sapiens 19gaggccaaca 10 20 10 DNA Homo sapiens 20tggggccgca 10 21 10 DNA Homo sapiens 21tccttggacc 10 22 10 DNA Homo sapiens 22ctgggcctga 10 23 11 DNA Homo sapiens 23agctggtttc c 11 24 10 DNA Homo sapiens 24gaggtgccgg 10 25 10 DNA Homo sapiens 25acaacgtcca 10 26 10 DNA Homo sapiens 26gtgcggagga 10 27 10 DNA Homo sapiens 27cgtcccggag 10 28 10 DNA Homo sapiens 28gtgctcattc 10 29 10 DNA Homo sapiens 29gctgactcag 10 30 10 DNA Homo sapiens 30agatgctgca 10 31 10 DNA Homo sapiens 31ctcagacagt 10 32 10 DNA Homo sapiens 32tccggccgcg 10 33 10 DNA Homo sapiens 33agccactgca 10 34 10 DNA Homo sapiens 34gcttttaagg 10 35 10 DNA Homo sapiens 35gggccaataa 10 36 10 DNA Homo sapiens 36aagggctctt 10 37 10 DNA Homo sapiens 37gtaagtgtac 10 38 10 DNA Homo sapiens 38tgtacctgta 10 39 10 DNA Homo sapiens 39aacgacctcg 10 40 10 DNA Homo sapiens 40agtttgttag 10 41 10 DNA Homo sapiens 41gactcgccca 10 42 10 DNA Homo sapiens 42gggccaataa 10 43 10 DNA Homo sapiens 43gggtttttat 10 44 10 DNA Homo sapiens 44agaaatacca 10 45 10 DNA Homo sapiens 45taccatcaat 10 46 10 DNA Homo sapiens 46ggattgtctg 10 47 10 DNA Homo sapiens 47tactagtcct 10 48 10 DNA Homo sapiens 48aatattgaga 10 49 10 DNA Homo sapiens 49gagggagttt 10 50 10 DNA Homo sapiens 50aagggcgcgg 10 51 10 DNA Homo sapiens 51ttcacaaagg 10 52 10 DNA Homo sapiens 52ctgcacttac 10 53 10 DNA Homo sapiens 53gatcccaact 10 54 10 DNA Homo sapiens 54gggaagcaga 10 55 10 DNA Homo sapiens 55gctttctcac 10 56 10 DNA Homo sapiens 56ttcattataa 10 57 10 DNA Homo sapiens 57taaggagctg 10 58 10 DNA Homo sapiens 58tgagggaata 10 59 10 DNA Homo sapiens 59gggatggcag 10 60 9 DNA Homo sapiens 60tcttctctg 9 61 10 DNA Homo sapiens 61gcaccttatt 10 62 10 DNA Homo sapiens 62actttaaact 10 63 10 DNA Homo sapiens 63ccattccact 10 64 10 DNA Homo sapiens 64tcaaatgcat 10 65 10 DNA Homo sapiens 65gaaaaatggt 10 66 10 DNA Homo sapiens 66actaacaccc 10 67 10 DNA Homo sapiens 67ttggggtttc 10 68 498 DNA Homo sapiens 68ttaaagcaaa gaattccccg gtcccagcca tgtccaacgt cccccacaag tcctcgctgc 60ccgagggcat ccgccctggc acggtgctga gaattcgcgg cttggttcct cccaatgcca 120gcaggttcca tgtaaacctg ctgtgcgggg aggagcaggg ctccgatgcc gccctgcatt 180tcaacccccg gctggacacg tcggaggtgg tcttcaacag caaggagcaa ggctcctggg 240gccgcgagga gcgcgggccg ggcgttcctt tccagcgcgg gcagcccttc gaggtgctca 300tcatcgcgtc agacgacggc ttcaaggccg tggttgggga cgcccagtac caccacttcc 360gccaccgcct gccgctggcg cgcgtgcgcc tggtggaggt gggcggggac gtgcagctgg 420actccgtgag gatcttctga gcagaagccc aggcggcccg gggccttggc tggcaaataa 480agcgttagcc cgcagcgc 498 69 993 DNA Homo sapiens 69cggcggcgcg cgatcgaggt cgggtcgccg tccagcctgc agcatgagcg cccccagcgc 60gacccccatc ttcgcgcccg gcgagaactg cagccccgcg tggggggcgg cgcccgcggc 120ctacgacgca gcggacacgc acctgcgcat cctgggcaag ccggtgatgg agcgctggga 180gaccccctat atgcacgcgc tggccgccgc cgcctcctcc aaagggggcc gggtcctgga 240ggtgggcttt ggcatggcca tcgcagcgtc aaaggtgcag gaggcgccca ttgatgagca 300ttggatcatc gagtgcaatg acggcgtctt ccagcggctc cgggactggg ccccacggca 360gacacacaag gtcatcccct tgaaaggcct gtgggaggat gtggcaccca ccctgcctga 420cggtcacttt gatgggatcc tgtacgacac gtacccactc tcggaggaga cctggcacac 480acaccagttc aacttcatca agaaccacgc ctttcgcctg ctgaagccgg ggggcgtcct 540cacctactgc aacctcacct cctgggggga gctgatgaag tccaagtact cagacatcac 600catcatgttt gaggagacgc aggtgcccgc gctgctggag gccggcttcc ggagggagaa 660catccgtacg gaggtgatgg cgctggtccc accggccgac tgccgctact acgccttccc 720acagatgatc acgcccctgg tgaccaaagg ctgagccccc accccggccc ggccacaccc 780atgccctccg ccgtgccttc ctggccggga gtccagggtg tcgcaccagc cctgggctga 840tcccagctgt gtgtcaccag aagctttccc ggcttctctg tgaggggtcc caccagccca 900gggctgatcc cagctgtgtg tcaccagcag ctttcccagc ttgctctgtg agggtcactg 960ctgcccactg cagggtgccc tgaggtgaag ccg 993 70 1670 DNA Homo sapiens 70ccagccgtcc attccggtgg aggcagaggc agtcctgggg ctctggggct cgggctttgt 60caccgggacc cgcagagcca gaaccactcg gcgccgctgg tgcatgggag gggagccggg 120ccaggagtaa gtaactcata cgggcgccgg ggacccgggt cggctggggg cttccaactc 180agagggagtg tgatttgcct gatcctcttc ggcgttgtcc tgctctgccg catccagccc 240tgtaccgcca tcccacttcc cgccgttccc atctgtgttc cgggtgggat cggtctggag 300gcggccgagg acttcccagg caggagctcg gggcggaggc gggtccgcgg cagaccaggg 360cagcgaggcg ctggccggca gggggcgctg cggtgccagc ctgaggctgg ctgctccgcg 420aggatacagc ggcccctgcc ctgtcctgtc ctgccctgcc ctgtcctgtc ctgccctgcc 480ctgccctgtc ctgtcctgcc ctgccctgcc ctgtgtcctc agacaatatg ttagccgtgc 540actttgacaa gccgggagga ccggaaaacc tctacgtgaa ggaggtggcc aagccgagcc 600cgggggaggg tgaagtcctc ctgaaggtgg cggccagcgc cctgaaccgg gcggacttaa 660tgcagagaca aggccagtat gacccacctc caggagccag caacattttg ggacttgagg 720catctggaca tgtggcagag ctggggcctg gctgccaggg acactggaag atcggggaca 780cagccatggc tctgctcccc ggtgggggcc aggctcagta cgtcactgtc cccgaagggc 840tcctcatgcc tatcccagag ggattgaccc tgacccaggc tgcagccatc ccagaggcct 900ggctcaccgc cttccagctg ttacatcttg tgggaaatgt tcaggctgga gactatgtgc 960taatccatgc aggactgagt ggtgtgggca cagctgctat ccaactcacc cggatggctg 1020gagctattcc tctggtcaca gctggctccc agaagaagct tcaaatggca gaaaagcttg 1080gagcagctgc tggattcaat tacaaaaaag aggatttctc tgaagcaacg ctgaaattca 1140ccaaaggtgc tggagttaat cttattctag actgcatagg cggatcctac tgggagaaga 1200acgtcaactg cctggctctt gatggtcgat gggttctcta tggtctgatg ggaggaggtg 1260acatcaatgg gcccctgttt tcaaagctac tttttaagcg aggaagtctg atcaccagtt 1320tgctgaggtc tagggacaat aagtacaagc aaatgctggt gaatgctttc acggagcaaa 1380ttctgcctca cttctccacg gagggccccc aacgtctgct gccggttctg gacagaatct 1440acccagtgac cgaaatccag gaggcccata gtacatggag gccaacaaga acataggcaa 1500gatcgtcctg gaactgcccc agtgaaggag gatgggggca ggacaggacg cggccacccc 1560aggcctttcc agagcaaacc tggagaagat tcacaataga caggccaaga aacccggtgc 1620ttcctccaga gccgtttaaa gctgatatga ggaaataaag agtgaactgg 1670 71 526 DNA Homo sapiens 71cagctacagc acagatcagc accatgaagc ttctcacggg cctggttttc tgctccttgg 60tcctgagtgt cagcagccga agcttctttt cgttccttgg cgaggctttt gatggggctc 120gggacatgtg gagagcctac tctgacatga gagaagccaa ttacatcggc tcagacaaat 180acttccatgc tcgggggaac tatgatgctg ccaaaagggg acctgggggt gcctgggccg 240cagaagtgat cagcaatgcc agagagaata tccagagact cacaggccat ggtgcggagg 300actcgctggc cgatcaggct gccaataaat ggggcaggag tggcagagac cccaatcact 360tccgacctgc tggcctgcct gagaaatact gagcttcctc ttcactctgc tctcaggaga 420cctggctatg agccctcggg gcagggattc aaagttagtg aggtctatgt ccagagaagc 480tgagatatgg catataatag gcatctaata aatgcttaag aggtgg 526 72 842 DNA Homo sapiens 72gcctcaaggg ctacgtcaac cacagcctgt ccgtcttcca caccaaggac ttccaggacc 60ctgatgggat tgagggctca gaaaacgtga ctctgtgcag atacagggac taccgcaatc 120ccccgattac aacttctccg agcagttctg gttcctcctg gccatccgcc tggccttcgt 180catcctcttt gagcacgtgg ccttgtgcat caagctcatc gccgcctggt tcgtgcccga 240catccctcag tcggtgaaga acaaggttct ggaggtgaag taccagaggc tgcgtgagaa 300gatgtggatg gaaggcagag gctgggtggg gtgggggctg gctctcggcc cccaatgcct 360gcccatccca ccccagcatc catcttcagt gccaggagca cagacgtgta gggccagagc 420ccgtccagag gccaccagga gctgagacag tgccaccacc agcacctccc acaaacccac 480cctgtgcgtg ttgaggggtg ctgtgagaag gctgtgccca tgtggggccg caggaatccc 540ctgtatgttc agggctgtga gctgccaccc tattccgcct gctccgtctt tgtggggctc 600tcaggcttgg cacagccctg acttgaactc tgggtgagcc tgggcaccca cagaactggg 660agtgagggct cctcaggcag ccacaaggca ggaaaactgg cgcaaatttc ctgggcctcc 720ctctgacttc tgggcgccag atcctgccgt gccccctacc tggctgttgg gggtgtcctg 780agcccacctc gctggcctgt tcccttcagc caacccgttt ctgcagtaaa attaagcctg 840tc 842 73 901 DNA Homo sapiens 73ggcgcatacc tggcccagga gcgagcccgt gcgcagatcg gctatgagga ccccatcaac 60cccacgtacg aggccaccaa cgccatgtac cacaggtgcc tggactacgt gttggaggag 120ctgaagcaca acgccaaggc caaggtgatg gtggcctccc acaatgagga cacagtgcgc 180ttcgcactgc gcaggatgga ggagctgggc ctgcatcctg ctgaccacca ggtgtacttt 240ggacagctgc taggcatgtg tgaccagatc agcttcccgc tgggccacgg ctggctaccc 300cgtgtacaag tacgtgccct atggccccgt gatggaggtg ctgccctact tgtccccgcc 360gtgccctgga agaacagcag cctcatgaag ggcacccatt cgggagcggc actggctgtg 420gctggagctc ttgaagcggc tccgaactgg caacctcttc catcgccctg cctagcaccc 480gccagcacac cctctagcct tccagcaccc cccgccccct gctccaggcc attcaaccaa 540caagctgcaa gccaaacccc aatccttcaa cacagattca ccttttttca ccccaccact 600ttgcagagct tgcttggagg tgaggtcagg tgcctcccag cccttgccca gagtatgggc 660actcaggtgt gggccgaacc tgatacctgc ctgggacagc cactggaaac ttttgggaac 720tctcctctga aatgtgtggg cccaaggccc ccacctctgt gacccccatg tccttggacc 780tagaggattg tccaccttct gccaaggcca gcccacacag cccgagcccc ttggggagca 840gtggccgggc tggggaggcc tgcctggtca ataaaccact gttcctgcaa aaaaaaaaaa 900a 901 74 1677 DNA Homo sapiens 74cacgcgcagc atagcagagt cgacactaga ggcatccaaa gaataccggc acgagcaggc 60ggcgcgggcg gcggttaaaa tgtcggttcc aggaccttac caggcggcca ctgggccttc 120ctccgcacca tccgcacctc catcctatga agagacagtg gctgttaaca gttattaccc 180cactcctcca gctcccatgc ctgggccaac tacggggctt gtgacggggc ctgatgggaa 240gggcatgaat cctccttcgt attataccca gccagcgccc atccccaata acaatccaat 300taccgtgcag acggtctacg tgcagcaccc catcaccttt ttggaccgcc ctatccaaat 360gtgttgtcct tcctgcaaca agatgatcgt gagtcagctg tcctataacg ccggtgctct 420gacctggctg tcctgcggga gcctgtgcct gctgggggtg catagcggcc tgctgcttca 480tccccttctg cgtggatgcc ctgcaggacg tggaccatta ctgtcccaac tgcagagctc 540tcctgggcac ctacaagcgt ttgtaggact cagccagacg tggagggagc cgggtgccgc 600aggaagtcct ttccacctct catccagctt cacgcctggt ggaggttctg ccctggtggt 660ctcacctctc cagggggccc accttcatgt cttcttttgg ggggaatacg tcgcaaaact 720aacaaatctc caaaccccag aaattgctgc ttggagtcgt gcataggact tgcaaagaca 780ttccccttga gtgtcagttc cacggtttcc tgcctccctg agaccctgag tcctgccatc 840taactgttga tcattgccct atccgaatat tttcctgtcg accccgggcc accagtggct 900cttttttcct gcttccatgg gcctttctgg tggcagtctc aaactgagga agccacagtt 960gcctcatttt tgaggctgtt ctccccagga gcttcggctg gaaccaggcc tttaggtggc 1020cttaccattt atctctatat ccggctcttt cccgttccct ggatggacaa aaatcttgcc 1080cttgacagga ctttaacagg gcttgggctt tgagattctg ttaacccgca ggacttcatt 1140aggcacacaa gattcacctt aatttctcta aatttttttt tttttaaaat accaagggaa 1200gggggctaat taacaaccca gtacaggaca tatccacaag ggtcggtaaa tggcatgcta 1260ggaaaaatag gggccttgga tcttattcac tggccctgtc ttccccttgg tttctcttgt 1320ggccagatct ttcagttgcc ccttttccat aacaggggat tttttttctt cataggagtt 1380aattattatg ggaacagttt tttatggacc tcccttttgg tctggaaata ccttttcgaa 1440cagaatttct tttttttaaa aaaaaacaga gatggggtct tactatgttg cccaggctgg 1500tgtcgaactc ctgggctcaa gcgatccttc tgccttggcc tcccgaagtg ctgggattgc 1560aggcataagc ttaccatgct gggcctgaac ataatttcaa gaggaggatt tataaaacca 1620ttttctgtaa tcaaatgatt ggtgtcattt tcccatttgc acaatgtagt ctcactt 1677 75 2608 DNA Homo sapiens 75agctcgccgg cctttggtct ccaggacttg tcccagcagc ccctcgaact gagaattaca 60ccatcggacc cctggctctg aggccttcag acttggactg tgtcacactg ccaggcttcc 120agggctccaa cttgcagacg gcctgttgtg ggacagtctc tgtaatcgcg aaagcaacca 180tggaagacct gggggaaaac accatggttt tatccaccct gagatctttg aacaacttca 240tctctcagcg tgtggaggga ggctctggac tggatatttc tacctcggcc ccaggttctc 300tgcagatgca gtaccagcag agcatgcagc tggaggaaag agcagagcag atccgttcga 360agtcccacct catccaggtg gagcgggaga aaatgcagat ggagctgagt cacaagaggg 420ctcgagtgga gctggagaga gcagccagca ccagtgccag gaactacgag cgtgaggtcg 480accgcaacca ggagctcctg acgcgcatcc ggcagcttca ggagcgggag gccggggcgg 540aggagaagat gcaggagcag ctggagcgca acaggcagtg tcagcagaac ttggatgctg 600ccagcaagag gctgcgtgag aaagaggaca gtctggccca ggctggcgag accatcaacg 660cactgaaggg gaggatctcg gaactgcagt ggagcgtgat ggaccaggag atgcgggtga 720agcgcctgga gtcggagaag caggacgtgc aggagcagct ggacctgcaa cacaaaaaat 780gccaggaagc caatcagaaa atccaggaac tccaggccag ccaagaagca agagcagacc 840acgagcagca gattaaggat ctggagcaga agctgtccct gcaagagcag gatgcagcga 900ttgtgaagaa catgaagtct gagctggtac ggctccctag gctggaacgg gagctggagc 960agctgcggga ggagagcgca ctgcgggaga tgagagagac caacgggctg ctccaggaag 1020agctggaagg gctgcagagg aagctggggc gccaggagaa gatgcaggag acgctggttg 1080gcttggagct ggagaacgag aggctgctgg ccaagctgca aagctgggag agactggacc 1140agaccatggg cctgagcatc aggactccag aagacctttc cagattcgtg gttgagctgc 1200agcagaggga gcttgccttg aaggacaaga acagcgccgt caccagcagc gcccgggggc 1260tggagaaggc caggcagcag ctgcaggagg agctccggca ggtcagcggc cagctgttgg 1320aggagaggaa gaagcgcgag acccacgagg cgctggcccg gaggctccag aaacgggtcc 1380tgctgctcac caaggagcgg gacggtatgc gggccatcct ggggtcctac gacagcgagc 1440tgaccccggc cgagtactca ccccagctga cgcggcgcat gcgggaggct gaggatatgg 1500tgcagaaggt gcacagccac agcgccgaga tggaggctca gctgtcgcag gccctggagg 1560agctgggagg ccagaaacaa agagcagaca tgctggagat ggagctgaag atgctgaagt 1620ctcagtccag ctctgccgaa cagagcttcc tgttctccag ggaggaggcg gacacgctca 1680ggttgaaggt cgaggagctg gaaggcgagc ggagtcggct ggaggaggaa aagaggatgc 1740tggaggcaca gctggagcgg cgagctctgc agggtgacta tgaccagagc aggaccaaag 1800tgctgcacat gagcctgaac cccaccagtg tggccaggca gcgcctgcgc gaggaccaca 1860gccagctgca ggcggagtgc gagcgactgc gcgggctcct gcgcgccatg gagagaggag 1920gcaccgtccc agccgacctt gaggctgccg ccgcgagtct gccatcgtcc aaggaggtgg 1980cagagctgaa gaagcaggtg gagagtgccg agctgaagaa ccagcggctc aaggaggttt 2040tccagaccaa gatccaggag ttccgcaagg cctgctacac gctcaccggc taccagatcg 2100acatcaccac ggagaaccag taccggctga cctcgctgta cgccgagcac ccaggcgact 2160gctcatcttc aaggccacca gcccctcggg ttccaagatg cagctactgg agacagagtt 2220ctcacacacc gtgggcgagc tcatcgaggt gcacctgcgg cgccaggaca gcatccctgc 2280cttcctcagc tcgctcaccc tcgagctctt cagccgccag accgtggcgt agcctgcagg 2340ctcgggggca tagccggagc cactctgctt ggcctgacct gcaggtcccc tgccccgcca 2400gccacaggct gggtgcacgt cctgcctctc cagccccaca gggcagcagc atgactgaca 2460gacacgctgg gacctacgtc gggcttcctg ctggggcggc cagcaccctc tccacgtgca 2520gaccccatgc gtcccggagc ctggtgtgtg ggcgtcggcc accagcctgg gttcctcacc 2580ttgtgaaata aaatcttctc ccctaaaa 2608 76 2326 DNA Homo sapiens 76aggccggaga ggaggcggtg cggcggtggc cgtgcggaga cccggtccag acgcctggcg 60gccgccggca cacaaggcgc tttctagctc cctcccccga gcgcacagcc cgcctccttc 120cgcggcgcct gcagtggcac ggattgctct gccctaccgt gacgcgctcc ggagacgctc 180tgcgggtcct ggacaccggg tccggcggcg tggggacgac agacggaggc gaacgcatcc 240ggtagccggt ccgcgagcca tcgttcgggg cgcagtcctc tccccggctg gccctccttt 300ctccggggca ttcgccaccg cttccctggg gctgagacga ccggttcgtc gcctccttgc 360ccgtgaccgt cgctagaact cagttgtgcg ttgcggccag tcgccactgc tgagtggaag 420caaaatgtca gtcagtgtgc atgagaaccg caagtccagg gccagcagcg gctccattaa 480catctatctg tttcacaagt cctcctacgc tgacagcgtc ctcactcacc tgaatctttt 540acgccagcag cgtctcttca ctgacgtcct tctccatgcc ggaaatagga ccttcccttg 600ccaccgggca gtgctggctg catgcagtcg ctactttgag gccatgttca gtggtggcct 660gaaagagagc caggacagtg aggtcaactt tgacaattcc atccacccag aagtcttgga 720gctgctgctt gactatgcgt actcctcccg ggtcattcat caattggaag gaaaatgcag 780aaattcgctc ctgggaagct tggtgacatg ctggagtttc aaggacatcc gggatgcatg 840tgcagagttc ctggaaaaga acctgcatcc caccaactgc ctgggcatgc tgctgctgtc 900tgatgcacac cagtgcacca agctgtacga actatcttgg agaatgtgtc tcagcaactt 960ccaaaccatc aggaagaatg aagatttcct ccagctgccc caggacatgg tagtgcaact 1020cttgtccagt gaagagctgg agacagagga tgaaaggctt gtgtacgagt ctgcaattaa 1080ctggatcagc tatgacctga agaagcgcta ttgctacctc ccagaactgt tgcagacagt 1140aacgcgggca cttctgccag ccatctatct catggagaat gtggccatgg aggaactcat 1200caccaagcag agaaagagta aggaaattgt ggaagaggcc atcaggtgca aactaaaaat 1260cctgcagaat gacggtgtgg taaccagcct ctgtgcccga cctcggaaaa ctggccatgc 1320cctcttcctt ctgggaggac agactttcat gtgtgacaag ttgtatctgg tagaccagaa 1380ggccaaagaa atcattccca aggctgacat tcccagccca agaaaagagt ttagtgcatg 1440tgcgattggc tgcaaagtgt acattactgg ggggcggggg tctgaaaatg gggtctcgaa 1500agatgtctgg gtttatgata ccctgcacga ggagtggtcc aaggctgccc ccatgctggt 1560ggccaggttt ggccatggct ctgctgaact gaagcactgc ctgtatgtgg ttggggggca 1620cacggccgca actggctgcc tcccggcctc cccctcagtc tctctaaagc aggtagaaca 1680ttatgacccc acaatcaaca aatggaccat ggcggcccca cgtccgagaa ggcgttacaa 1740ctgcgcacag gtagtgagtg ccaaacttaa gttatttgct ttcggaggta ccagtgtcag 1800tcatgacaag ctccccaaag ttcagtgtta cgatcagtgt gaaaacaggt ggactgtacc 1860ggccacctgt ccccagccct ggcgtataca cagccaagca agctgtcctg ggggaaccca 1920ggatttttta ttatgggggg tgatacagaa tttctctgcc tgcttctgct tataaattcg 1980caacagtgag acttaccagt ggaccaaagg tgggagatgt gacagcaaag cgcatgagct 2040gccatgctgt tggcctctgg aaacaaactc ttacgtggtt ggaggatact ttgggcattc 2100agcgatgcaa gactttggac tgctacgatc caacattaga cgtgtggaac agcatcacca 2160ctgtcccgta ctcgctgatt cctactgcat tttgtcagca cctggaaaca tctgccttct 2220taaatgcagt acattctaaa gagaagatga gcatgagctc actccatcac tcgatgagat 2280aatatgagat ttctacttcg gagaggccaa gtctaatgaa gagaaa 2326 77 2302 DNA Homo sapiens 77ctaaatcaag ctggagtcat gagggtagtg ggctaagtcg agggtccagc ctcttctgcc 60aggaagccct tcttgctttt gagagagggc tgtgaccacc ccccatcctt ctccctacac 120tcccagccaa cctagtgccc aagcagctaa acttggcttc cttctaatcc tggaaaaccc 180tgtacccctc ctcctcaatc tggccctctc cacatgcaca ccctgagaac acacacagac 240acacaacaca cacacataca cacccctgaa cacacacaca gacacacata cacccatgat 300gtgagcaaac acacacacgt gcgccttcat agcccagcca aggcatcgca ggcagggtgt 360gctgcctgag atggcacctc cctttcagcc attcttcaag aatgggccac acacagctag 420aagtcctctc ccagctagaa gtcctgtccc actctcctgg cctgacaaga tgagctctcc 480tgggaccttg ctctagggca ctctgcctct accctaggac actggaatgc cctgggagcc 540ccctccctgc aaccagcctg agttcagccc cacggacaaa gggacacaca gcccccaatg 600gagaccattg taagtggtgg ggctgggaga ggaggaacag aaggaaagcc atagcgctct 660cttgcccctt ggcatgtacc ccaaggcctg atggccactg ggctcagcct gtcccccact 720cctgcctgct tcccggtgag ctgcccccga cacgtgcagc ccgggctgcc tccagggtct 780ggctgagtgg gatcaggtgg ccctccaact cagcacagga aataagtaga aacatttcag 840caggccacct cccctcatct tccccgccct gtccagcgcc ctggcaaagg ctgacaactg 900gctgtcttgg ggccgaacag ccctgcctgc tctgagggcc acagcctgtg ctgcataccc 960accgcccagc ttctccctga gggcccacca gcctgtgctg catacccacc acccagcttc 1020tccctgaggg cccaccagcc tgtgctgtac accccgttag tccctgatcc caaccttctc 1080cctcctgcca gcacaccgat gcacacaccg gaagtggcga gcccaagccc tggggacagg 1140tgtagggaga aaagcagccc caggcctcag actcgctctc ccatcactgg catagagtgg 1200gaggatggct ggagggtgtc tataggtaca gcccgctctg gctgctgcca ggtgggcccc 1260tgccaggggt cctcacccct gtccaccctg tgcctggctg tccctgcacc cagatacagc 1320aacatggcct gtacccagca gagtggtggc aaccaccatg gttacagcgg atgccccgag 1380actctgcttg gtaaacgtgg cagagcagaa tgggaggctg ggaccctgag gaagggcccc 1440tctcctggca tctgtctctt gctacctaag cctgtgcctc tccctaaaga gctgcctccc 1500tgctgccgag ccctggtctg gccacgagcc actactgcct cccacaggca ccactgcctc 1560ccgctgctgc ccacaggtgg tgccgccaat gggcagtgcc tccaggccga agccttcaat 1620cccccatctt gagccagggc ctaaatcctc ttaatagtga tggttggttt tgtcctccca 1680ttaactgcag gtgggatttc cacctggggg aatgaggctt gcgttgttcg ggcgtctgct 1740ggccctgaga catccagtct tccacactca actgtgggat gggagggtgg cgtggcttta 1800ccccatggag gctgttccag ggctctgggc acacagctgt gctcacacaa aatactgggt 1860ggcttggttt agagctaatt gtagtggaag cctgcaaggt tgaggggtga aggggagggg 1920gcttgcaagg tccaggtaaa gatctggaaa gacagaacgt acagcttgga gggcaagggg 1980gactctaaag tgcaaggaga tttacagttg ggaaaggagg cagtggcaga ggggttgagg 2040gacaggggcc cttaagtcca gcgaggaaag ctcggtgtgg ggcccgctct acgctccgtt 2100tggggtgacc tggaacgcct cttctcccag ctccctccag ccatcagcag cctcttgtca 2160agcttctgcc tcgccccagt ctatccccaa ccccaaatca agaccacctt tcttcaacgg 2220tcactattta ttctttgttc ctttttcttt tgtgtaagaa acattcacaa aaaccagtgc 2280caaaaccatc aaaaaaaaaa aa 2302 78 1729 DNA Homo sapiens 78tggccagaga tgcctgccca cagcctggtg atgagcagcc cggccctccc ggccttcctg 60ctctgcagca cgctgctggt catcaagatg tacgtggtgg ccatcatcac gggccaagtg 120aggctgcgga agaaggcctt tgccaacccc gaggatgccc tgagacacgg aggaggcccc 180cagtattgca ggagcgaccc cgacgtggaa cgctgcctca gggcccaccg gaacgacatg 240gagaccatct accccttcct tttcctgggc ttcgtctact cctttctggg tcctaaccct 300tttgtcgcct ggatgcactt cctggtcttc ctcgtgggcc gtgtggcaca caccgtggcc 360tacctgggga agctgcgggc acccatccgc tccgtgacct acaccctggc ccagctcccc 420tgcgcctcca tggctctgca gatcctctgg gaagcggccc gccacctgtg accagcagct 480gatgcctcct tggccaccag accatgggcc aagagccgcc gtggctatac ctggggactt 540gatgttcctt ccagattgtg gtgtgggccc tgagtcctgg tttcctggca gcctgctgcg 600cgtgtgggtc tctgggcaca gtgggcctgt gtgtgtgccc gtgtgtgtgt atgtgtgtgt 660gtatgtttct tagccccttg gattcctgca cgaagtggct gatgggaacc atttcaagac 720agattgtgaa gattgataga aaatccttca gctaaagtaa cagagcatca aaaacatcac 780tccctctccc tccctaacag tgaaaagaga gaagggagac tctatttaag attcccaaac 840ctaatgatca tctgaatccc gggctaagaa tgcagacttt tcagactgac cccagaaatt 900ctggcccagc caatctagag gcaagcctgg ccatctgtat tttttttttc caagacagag 960tcttgctctc gttgcccaag ctggagtgaa gtggtacaat ctggctcact gcagcctccg 1020cctcccgggt tcaagcgatt ctcccgcctc agcctcctga gtagctggga ttacaggcgc 1080gtatcaccat acccagctaa tttttgtatt tttagtagag acgggttcac catgttgccc 1140aggagggtct cgaactcctg gcctcaagtg atccacgcct cggcctccca aagtgctggg 1200atgacaggca tgaatcactg tgctcagcca ccatctggag tttaaaagga cctcccatgt 1260gagtccctgt gtggccaggc cagggacccc tgccagttct atgtggaagc aaggctgggg 1320tcttgggttc ctgtatggtg gaagctgggt gagccaagga cagggctggc tcctctgccc 1380ccgctgacgc ttcccttgcc gttggctttg gatgtctttg ctgcagtctt ctctctggct 1440caggtgtggg tgggaggggc ccacaggaag ctcagccttc tcctcccaag gtttgagtcc 1500ctccaaaggg cagtgggtgg aggaccggga gctttgggtg accagccact caaaggaact 1560ttctggtccc ttcagtatct tcaaggtttg gaaactgcaa atgtcccctg atggggaatc 1620ctgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgttt tctcctagac 1680ccgtgacctg agatgtgtga tttttagtca ttaaatggaa gtgtctgcc 1729 79 136 PRT Homo sapiens 79Met Ser Asn Val Pro His Lys Ser Ser Leu Pro Glu Gly Ile Arg Pro 1 5 10 15Gly Thr Val Leu Arg Ile Arg Gly Leu Val Pro Pro Asn Ala Ser Arg 20 25 30Phe His Val Asn Leu Leu Cys Gly Glu Glu Gln Gly Ser Asp Ala Ala 35 40 45Leu His Phe Asn Pro Arg Leu Asp Thr Ser Glu Val Val Phe Asn Ser 50 55 60Lys Glu Gln Gly Ser Trp Gly Arg Glu Glu Arg Gly Pro Gly Val Pro65 70 75 80Phe Gln Arg Gly Gln Pro Phe Glu Val Leu Ile Ile Ala Ser Asp Asp 85 90 95Gly Phe Lys Ala Val Val Gly Asp Ala Gln Tyr His His Phe Arg His 100 105 110Arg Leu Pro Leu Ala Arg Val Arg Leu Val Glu Val Gly Gly Asp Val 115 120 125Gln Leu Asp Ser Val Arg Ile Phe 130 135 80 236 PRT Homo sapiens 80Met Ser Ala Pro Ser Ala Thr Pro Ile Phe Ala Pro Gly Glu Asn Cys 1 5 10 15Ser Pro Ala Trp Gly Ala Ala Pro Ala Ala Tyr Asp Ala Ala Asp Thr 20 25 30His Leu Arg Ile Leu Gly Lys Pro Val Met Glu Arg Trp Glu Thr Pro 35 40 45Tyr Met His Ala Leu Ala Ala Ala Ala Ser Ser Lys Gly Gly Arg Val 50 55 60Leu Glu Val Gly Phe Gly Met Ala Ile Ala Ala Ser Lys Val Gln Glu65 70 75 80Ala Pro Ile Asp Glu His Trp Ile Ile Glu Cys Asn Asp Gly Val Phe 85 90 95Gln Arg Leu Arg Asp Trp Ala Pro Arg Gln Thr His Lys Val Ile Pro 100 105 110Leu Lys Gly Leu Trp Glu Asp Val Ala Pro Thr Leu Pro Asp Gly His 115 120 125Phe Asp Gly Ile Leu Tyr Asp Thr Tyr Pro Leu Ser Glu Glu Thr Trp 130 135 140His Thr His Gln Phe Asn Phe Ile Lys Asn His Ala Phe Arg Leu Leu145 150 155 160Lys Pro Gly Gly Val Leu Thr Tyr Cys Asn Leu Thr Ser Trp Gly Glu 165 170 175Leu Met Lys Ser Lys Tyr Ser Asp Ile Thr Ile Met Phe Glu Glu Thr 180 185 190Gln Val Pro Ala Leu Leu Glu Ala Gly Phe Arg Arg Glu Asn Ile Arg 195 200 205Thr Glu Val Met Ala Leu Val Pro Pro Ala Asp Cys Arg Tyr Tyr Ala 210 215 220Phe Pro Gln Met Ile Thr Pro Leu Val Thr Lys Gly225 230 235 81 322 PRT Homo sapiens 81Met Leu Ala Val His Phe Asp Lys Pro Gly Gly Pro Glu Asn Leu Tyr 1 5 10 15Val Lys Glu Val Ala Lys Pro Ser Pro Gly Glu Gly Glu Val Leu Leu 20 25 30Lys Val Ala Ala Ser Ala Leu Asn Arg Ala Asp Leu Met Gln Arg Gln 35 40 45Gly Gln Tyr Asp Pro Pro Pro Gly Ala Ser Asn Ile Leu Gly Leu Glu 50 55 60Ala Ser Gly His Val Ala Glu Leu Gly Pro Gly Cys Gln Gly His Trp65 70 75 80Lys Ile Gly Asp Thr Ala Met Ala Leu Leu Pro Gly Gly Gly Gln Ala 85 90 95Gln Tyr Val Thr Val Pro Glu Gly Leu Leu Met Pro Ile Pro Glu Gly 100 105 110Leu Thr Leu Thr Gln Ala Ala Ala Ile Pro Glu Ala Trp Leu Thr Ala 115 120 125Phe Gln Leu Leu His Leu Val Gly Asn Val Gln Ala Gly Asp Tyr Val 130 135 140Leu Ile His Ala Gly Leu Ser Gly Val Gly Thr Ala Ala Ile Gln Leu145 150 155 160Thr Arg Met Ala Gly Ala Ile Pro Leu Val Thr Ala Gly Ser Gln Lys 165 170 175Lys Leu Gln Met Ala Glu Lys Leu Gly Ala Ala Ala Gly Phe Asn Tyr 180 185 190Lys Lys Glu Asp Phe Ser Glu Ala Thr Leu Lys Phe Thr Lys Gly Ala 195 200 205Gly Val Asn Leu Ile Leu Asp Cys Ile Gly Gly Ser Tyr Trp Glu Lys 210 215 220Asn Val Asn Cys Leu Ala Leu Asp Gly Arg Trp Val Leu Tyr Gly Leu225 230 235 240Met Gly Gly Gly Asp Ile Asn Gly Pro Leu Phe Ser Lys Leu Leu Phe 245 250 255Lys Arg Gly Ser Leu Ile Thr Ser Leu Leu Arg Ser Arg Asp Asn Lys 260 265 270Tyr Lys Gln Met Leu Val Asn Ala Phe Thr Glu Gln Ile Leu Pro His 275 280 285Phe Ser Thr Glu Gly Pro Gln Arg Leu Leu Pro Val Leu Asp Arg Ile 290 295 300Tyr Pro Val Thr Glu Ile Gln Glu Ala His Ser Thr Trp Arg Pro Thr305 310 315 320Arg Thr 82 122 PRT Homo sapiens 82Met Lys Leu Leu Thr Gly Leu Val Phe Cys Ser Leu Val Leu Ser Val 1 5 10 15Ser Ser Arg Ser Phe Phe Ser Phe Leu Gly Glu Ala Phe Asp Gly Ala 20 25 30Arg Asp Met Trp Arg Ala Tyr Ser Asp Met Arg Glu Ala Asn Tyr Ile 35 40 45Gly Ser Asp Lys Tyr Phe His Ala Arg Gly Asn Tyr Asp Ala Ala Lys 50 55 60Arg Gly Pro Gly Gly Ala Trp Ala Ala Glu Val Ile Ser Asn Ala Arg65 70 75 80Glu Asn Ile Gln Arg Leu Thr Gly His Gly Ala Glu Asp Ser Leu Ala 85 90 95Asp Gln Ala Ala Asn Lys Trp Gly Arg Ser Gly Arg Asp Pro Asn His 100 105 110Phe Arg Pro Ala Gly Leu Pro Glu Lys Tyr 115 120 83 253 PRT Homo sapiens 83Gly Ala Tyr Leu Ala Gln Glu Arg Ala Arg Ala Gln Ile Gly Tyr Glu 1 5 10 15Asp Pro Ile Asn Pro Thr Tyr Glu Ala Thr Asn Ala Met Tyr His Arg 20 25 30Cys Leu Asp Tyr Val Leu Glu Glu Leu Lys His Asn Ala Lys Ala Lys 35 40 45Val Met Val Ala Ser His Asn Glu Asp Thr Val Arg Phe Ala Leu Arg 50 55 60Arg Met Glu Glu Leu Gly Leu His Pro Ala Asp His Gln Val Tyr Phe65 70 75 80Gly Gln Leu Leu Gly Met Cys Asp Gln Ile Ser Phe Pro Leu Gly His 85 90 95Gly Trp Leu Pro Arg Val Gln Val Arg Ala Leu Trp Pro Arg Asp Gly 100 105 110Gly Ala Ala Leu Leu Val Pro Ala Val Pro Trp Lys Asn Ser Ser Leu 115 120 125Met Lys Gly Thr His Ser Gly Ala Ala Leu Ala Val Ala Gly Ala Leu 130 135 140Glu Ala Ala Pro Asn Trp Gln Pro Leu Pro Ser Pro Cys Leu Ala Pro145 150 155 160Ala Ser Thr Pro Ser Ser Leu Pro Ala Pro Pro Ala Pro Cys Ser Arg 165 170 175Pro Phe Asn Gln Gln Ala Ala Ser Gln Thr Pro Ile Leu Gln His Arg 180 185 190Phe Thr Phe Phe His Pro Thr Thr Leu Gln Ser Leu Leu Gly Gly Glu 195 200 205Val Arg Cys Leu Pro Ala Leu Ala Gln Ser Met Gly Thr Gln Val Trp 210 215 220Ala Glu Pro Asp Thr Cys Leu Gly Gln Pro Leu Glu Thr Phe Gly Asn225 230 235 240Ser Pro Leu Lys Cys Val Gly Pro Arg Pro Pro Pro Leu 245 250 84 228 PRT Homo sapiens 84Met Ser Val Pro Gly Pro Tyr Gln Ala Ala Thr Gly Pro Ser Ser Ala 1 5 10 15Pro Ser Ala Pro Pro Ser Tyr Glu Glu Thr Val Ala Val Asn Ser Tyr 20 25 30Tyr Pro Thr Pro Pro Ala Pro Met Pro Gly Pro Thr Thr Gly Leu Val 35 40 45Thr Gly Pro Asp Gly Lys Gly Met Asn Pro Pro Ser Tyr Tyr Thr Gln 50 55 60Pro Ala Pro Ile Pro Asn Asn Asn Pro Ile Thr Val Gln Thr Val Tyr65 70 75 80Val Gln His Pro Ile Thr Phe Leu Asp Arg Pro Ile Gln Met Cys Cys 85 90 95Pro Ser Cys Asn Lys Met Ile Val Ser Gln Leu Ser Tyr Asn Ala Gly 100 105 110Ala Leu Thr Trp Leu Ser Cys Gly Ser Leu Cys Leu Leu Gly Val His 115 120 125Ser Gly Leu Leu Leu His Pro Leu Leu Arg Gly Cys Pro Ala Gly Arg 130 135 140Gly Pro Leu Leu Ser Gln Leu Gln Ser Ser Pro Gly His Leu Gln Ala145 150 155 160Phe Val Gly Leu Ser Gln Thr Trp Arg Glu Pro Gly Ala Ala Gly Ser 165 170 175Pro Phe His Leu Ser Ser Ser Phe Thr Pro Gly Gly Gly Ser Ala Leu 180 185 190Val Val Ser Pro Leu Gln Gly Ala His Leu His Val Phe Phe Trp Gly 195 200 205Glu Tyr Val Ala Lys Leu Thr Asn Leu Gln Thr Pro Glu Ile Ala Ala 210 215 220Trp Ser Arg Ala225 85 803 PRT Homo sapiens 85Met Glu Asp Leu Gly Glu Asn Thr Met Val Leu Ser Thr Leu Arg Ser 1 5 10 15Leu Asn Asn Phe Ile Ser Gln Arg Val Glu Gly Gly Ser Gly Leu Asp 20 25 30Ile Ser Thr Ser Ala Pro Gly Ser Leu Gln Met Gln Tyr Gln Gln Ser 35 40 45Met Gln Leu Glu Glu Arg Ala Glu Gln Ile Arg Ser Lys Ser His Leu 50 55 60Ile Gln Val Glu Arg Glu Lys Met Gln Met Glu Leu Ser His Lys Arg65 70 75 80Ala Arg Val Glu Leu Glu Arg Ala Ala Ser Thr Ser Ala Arg Asn Tyr 85 90 95Glu Arg Glu Val Asp Arg Asn Gln Glu Leu Leu Thr Arg Ile Arg Gln 100 105 110Leu Gln Glu Arg Glu Ala Gly Ala Glu Glu Lys Met Gln Glu Gln Leu 115 120 125Glu Arg Asn Arg Gln Cys Gln Gln Asn Leu Asp Ala Ala Ser Lys Arg 130 135 140Leu Arg Glu Lys Glu Asp Ser Leu Ala Gln Ala Gly Glu Thr Ile Asn145 150 155 160Ala Leu Lys Gly Arg Ile Ser Glu Leu Gln Trp Ser Val Met Asp Gln 165 170 175Glu Met Arg Val Lys Arg Leu Glu Ser Glu Lys Gln Asp Val Gln Glu 180 185 190Gln Leu Asp Leu Gln His Lys Lys Cys Gln Glu Ala Asn Gln Lys Ile 195 200 205Gln Glu Leu Gln Ala Ser Gln Glu Ala Arg Ala Asp His Glu Gln Gln 210 215 220Ile Lys Asp Leu Glu Gln Lys Leu Ser Leu Gln Glu Gln Asp Ala Ala225 230 235 240Ile Val Lys Asn Met Lys Ser Glu Leu Val Arg Leu Pro Arg Leu Glu 245 250 255Arg Glu Leu Glu Gln Leu Arg Glu Glu Ser Ala Leu Arg Glu Met Arg 260 265 270Glu Thr Asn Gly Leu Leu Gln Glu Glu Leu Glu Gly Leu Gln Arg Lys 275 280 285Leu Gly Arg Gln Glu Lys Met Gln Glu Thr Leu Val Gly Leu Glu Leu 290 295 300Glu Asn Glu Arg Leu Leu Ala Lys Leu Gln Ser Trp Glu Arg Leu Asp305 310 315 320Gln Thr Met Gly Leu Ser Ile Arg Thr Pro Glu Asp Leu Ser Arg Phe 325 330 335Val Val Glu Leu Gln Gln Arg Glu Leu Ala Leu Lys Asp Lys Asn Ser 340 345 350Ala Val Thr Ser Ser Ala Arg Gly Leu Glu Lys Ala Arg Gln Gln Leu 355 360 365Gln Glu Glu Leu Arg Gln Val Ser Gly Gln Leu Leu Glu Glu Arg Lys 370 375 380Lys Arg Glu Thr His Glu Ala Leu Ala Arg Arg Leu Gln Lys Arg Val385 390 395 400Leu Leu Leu Thr Lys Glu Arg Asp Gly Met Arg Ala Ile Leu Gly Ser 405 410 415Tyr Asp Ser Glu Leu Thr Pro Ala Glu Tyr Ser Pro Gln Leu Thr Arg 420 425 430Arg Met Arg Glu Ala Glu Asp Met Val Gln Lys Val His Ser His Ser 435 440 445Ala Glu Met Glu Ala Gln Leu Ser Gln Ala Leu Glu Glu Leu Gly Gly 450 455 460Gln Lys Gln Arg Ala Asp Met Leu Glu Met Glu Leu Lys Met Leu Lys465 470 475 480Ser Gln Ser Ser Ser Ala Glu Gln Ser Phe Leu Phe Ser Arg Glu Glu 485 490 495Ala Asp Thr Leu Arg Leu Lys Val Glu Glu Leu Glu Gly Glu Arg Ser 500 505 510Arg Leu Glu Glu Glu Lys Arg Met Leu Glu Ala Gln Leu Glu Arg Arg 515 520 525Ala Leu Gln Gly Asp Tyr Asp Gln Ser Arg Thr Lys Val Leu His Met 530 535 540Ser Leu Asn Pro Thr Ser Val Ala Arg Gln Arg Leu Arg Glu Asp His545 550 555 560Ser Gln Leu Gln Ala Glu Cys Glu Arg Leu Arg Gly Leu Leu Arg Ala 565 570 575Met Glu Arg Gly Gly Thr Val Pro Ala Asp Leu Glu Ala Ala Ala Ala 580 585 590Ser Leu Pro Ser Ser Lys Glu Val Ala Glu Leu Lys Lys Gln Val Glu 595 600 605Ser Ala Glu Leu Lys Asn Gln Arg Leu Lys Glu Val Phe Gln Thr Lys 610 615 620Ile Gln Glu Phe Arg Lys Ala Cys Tyr Thr Leu Thr Gly Tyr Gln Ile625 630 635 640Asp Ile Thr Thr Glu Asn Gln Tyr Arg Leu Thr Ser Leu Tyr Ala Glu 645 650 655His Pro Gly Asp Cys Ser Ser Ser Arg Pro Pro Ala Pro Arg Val Pro 660 665 670Arg Cys Ser Tyr Trp Arg Gln Ser Ser His Thr Pro Trp Ala Ser Ser 675 680 685Ser Arg Cys Thr Cys Gly Ala Arg Thr Ala Ser Leu Pro Ser Ser Ala 690 695 700Arg Ser Pro Ser Ser Ser Ser Ala Ala Arg Pro Trp Arg Ser Leu Gln705 710 715 720Ala Arg Gly His Ser Arg Ser His Ser Ala Trp Pro Asp Leu Gln Val 725 730 735Pro Cys Pro Ala Ser His Arg Leu Gly Ala Arg Pro Ala Ser Pro Ala 740 745 750Pro Gln Gly Ser Ser Met Thr Asp Arg His Ala Gly Thr Tyr Val Gly 755 760 765Leu Pro Ala Gly Ala Ala Ser Thr Leu Ser Thr Cys Arg Pro His Ala 770 775 780Ser Arg Ser Leu Val Cys Gly Arg Arg Pro Pro Ala Trp Val Pro His785 790 795 800Leu Val Lys 86 516 PRT Homo sapiens 86Met Ser Val Ser Val His Glu Asn Arg Lys Ser Arg Ala Ser Ser Gly 1 5 10 15Ser Ile Asn Ile Tyr Leu Phe His Lys Ser Ser Tyr Ala Asp Ser Val 20 25 30Leu Thr His Leu Asn Leu Leu Arg Gln Gln Arg Leu Phe Thr Asp Val 35 40 45Leu Leu His Ala Gly Asn Arg Thr Phe Pro Cys His Arg Ala Val Leu 50 55 60Ala Ala Cys Ser Arg Tyr Phe Glu Ala Met Phe Ser Gly Gly Leu Lys65 70 75 80Glu Ser Gln Asp Ser Glu Val Asn Phe Asp Asn Ser Ile His Pro Glu 85 90 95Val Leu Glu Leu Leu Leu Asp Tyr Ala Tyr Ser Ser Arg Val Ile His 100 105 110Gln Leu Glu Gly Lys Cys Arg Asn Ser Leu Leu Gly Ser Leu Val Thr 115 120 125Cys Trp Ser Phe Lys Asp Ile Arg Asp Ala Cys Ala Glu Phe Leu Glu 130 135 140Lys Asn Leu His Pro Thr Asn Cys Leu Gly Met Leu Leu Leu Ser Asp145 150 155 160Ala His Gln Cys Thr Lys Leu Tyr Glu Leu Ser Trp Arg Met Cys Leu 165 170 175Ser Asn Phe Gln Thr Ile Arg Lys Asn Glu Asp Phe Leu Gln Leu Pro 180 185 190Gln Asp Met Val Val Gln Leu Leu Ser Ser Glu Glu Leu Glu Thr Glu 195 200 205Asp Glu Arg Leu Val Tyr Glu Ser Ala Ile Asn Trp Ile Ser Tyr Asp 210 215 220Leu Lys Lys Arg Tyr Cys Tyr Leu Pro Glu Leu Leu Gln Thr Val Thr225 230 235 240Arg Ala Leu Leu Pro Ala Ile Tyr Leu Met Glu Asn Val Ala Met Glu 245 250 255Glu Leu Ile Thr Lys Gln Arg Lys Ser Lys Glu Ile Val Glu Glu Ala 260 265 270Ile Arg Cys Lys Leu Lys Ile Leu Gln Asn Asp Gly Val Val Thr Ser 275 280 285Leu Cys Ala Arg Pro Arg Lys Thr Gly His Ala Leu Phe Leu Leu Gly 290 295 300Gly Gln Thr Phe Met Cys Asp Lys Leu Tyr Leu Val Asp Gln Lys Ala305 310 315 320Lys Glu Ile Ile Pro Lys Ala Asp Ile Pro Ser Pro Arg Lys Glu Phe 325 330 335Ser Ala Cys Ala Ile Gly Cys Lys Val Tyr Ile Thr Gly Gly Arg Gly 340 345 350Ser Glu Asn Gly Val Ser Lys Asp Val Trp Val Tyr Asp Thr Leu His 355 360 365Glu Glu Trp Ser Lys Ala Ala Pro Met Leu Val Ala Arg Phe Gly His 370 375 380Gly Ser Ala Glu Leu Lys His Cys Leu Tyr Val Val Gly Gly His Thr385 390 395 400Ala Ala Thr Gly Cys Leu Pro Ala Ser Pro Ser Val Ser Leu Lys Gln 405 410 415Val Glu His Tyr Asp Pro Thr Ile Asn Lys Trp Thr Met Ala Ala Pro 420 425 430Arg Pro Arg Arg Arg Tyr Asn Cys Ala Gln Val Val Ser Ala Lys Leu 435 440 445Lys Leu Phe Ala Phe Gly Gly Thr Ser Val Ser His Asp Lys Leu Pro 450 455 460Lys Val Gln Cys Tyr Asp Gln Cys Glu Asn Arg Trp Thr Val Pro Ala465 470 475 480Thr Cys Pro Gln Pro Trp Arg Ile His Ser Gln Ala Ser Cys Pro Gly 485 490 495Gly Thr Gln Asp Phe Leu Leu Trp Gly Val Ile Gln Asn Phe Ser Ala 500 505 510Cys Phe Cys Leu 515 87 153 PRT Homo sapiens 87Met Pro Ala His Ser Leu Val Met Ser Ser Pro Ala Leu Pro Ala Phe 1 5 10 15Leu Leu Cys Ser Thr Leu Leu Val Ile Lys Met Tyr Val Val Ala Ile 20 25 30Ile Thr Gly Gln Val Arg Leu Arg Lys Lys Ala Phe Ala Asn Pro Glu 35 40 45Asp Ala Leu Arg His Gly Gly Gly Pro Gln Tyr Cys Arg Ser Asp Pro 50 55 60Asp Val Glu Arg Cys Leu Arg Ala His Arg Asn Asp Met Glu Thr Ile65 70 75 80Tyr Pro Phe Leu Phe Leu Gly Phe Val Tyr Ser Phe Leu Gly Pro Asn 85 90 95Pro Phe Val Ala Trp Met His Phe Leu Val Phe Leu Val Gly Arg Val 100 105 110Ala His Thr Val Ala Tyr Leu Gly Lys Leu Arg Ala Pro Ile Arg Ser 115 120 125Val Thr Tyr Thr Leu Ala Gln Leu Pro Cys Ala Ser Met Ala Leu Gln 130 135 140Ile Leu Trp Glu Ala Ala Arg His Leu145 150 88 155 PRT Rat 88Met Ala Asp Leu Lys Gln Leu Met Asp Asn Glu Val Leu Met Ala Phe 1 5 10 15Thr Ser Tyr Ala Thr Ile Ile Leu Ala Lys Met Met Phe Leu Ser Ser 20 25 30Ala Thr Ala Phe Gln Arg Leu Thr Asn Lys Val Phe Ala Asn Pro Glu 35 40 45Asp Cys Ala Gly Phe Gly Lys Gly Glu Asn Ala Lys Lys Phe Leu Arg 50 55 60Thr Asp Glu Lys Val Glu Arg Val Arg Arg Ala His Leu Asn Asp Leu65 70 75 80Glu Asn Ile Val Pro Phe Leu Gly Ile Gly Leu Leu Tyr Ser Leu Ser 85 90 95Gly Pro Asp Leu Ser Thr Ala Leu Ile His Phe Arg Ile Phe Val Gly 100 105 110Ala Arg Ile Tyr His Thr Ile Ala Tyr Leu Thr Pro Leu Pro Gln Pro 115 120 125Asn Arg Gly Leu Ala Phe Phe Val Gly Tyr Gly Val Thr Leu Ser Met 130 135 140Ala Tyr Arg Leu Leu Arg Ser Arg Leu Tyr Leu145 150 155 89 324 PRT Vigna 89Met Val Lys Ala Ile Arg Val His Glu Gln Gly Gly Pro Gln Val Leu 1 5 10 15Lys Trp Glu Asp Val Glu Ile Gly Glu Pro Lys Glu Gly Glu Val Arg 20 25 30Val Arg Asn Lys Ala Val Gly Val Asn Phe Ile Asp Val Tyr Phe Arg 35 40 45Lys Gly Val Tyr Lys Pro Pro Ser Phe Pro Phe Thr Pro Gly Met Glu 50 55 60Ala Val Gly Val Val Thr Ala Val Gly Ala Gly Leu Thr Gly Arg Gln65 70 75 80Val Gly Asp Leu Val Ala Tyr Ala Gly Gln Pro Met Gly Ser Tyr Ala 85 90 95Glu Glu Gln Ile Leu Pro Ala Asn Lys Val Val Pro Val Pro Ser Ser 100 105 110Ile Asp Pro Pro Ile Ala Ala Ser Ile Met Leu Lys Gly Met Thr Thr 115 120 125His Phe Leu Val Arg Arg Cys Phe Lys Val Glu Pro Gly His Thr Ile 130 135 140Leu Val His Ala Ala Ala Gly Gly Val Gly Ser Leu Leu Cys Gln Trp145 150 155 160Ala Asn Ala Leu Gly Ala Thr Val Ile Gly Thr Val Ser Asn Lys Glu 165 170 175Lys Ala Ala Gln Ala Lys Glu Asp Gly Cys His His Val Ile Ile Tyr 180 185 190Lys Glu Glu Asp Phe Val Ala Arg Val Asn Glu Ile Thr Ser Gly Asn 195 200 205Gly Val Glu Val Val Tyr Asp Ser Val Gly Lys Asp Thr Phe Glu Gly 210 215 220Ser Leu Ala Cys Leu Lys Leu Arg Gly Tyr Met Val Ser Phe Gly Gln225 230 235 240Ser Ser Gly Ser Pro Asp Pro Val Pro Leu Ser Ser Leu Ala Ala Lys 245 250 255Ser Leu Phe Leu Thr Arg Pro Ser Leu Met Gln Tyr Val Val Thr Arg 260 265 270Asp Glu Leu Leu Glu Ala Ala Gly Glu Leu Phe Ala Asn Val Ala Ser 275 280 285Gly Val Leu Lys Val Arg Val Asn His Thr Tyr Pro Leu Ser Glu Ala 290 295 300Ala Lys Ala His Glu Asp Leu Glu Asn Arg Lys Thr Ser Gly Ser Ile305 310 315 320Val Leu Ile Pro 90 359 PRT Homo sapiens 90Met Ile Trp Gly His Phe Ser Leu Leu Cys Val Val Asp Ser Leu Gly 1 5 10 15Gly Glu Glu Met Ala Asp Ser Val Lys Thr Phe Leu Gln Asp Leu Ala 20 25 30Arg Gly Ile Lys Asp Ser Ile Trp Gly Ile Cys Thr Ile Ser Lys Leu 35 40 45Asp Ala Arg Ile Gln Gln Lys Arg Glu Glu Gln Arg Arg Arg Arg Ala 50 55 60Ser Ser Val Leu Ala Gln Arg Arg Pro Gln Ser Ile Glu Arg Lys Gln65 70 75 80Glu Ser Glu Pro Arg Ile Val Ser Arg Ile Phe Gln Cys Cys Ala Trp 85 90 95Asn Gly Gly Val Phe Trp Phe Ser Leu Leu Leu Phe Tyr Arg Val Phe 100 105 110Ile Pro Val Leu Gln Ser Val Thr Ala Arg Ile Ile Gly Asp Pro Ser 115 120 125Leu His Gly Asp Val Trp Ser Trp Leu Gly Phe Phe Leu Thr Ser Ile 130 135 140Phe Ser Ala Val Trp Val Leu Pro Leu Phe Val Leu Ser Lys Val Val145 150 155 160Asn Ala Ile Trp Phe Gln Asp Ile Ala Asp Leu Ala Phe Glu Val Ser 165 170 175Gly Arg Lys Pro His Pro Phe Pro Ser Val Ser Lys Ile Ile Ala Asp 180 185 190Met Leu Phe Asn Leu Leu Leu Gln Ala Leu Phe Leu Ile Gln Gly Met 195 200 205Phe Val Ser Leu Phe Pro Ile His Leu Val Gly Gln Leu Val Ser Leu 210 215 220Leu His Met Ser Leu Leu Tyr Ser Leu Tyr Cys Phe Glu Tyr Arg Trp225 230 235 240Phe Asn Lys Gly Ile Glu Met His Gln Arg Leu Ser Asn Ile Glu Arg 245 250 255Asn Trp Pro Tyr Tyr Phe Gly Phe Gly Leu Pro Leu Ala Phe Leu Thr 260 265 270Ala Met Gln Ser Ser Tyr Ile Ile Ser Gly Cys Leu Phe Ser Ile Leu 275 280 285Phe Pro Leu Phe Ile Ile Ser Ala Asn Glu Ala Lys Thr Pro Gly Lys 290 295 300Ala Tyr Leu Phe Gln Leu Arg Leu Phe Ser Leu Val Val Phe Leu Ser305 310 315 320Asn Arg Leu Phe His Lys Thr Val Tyr Leu Gln Ser Ala Leu Ser Ser 325 330 335Ser Thr Ser Ala Glu Lys Phe Pro Ser Pro His Pro Ser Pro Ala Lys 340 345 350Leu Lys Ala Thr Ala Gly His 355 91 315 PRT C. elegans 91Met Val Lys Phe Gln Ile Ile Ala Arg Asp Phe Tyr His Gly Phe Ile 1 5 10 15Asp Ser Phe Lys Gly Ile Thr Phe Val Arg Arg Ile Arg Glu Glu Glu 20 25 30Ala Lys Glu Val Lys Val Glu Pro Pro Lys Pro Val Glu Arg Thr Val 35 40 45Leu Met Met Arg Arg Glu Lys Gln Gly Ile Phe Lys Arg Pro Pro Glu 50 55 60Pro Pro Lys Lys Lys Asp Ser Phe Leu Lys Lys Leu Trp Gln Ile Tyr65 70 75 80Ala Met Asn Ile Gly Phe Leu Val Leu Trp Gln Val Cys Ile Leu Ile 85 90 95Leu Gly Leu Phe Phe Ser Phe Phe Asp Arg Thr Asp Leu Gly His Asn 100 105 110Ile Gly Tyr Ile Leu Ile Ile Pro Ile Phe Phe Ala Ser Arg Ile Ile 115 120 125Gln Ala Leu Trp Phe Ser Asp Ile Ser Gly Ala Cys Met Arg Ala Leu 130 135 140Lys Leu Pro Pro Pro Pro Val Val Pro Phe Ser Ser Met Leu Ala Gly145 150 155 160Thr Leu Ile Ser Ala Leu His Gln Ile Phe Phe Leu Ile Gln Gly Met 165 170 175Leu Ser Gln Tyr Leu Pro Ile Pro Leu Ile Thr Pro Val Ile Val Tyr 180 185 190Leu His Met Ala Leu Leu Asn Ser Met Tyr Cys Phe Asp Tyr Phe Phe 195 200 205Asp Gly Tyr Asn Leu Ser Phe Leu Arg Arg Lys Asp Ile Phe Glu Ser 210 215 220His Trp Pro Tyr Phe Leu Gly Phe Gly Thr Pro Leu Ala Leu Ala Cys225 230 235 240Ser Ile Ser Ser Asn Met Phe Val Asn Ser Val Ile Phe Ala Leu Leu 245 250 255Phe Pro Phe Phe Ile Ile Thr Ser Tyr Pro Ala Asn Trp Asn Arg Lys 260 265 270Tyr Glu Glu Glu Ile Pro Lys Ile Ala Phe Cys Arg Ile Ser Tyr Met 275 280 285Phe Thr Glu Leu Val Gly Lys Phe Val Lys Ser Ile Thr Pro Thr Asn 290 295 300Asn Pro Thr Ala Ala Arg Asn Asn Ala Gln Asn305 310 315 92 83 PRT Homo sapiens 92Ile Gly Tyr Glu Asp Pro Ile Asn Pro Thr Tyr Glu Ala Thr Asn Ala 1 5 10 15Met Tyr His Arg Cys Leu Asp Tyr Val Leu Glu Glu Leu Lys His Asn 20 25 30Ala Lys Ala Lys Val Met Val Ala Ser His Asn Glu Asp Thr Val Arg 35 40 45Phe Ala Leu Arg Arg Met Glu Glu Leu Gly Leu His Pro Ala Asp His 50 55 60Gln Val Tyr Phe Gly Gln Leu Leu Gly Met Cys Asp Gln Ile Ser Phe65 70 75 80Pro Leu Gly 93 89 PRT Drosophila 93Ile Gly Tyr Glu Asp Pro Val Asn Pro Thr Phe Glu Ala Thr Thr Asp 1 5 10 15Met Tyr His Arg Leu Ser Glu Cys Leu Arg Arg Ile Lys Leu Met Lys 20 25 30Asp Cys Asp Asp Asp Ala Arg Lys Ile Gly Ile Met Val Ala Ser His 35 40 45Asn Glu Asp Thr Val Arg Phe Ala Ile Gln Gln Met Lys Glu Ile Gly 50 55 60Ile Ser Pro Glu Asp Lys Val Ile Cys Phe Gly Gln Leu Leu Gly Met65 70 75 80Cys Asp Tyr Ile Thr Phe Pro Leu Gly 85

Claims

1. A method of screening for cancer or p53 status in a sample suspected of being neoplastic, comprising the steps of:comparing the level of transcription of an RNA transcript in a first sample of a first tissue to the level of transcription of the transcript in a second sample of a second tissue, wherein the first tissue is a human tissue suspected of being neoplastic and the second tissue is a normal human tissue, wherein the first and second tissue are of the same tissue type, and wherein the transcript is identified by a nucleic acid consisting of a tag set forth as SEQ ID NO:19, and wherein the tag is located 3′ of the 3′-most site for a NlaIII restriction endonuclease in a cDNA reverse transcribed from the transcript; categorizing the first sample as likely to be neoplastic or likely to have a mutant p53 when transcription is found to be the same or lower in the first sample than in the second sample.

2. A DNA construct for screening drugs as anti-neoplastic agents comprising:a reporter gene under the control of a PIG-3 promoter, wherein the reporter gene is 3′ and covalently linked to the PIG-3 promoter, wherein the PIG-3 promoter comprises the sequence CAGCTTGCCCACCCATGCTC (SEQ ID NO:1).

3. A method of screening for cancer or p53 status in a human sample suspected of being neoplastic, comprising the steps of:treating cells of the sample with a DNA-damaging agent; comparing the level of transcription of an RNA transcript in cells of the sample to the level of transcription of the transcript in cells of the sample which are not subject to said treating, wherein the transcript is identified by a nucleic acid consisting of a tag set forth as SEQ ID NO:19, and wherein the tag is located 3′ of the 3′-most site for a NlaIII restriction endonuclease in a cDNA reverse transcribed from the transcript; categorizing the sample as likely to be neoplastic or likely to have a mutant p53 when transcription is found to be the same or lower in the treated cells than in the untreated cells.

4. The method of claim 1 wherein the first and second samples are treated with a DNA-damaging agent prior to said step of comparing.