Patent Application
20060292577
Not applicable.
Not applicable.
The field of this invention is the area of molecular biology, in particular, as related to the molecular biology of neoplastic and diseased cells, as specifically related to splice variant transcripts for a cell surface marker characteristic of neoplastic and certain other diseased cell states.
There is a unique, growth-related family of cell surface hydroquinone or NADH oxidases with protein disulfide-thiol interchange activity referred to as ECTO-NOX proteins (for cell surface NADH oxidases) (1, 2). One member of the ECTO-NOX family, designated tNOX (for tumor associated) is specific to the surfaces of cancer cells and the sera of cancer patients (3, 4). The presence of the tNOX protein has been demonstrated for several human tumor tissues (mammary carcinoma, prostate cancer, neuroblastoma, colon carcinoma and melanoma) (5), and serum analyses suggest a much broader association with human cancer (6, 7).
NOX proteins are ectoproteins anchored in the outer leaflet of the plasma membrane (8). As is characteristic of other examples of ectoproteins (sialyl and galactosyl transferase, dipeptidylamino peptidase IV, etc.), the NOX proteins are shed. They appear in soluble form in conditioned media of cultured cells (5) and in patient sera (6, 7). The serum form of tNOX from cancer patients exhibits the same degree of drug responsiveness as does the membrane-associated form. Drug-responsive tNOX activities are seen in sera of a variety of human cancer patients, including patients with leukemia, lymphomas or solid tumors (prostate, breast, colon, lung, pancreas, ovarian, liver) (6, 7). An extreme stability and protease resistance of the tNOX protein (9) may help explain its ability to accumulate in sera of cancer patients to readily detectable levels. In contrast, no drug-responsive NOX activities have been found in the sera of healthy volunteers (6, 7).
While the basis for the cancer specificity of cell surface tNOX has not been determined, the concept is strongly supported by several lines of evidence. A drug responsive tNOX activity has been rigorously determined to be absent from plasma membranes of non-transformed human and animal cells and tissues (3). The tNOX proteins lack a transmembrane binding domain (10) and are released from the cell surface by brief treatment at low pH (9). A drug-responsive tNOX activity has not been detected in sera from healthy volunteers or patients with diseases other than cancer (6, 7). Several tNOX antisera have identified the immunoreactive band at 34 kDa (the processed molecular weight of the cell surface form of tNOX) with Western blot analysis or immunoprecipitation when using non-transformed cells and tissues or sera from healthy volunteers or patients with disorders other than cancers as antigen source (5, 10, 11). Those antisera include a monoclonal antibody (5), single-chain variable region fragment (scFv) recombinant antibody derived from DNA of the monoclonal hybridoma (5), polyclonal antisera to expressed tNOX (11) and polyclonal peptide antisera to the conserved adenine nucleotide binding region of tNOX (11).
tNOX cDNA has been cloned (GenBank Accession No. AF207881; 11; U.S. Patent Publication 2003-0207340 A1). The derived molecular weight from the open reading frame was 70.1 kDa. Identified functional motifs include a quinone binding site, an adenine nucleotide binding site, and a CXXXXC cysteine pair as a potential protein disulfide-thiol interchange site based on site-directed mutagenesis (11). Based on available genomic information (12) the tNOX gene is located on chromosome X, and it is comprised of multiple exons (thirteen) (See
Because cancer and certain viral, protozoan and parasite infections pose significant threats to human health and because cancer and such infections result in significant economic costs, there is a long-felt need in the art for an effective, economical and technically simple system in which to assay for or model for, identifying inhibitors of the aforementioned disease states.
The present invention provides a cancer-specific alternatively spliced tumor-specific plasma membrane NADH oxidase/thiol interchange protein transcript termed E4mtNOX herein). This cDNA sequence, spliced such that exon 4 is not present, is shown in SEQ ID NO:10, and the resulting 44.2 kDa cancer-specific E4mtNOX-encoded primary translation product expressed therefrom (initiation at M231 of SEQ ID NO:13) has the amino acid sequence given in SEQ ID NO:15. A second cancer-specific, alternatively spliced tNOX sequence, lacking exon 5 (E5mtNOX) has the coding sequence given in SEQ ID NO:11. Also within the scope of the present invention are coding sequences which are synonymous with the specifically exemplified E4mtNOX coding sequence. Also contemplated are sequences which encode a neoplastic cell surface marker and which coding sequences hybridize under stringent conditions to the specifically exemplified full length or partial sequence. The cell surface E4mtNOX protein is characteristic of neoplastic conditions, as are the particular mRNAs. The recombinant E4mtNOX protein is useful in preparing antigen for use in generation of monoclonal antibodies or antisera for diagnosis of neoplastic conditions, including cancers. The E4mtNOX protein is expressed in all cancerous (neoplastic cells), and thus, it can serve as a “pancancer” marker.
Within the scope of the present invention are non-naturally occurring recombinant DNA molecules comprising the alternately spliced coding sequence termed E4mtNOX herein, and optionally further comprising a heterologous sequence such as a tag sequence which facilitates purification. Such a tag sequence can be, but is not limited to, a polyhistidine (His tag) sequence, a flagellar antigen tag sequence (Flag tag), a glutathione S-transferase tag sequence (GST tag) or biotin binding sequence (Strep tag). Also provided herein are methods for recombinantly producing a recombinant NADH oxidase/protein disulfide-thiol interchange active polypeptide in a host cell (bacterial, yeast, mammalian) using the alternately spliced sequence and recombinant DNA molecules provided herein.
The present invention further provides a method for determining neoplasia in a mammal, including a human, said method comprising the steps of detecting the presence, in a biological sample from a mammal, of an alternatively spliced NADH oxidase/protein disulfide thiol interchange protein ribonucleic acid molecule (lacking exon 4 or exon 5) uniquely associated with neoplastic cells as compared to a ribonucleic acid molecule encoding a NADH oxidase associated with normal cells, wherein the step of detecting is carried out using hybridization under stringent conditions or using a polymerase chain reaction in which a perfect match of primer to template is required, where a hybridization probe or forward RT-PCR primer comprising of at least 15 consecutive nucleotides as given in SEQ ID NO:4 or SEQ ID NO:6 and correlating the result obtained with said sample in step (a), where the presence of the ribonucleic acid molecule in the biological sample is indicative of the presence of neoplasia. These SEQ ID NO:4 and SEQ ID NO:6 probes and primers comprise sequences that encompass the splice junctions of the alternately spliced tNOX cDNA, as taught herein. Either of these forward primers can be used in conjunction with a primer downstream of the cancer-specific splice junction, for example a primer from exon 8, as specifically exemplified a primer characterized by the nucleotide sequence given in SEQ ID NO:5. Alternatively, a forward primer matching a sequence upstream of exon 4 or exon 5 can be used, with the size(s) of amplification products revealing whether the exon 4 or exon 5 sequences are missing from the transcript template.
The present invention further provides additional RT-PCT (reverse transcriptase polymerase chain reaction) methods for assessment of neoplasia in a patient sample, for example, in biopsy material, based on the cancer-specific transcription products corresponding in sequence to E4mtNOX and E5mtNOX. A forward primer is chosen to comprise a nucleotide sequence of at least 15, preferably 20-25 contiguous nucleotides, from SEQ ID NO:12, nucleotides 1-362, preferably from 1-330, when an E4tNOX or E5mtNOPX transcript is to be detected. The reverse primer is designed to contain at least 15, preferably 20-25, contiguous, complementary nucleotides (from SEQ ID NO:11) downstream of the missing exon 4 or exon 5 portions of the transcript to be detected, such that the amplification product is 208 nucleotides shorter in a cancer (neoplastic) cell or tissue than in a normal cell or tissue when the exon 4-minus product is detected and such that the amplification product is 233 nucleotides shorter when the exon 5-minus product is detected. A specifically exemplified reverse primer is given in SEQ ID NO:5; it is from exon 8. The use of a reverse primer downstream of both the exon 4 and exon 5 sequences allows the detection of both the E4mtNOX and E5tNOX transcripts in a single RT-PCR assay. Exon 4 corresponds to nucleotides 363 to 571 of SEQ ID NO:12, and exon 5 corresponds to nucleotides 572 to 805 of SEQ ID NO:12.
Also within the scope of the present invention are antisense oligonucleotides which block the expression of the cancer-specific tNOX protein and restore the normal phenotype of neoplastic cells into which one or more of those antisense oligonucleotides has been introduced. Specifically exemplified antisense nucleic acid molecules comprise sequences as set forth in SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:25 and SEQ ID NO:27.
Full length tNOX mRNA is transcriptionally expressed in human non-cancer cells and tissues as well as in neoplastic cells and tissues. HeLa cells (human cervical carcinoma cell line) express the full length mRNA and the functional 34 kDa tNOX protein. To investigate whether the full length tNOX mRNA is cancer specific, human normal cell lines and tissues were examined by RT-PCR and Northern blot in separate experiments. Commercially available normal human tissue mRNA preparations were probed using the tNOX cDNA sequence in Northern blots. Of the eight different normal human tissues examined, all exhibited actively transcribed full length tNOX mRNA (3.8 Kb as shown in
The full length tNOX cDNA sequence is identical in both cancer cells and non-cancer cells. RT-PCR for both cancer and non-cancer cells was carried out using probes covering the 5′UTR and the 3′ end of the tNOX cDNA ORF. The PCR products were cloned into a T7 vector and sequenced. Sequencing results showed no differences in the full length tNOX cDNA sequence between cancer and non-cancer cell lines.
5′-RACE showed there was no upstream ATG beyond that previously found. The tNOX cDNA ORF is 1.83 kb, and it is predicted to encode a protein of 610 amino acids, but there was no naturally expressed 71 kDa tNOX (610 amino acids deduced from tNOX ORF) observed in either cancer or non-cancer cells. The tNOX cDNA sequence has a rare short 22 nucleotide 5′-UTR. Without wishing to be bound by any particular theory, it is believed that this explains why translation does not start from the first ATG. On the other hand, it raises the question whether there is more sequence in the 5′-UTR and/or whether there is another ATG downstream of the current 5′ end in cancer cells.
In 5′-RACE, mRNAs of BT-20 and MCF-10A cells were prepared following standard mRNA preparation protocols. The ˜240 bp band in the result of 5′-RACE corresponded to exon 1 and exon 2 of tNOX cDNA (
Expression of full length tNOX cDNA in non-cancer cells did not result in expression of 34 kDa processed tNOX protein—full-length tNOX cDNA inserted in a mammalian expression vector was expressed in mammalian cells to determine if a 34 kDa tNOX protein was produced. The full length tNOX cDNA was first cloned into the mammalian expression vector pcDNA 3.1. The constructs were then transfected into COS and MCF-10A cells (wild type COS and MCF-10A cells do not express tNOX protein as determined by Western blots and enzymatic assay of drug-response NOX activity). The wild type COS and MCF-10A cells were collected, and the proteins were separated by SDS-PAGE and visualized by Western blotting. A peptide antibody specific for the quinone binding region of tNOX was used in the Western blot.
The results showed that the full-length tNOX cDNA directed expression of a protein of around 71 kDa under the control of a vector promoter in COS and MCF-10A cells. There was no processed form of tNOX protein around 34 kDa observed in the transfectants (
To study cancer specific expression of tNOX, MCF-10A and BT-20 plasma membranes were purified. About 0.05 mg samples of plasma membrane proteins of MCF-10A and BT-20 were analyzed by SDS-PAGE (10%) and by Western blot analysis.
For Western blot analysis, proteins were reacted with peptide antibody specific for the tNOX C-terminus containing the conserved adenine nucleotide binding region (KQEMTGVGASLEKRW) (SEQ ID NO:9). Detection was with phosphatase and BCIP and NBT. The 34 kDa protein was present only in BT-20 plasma membranes and not in MCF-10A plasma membranes (
There were several other bands present in both MCF-10A and BT-20 plasma membranes. Without wishing to be bound by theory, it is believed those bands have sequences homologous to the adenine nucleotide binding region of tNOX that bind the peptide antibody and therefore cross-react with that antibody.
Splice variants of tNOX were found in cancer cells, while fill length tNOX mRNA was present in normal and cancer cells. The tNOX protein is present only on the surface of cancer cells. RT-PCR of MCF-10A, BT-20 and HeLa cells showed an Exon 4 minus form (band 2) and an Exon 5 minus form product (band 3) in both BT-20 and HeLa cells, which are cancer cells, but not in the non-cancer MCF-10A cells (
When the NCBI database was searched, two other possible splice variants were found: AK000353 (from a hepatoma cell line) and AL133207 (gene located on chromosome X). Both have the same sequence as exon 2 to exon 8 of tNOX. AK000353 has a 234 bp sequence before exon 2 that is different from that of tNOX. AL133207 has a 348 bp sequence before exon 2 that differs from that of tNOX. Nucleotide 31 to 234 of AK000353 is the same as nucleotide 1 to 204 of AL133207. The presence of these two splice variants were established by RT-RCR (
Additional evidence for a cancer cell-specific expression of exon 4 minus mRNA was provided using exon 4 minus-specific probes generated to the exon 4 minus-specific sequences at the splice junction between exon 3 and exon 5. RNA preparations from HeLa human cervical carcinoma and BT-20 human mammary carcinoma cells clearly contained the expected 670 bp cancer specific product indicative of the absence of exon 4 (
Expression of Exon 4 minus and Exon 5 minus tNOX transcripts (E4mtNOX and E5mtNOX, respectively) in COS cells has been studied. The Exon 5 minus cDNA yielded an open reading frame encoding a deduced amino acid sequence of 532 amino acids and a predicted molecular weight of 60.7 kDa (
Transport of the 34 kDa tNOX protein to plasma membrane was studied. The functional 34 kDa tNOX protein is on the outer leaflet of cancer cell plasma membranes (7). For the 34 kDa protein expressed from exon 4 minus splice variant is functional, it must reach the plasma membrane. To investigate the sub-cellular localization of the 34 kDa tNOX protein expressed in exon 4 minus COS transfectants, plasma membranes and internal membranes of the transfectants were prepared, the proteins were resolved on SDS-PAGE and then analyzed by Western blotting. Plasma membranes and internal membranes of the exon 4 minus transfectants, along with a whole cell preparation, were probed with a peptide antibody specific for the quinone binding motif of the tNOX protein. The whole cell preparation exhibited 52, 47 and 34 kDa bands. The internal membrane preparation exhibited only 52 and 47 kDa bands. The plasma membrane preparation exhibited only the 34 kDa band. The 52 kDa protein remained in cytoplasm and did not reach the plasma membrane. By contrast, the 34 kDa protein was delivered to the plasma membrane, either as a single product of the exon 4 minus mRNA or a processed form of the 47 or 52 kDa protein (
Mutation of Met 231 (of SEQ ID NO:13) blocked translational expression of the exon 4 minus splice variant. In the previous overexpression experiments, the 52, 47 and 34 kDa proteins were observed in exon 4 minus splice variant transfectants. The hypothesis to explain the expression of these proteins is that a downstream Met is codon used in the exon 4 minus mRNA during translation (see details about downstream translation initiation below). With our knowledge of the translation initiation and membrane insertion sequence required for the expression of the 34 kDa protein and considering the deletion of exon 4, possible downstream methionine codons include those encoding Met 220, Met 231 and Met314 (With reference to SEQ ID NO:13). Site-directed mutagenesis of the cloned E4m sequence to replace methionine codons with alanine codons (Met220Ala, Met231Ala, and Met314Ala) was carried out. Preliminary results showed that in Met231Ala transfected COS cells, the 47 kDa band was missing (
The size of the tNOX protein translated in HeLa cell endoplasmic reticulum was determined. HeLa cells were fractionated into endoplasmic reticulum- and plasma membrane-rich fractions by aqueous two-phase partition (17). A recombinant single chain variable region antibody carrying a His tag (partially purified by nickel agarose precipitation) was used to concentrate and identify tNOX related proteins. The endoplasmic reticulum-enriched fraction contained a 47 kDa band absent from the plasma membrane (
Confocal microscopy was used to determine the subcellular localization of E4m tNOX-EGFP and full length tNOX-EGFP expressed in COS cells. The tNOX constructs tagged with EGFP at the C terminus were expressed under the control of the cytomegalovirus promoter. Fluorescence microscopy revealed that the E4mtNOX-EGFP fusion protein was localized to plasma membrane. In contrast, full length tNOX-EGFP fusion protein was retained in internal membranes (
EGFP and E4m-EGFP (expressed in COS cells) proteins were analyzed by PAGE and Western blotting using the anti-GFP antibody and peptide Ab (a polyclonal peptide antibody to the conserved adenine nucleotide binding region of tNOX). An approximately 74 kDa band was seen in E4m-EGFP, consistent with the predicted molecular mass of the E4m tNOX (47 kDa) plus EGFP (27 kDa) fusion protein (
A 34 kDa tNOX protein is found on the surface of cancer cells (1, 2, 8). It is shed into the sera of cancer patients (6, 7) as well as the media of cultured cancer cells (14). A characteristic of the tNOX protein is its drug-response to vanilloids as capsaicin (3) and anti-tumor drugs such as anti-tumor sulfonylureas (15), EGCg (16), and adriamycin (17). It exists in all of the solid cancer tissues, sera of cancer patients, and on the surface of cancer cells (6,7), but not in normal human tissues, sera of healthy volunteers, or non-cancer cells (10). This study was undertaken in part to determine the mechanism for the cancer cell specificity of the 34 kDa cell surface tNOX.
Taken together, the findings indicate that the exon 4 minus splice variant tNOX transcript (E4mtNOX) is unique to neoplastic cells and tissue. First, the exon 4 minus tNOX mRNA exists only in cancer cell lines, as thus far investigated. Second, overexpression experiments show that the exon 4 minus transcript generates the 34 kDa tNOX protein that reacts with a peptide antibody specific for tNOX. In exon 4 minus tNOX cDNA transfected COS cells, at least three proteins (52, 47 and 34 kDa) are expressed that react with the tNOX peptide antibody. The three expressed proteins may be either the result of multiple translation initiation sites, including alternative downstream translation initiation sites, or the 34 kDa protein is a proteolytically processed form originating either from the 52 kDa or 47 kDa protein. The Western blot of transfectants' plasma membranes support the latter hypothesis. The 34 kDa protein is only seen in plasma membranes but not in internal membranes, while the 52 kDa protein exists only in internal membranes and is not found in plasma membranes.
As stated above, there is no evidence of physiological translation of the full length tNOX mRNA despite its widespread transcription. In the cDNA sequence of tNOX, the first ATG starts at nucleotide 23. There are only 22 nucleotides in the 5′UTR. This short 5′UTR makes the first ATG a less likely translation initiation site because of the potential restrictions on ribosome binding.
The scanning mechanism is the current model for translation initiation in eukaryotes (18). After recognition of the cap structure of mRNA, the translation initiation complex forms around 21-24 nucleotides, then begins the scanning for a Met codon. The “first AUG” rule holds for about 90% of the hundreds of eukaryotic mRNAs that have been analyzed (19). Usually the first Met codon starts at about 50-150 nucleotides downstream of the cap (20). In the tNOX sequence, the first Met encoded at nucleotide 23 is believed to be a non-functional initiation sequence for translation. Sequence elements in 5′ UTR are involved in regulation of translation (21). The first ATG does not always function as an initiator codon (19, 22). A 5′UTR length is usually smaller than 200 bp because of the limitation of the scanning capacity of the ribosome (19). If the first ATG of full length tNOX mRNA functions as the initiator codon, the 5′UTR of tNOX is only 22 nucleotides, shorter than most functional 5′UTRs (23). Thus it is believed that the initiator ATG is a downstream ATG. A search for a Kozak sequence (A/G)XXAUGG (19) including a downstream initiation site uncovers a Met codon at nucleotide 168 (exon 2), nucleotide 543 (exon 4), nucleotide 713 (exon 5), nucleotide 1155 (exon 8). By deleting an upstream portion of the message through alternative splicing, it may be possible for the scanning ribosome to reach a downstream methionine codon to begin initiation thus to ultimately generate the cell surface 34 kDa form. Another possible explanation is that deletion of exon 4 disrupts the 2nd structure of tNOX mRNA, thus hindering ribosome binding. There may be cancer specific RNA binding proteins involved in this process as well, which increase or decrease the possibility of ribosome binding for downstream translation initiation. Because the processed tNOX protein is on the extracellular side of plasma membrane, the actual translation initiation site must be followed by a membrane insertion sequence. The exon 4 minus splice variant results in a 207 nucleotide deletion. There is no frame shift after the deletion of exon 4. The result is a 68 amino acid deletion and one amino acid change. As discussed above with respect to alternative translation initiation sites, the deletion of exon 4 results in deletion of one Met (at the Met codon beginning at nucleotide 543 of SEQ ID NO:14) in exon 4 and brings the possible downstream Met closer to 5′ end. The apparent 52 kDa protein on Western blots may be a processed form that initiates from Met-1 or from a downstream Met. The apparent 34 kDa protein observed on western blots might have come from multiple translation initiation (i.e. starts from a further downstream Met codon), or it could be a processed form from the 52 kDa protein. There are two candidate leader sequences, L236 through S248 and Q323 through H340, with reference to SEQ ID NO:13.
tNOX mRNA synthesis is down-regulated in HeLa cells transfected with antisense oligonucleotides. By using real-time quantitative PCR, E4AS and E5AS were tested for their ability to down-regulate the tNOX mRNA in HeLa cells that were transfected with antisense. As expected, E4AS mediated the down-regulation of full length tNOX mRNA (
HeLa cells transfected with E5AS were no longer inhibited by EGCg or Capsibiol-T. In vitro cytotoxicity results showed that E5AS transfection could eliminate the inhibition of EGCg (
E5AS induced cell death of HeLa cells The effects of antisense oligonucleotides on HeLa cell viability were tested. As show in
The amino acids which occur in the various amino acid sequences referred to in the specification have their usual three- and one-letter abbreviations routinely used in the art: A, Ala, Alanine; C, Cys, Cysteine; D, Asp, Aspartic Acid; E, Glu, Glutamic Acid; F, Phe, Phenylalanine; G, Gly, Glycine; H, His, Histidine; I, Ile, Isoleucine; K, Lys, Lysine; L, Leu, Leucine; M, Met, Methionine; N, Asn, Asparagine; P, Pro, Proline; Q, GIn, Glutamine; R, Arg, Arginine; S, Ser, Serine; T, Thr, Threonine; V, Val, Valine; W, Try, Tryptophan; Y, Tyr, Tyrosine; BCIP, 5-bromo-4-chloro-3-indolyl-phosphate; NBT, nitroblue tetrazolium.
As used herein, neoplasia describes a disease state of a human or an animal in which there are cells and/or tissues which proliferate abnormally. Neoplastic conditions include, but are not limited to, cancers, sarcomas, tumors, leukemias, lymphomas, and the like. The cell surface NADH oxidase/protein disulfide-thiol exchange protein of the present invention is characteristic of neoplastic cells and tissue.
The cell surface marker which is characteristic of diseased cells is described in U.S. Pat. No. 5,605,810, issued Feb. 25, 1997, which is incorporated by reference herein, the full length cDNA sequence is presented in WO 01/032673 and in numerous scientific publications of which D. James Morre is sole author or a coauthor. This NADH oxidase/thiol interchange protein is found in the plasma membrane of neoplastic cells and cells infected with viruses, especially retroviruses and protozoan parasites. This neoplasia-specific transcript lacking exon 4, as characterized by the nucleotide sequence set forth in SEQ ID NO:10, is termed E4mtNOX herein (alternatively spliced tumor NADH oxidase, lacking exon 4). The E4mtNOX transcript gives rise to 46 kDa and 44 kDa proteins (see Tables 1 and 2 and SEQ ID NO:14 and SEQ ID NO:15). The cell surface 34 kDa protein, processed from a E4mtNOX translation product, is shed into serum and urine in cancer patients, but purification is relatively difficult. Therefore, it was a goal of the present work to obtain a cDNA clone encoding E4m tNOX for use in recombinant production of this protein and for use of the E4mtNOX and E5mtNOX spliced coding sequences or portions thereof in probes and primers for the detection of cancer-specific, alternatively spliced tNOX transcripts. A cDNA for the “full length” message has been described (see, e.g., WO 01/032673 or US 2003-0207340 A1) and its sequence is also available on the website of the National Center for Biotechnology Information, Accession No. AF207881. See also the cDNA sequence set forth in SEQ ID NO:12 herein. The corresponding genomic sequence is available within Accession No. AL049733.
As used herein, neoplasia describes a disease state of a human or an animal in which there are cells and/or tissues which proliferate abnormally. Neoplastic conditions include, but are not limited to, cancers, sarcomas, tumors, leukemias, lymphomas, and the like. The cell surface NADH oxidase/protein disulfide-thiol interchange protein of the present invention characterizes neoplastic cells and tissue as well as certain virus-infected cells (for example, human immunodeficiency virus, feline immunodeficiency virus, etc).
A protein is considered an isolated protein if it is a protein isolated from a host cell in which it is recombinantly produced. It can be purified or it can simply be free of other proteins and biological materials with which it is associated in nature.
An isolated nucleic acid is a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes. The term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding or noncoding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Specifically excluded from this definition are nucleic acids present in mixtures of (i) DNA molecules, (ii) transformed or transfected cells, and (iii) cell clones, e.g., as these occur in a DNA library such as a cDNA or genomic DNA library.
As used herein expression directed by a particular sequence is the transcription of an associated downstream sequence. If appropriate and desired for the associated sequence, there the term expression also encompasses translation (protein synthesis) of the transcribed RNA.
In the present context, a promoter is a DNA region which includes sequences sufficient to cause transcription of an associated (downstream) sequence. The promoter may be regulated, i.e., not constitutively acting to cause transcription of the associated sequence. If inducible, there are sequences present which mediate regulation of expression so that the associated sequence is transcribed only when an inducer molecule is present in the medium in or on which the organism is cultivated. In the present context, a transcription regulatory sequence includes a promoter sequence and can further include cis-active sequences for regulated expression of an associated sequence in response to environmental signals.
One DNA portion or sequence is downstream of second DNA portion or sequence when it is located 3′ of the second sequence. One DNA portion or sequence is upstream of a second DNA portion or sequence when it is located 5′ of that sequence.
One DNA molecule or sequence and another are heterologous to another if the two are not derived from the same ultimate natural source. The sequences may be natural sequences, or at least one sequence can be designed by man, as in the case of a multiple cloning site region. The two sequences can be derived from two different species or one sequence can be produced by chemical synthesis provided that the nucleotide sequence of the synthesized portion was not derived from the same organism as the other sequence.
An isolated or substantially pure nucleic acid molecule or polynucleotide is a polynucleotide which is substantially separated from other polynucleotide sequences which naturally accompany a native transcription regulatory sequence. The term embraces a polynucleotide sequence which has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates, chemically synthesized analogues and analogues biologically synthesized by heterologous systems.
A polynucleotide is said to encode a polypeptide if, in its native state or when manipulated by methods known to those skilled in the art, it can be transcribed and/or translated to produce the polypeptide or a fragment thereof. The anti-sense strand of such a polynucleotide is also said to encode the sequence.
A nucleotide sequence is operably linked when it is placed into a functional relationship with another nucleotide sequence. For instance, a promoter is operably linked to a coding sequence if the promoter effects its transcription or expression. Generally, operably linked means that the sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame. However, it is well known that certain genetic elements, such as enhancers, may be operably linked even at a distance, i.e., even if not contiguous.
The term recombinant polynucleotide refers to a polynucleotide which is made by the combination of two otherwise separated segments of sequence accomplished by the artificial manipulation of isolated segments of polynucleotides by genetic engineering techniques or by chemical synthesis. In so doing one may join together polynucleotide segments of desired functions to generate a desired combination of functions.
Polynucleotide probes include an isolated polynucleotide attached to a label or reporter molecule and may be used to identify and isolate other sequences, for example, those from other mammalian species. Probes comprising synthetic oligonucleotides or other polynucleotides may be derived from naturally occurring or recombinant single or double stranded nucleic acids or be chemically synthesized. Polynucleotide probes may be labeled by any of the methods known in the art, e.g., random hexamer labeling, nick translation, or the Klenow fill-in reaction.
Large amounts of the polynucleotides may be produced by replication in a suitable host cell. Natural or synthetic DNA fragments coding for a protein of interest are incorporated into recombinant polynucleotide constructs, typically DNA constructs, capable of introduction into and replication in a prokaryotic or eukaryotic cell. Usually the construct is suitable for replication in a unicellular host, such as COS cells or a bacterium, but a multicellular eukaryotic host may also be appropriate, with or without integration within the genome of the host cell. Commonly used prokaryotic hosts include strains of Escherichia coli, although other prokaryotes, such as Bacillus subtilis or a pseudomonad, may also be used. Eukaryotic host cells include yeast, fungi, plant, insect, amphibian and avian species, but the regulated expression of a protein of interest a mammalian cell is preferred. Such factors as ease of manipulation, ability to appropriately glycosylate expressed proteins, degree and control of protein expression, ease of purification of expressed proteins away from cellular contaminants or other factors influence the choice of the host cell.
The polynucleotides may also be produced by chemical synthesis, e.g., by the phosphoramidite method described by Beaucage and Caruthers (1981) Tetra. Lefts. 22: 1859-1862 or the triester method according to Matteuci et al. (1981) J. Am. Chem. Soc. 103: 3185, and may be performed on commercial automated oligonucleotide synthesizers. A double-stranded fragment may be obtained from the single stranded product of chemical synthesis either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
DNA constructs prepared for introduction into a prokaryotic or eukaryotic host will typically comprise a replication system (i.e. vector) recognized by the host, including the intended DNA fragment encoding the desired polypeptide, and will preferably also include transcription and translational initiation regulatory sequences operably linked to the polypeptide-encoding segment. Expression systems (expression vectors) may include, for example, an origin of replication or autonomously replicating sequence (ARS) and expression control sequences, a promoter, an enhancer and necessary processing information sites, such as ribosome-binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, and mRNA stabilizing sequences. Signal peptides may also be included where appropriate from secreted polypeptides of the same or related species, which allow the protein to cross and/or lodge in cell membranes or be secreted from the cell.
An appropriate promoter and other necessary vector sequences will be selected so as to be functional in the host. Examples of workable combinations of cell lines and expression vectors are described in Sambrook et al. (1989) vide infra; Ausubel et al. (Eds.) (1995) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York; and Metzger et al. (1988) Nature, 334: 31-36. Many useful vectors for expression in bacteria, yeast, fungal, mammalian, insect, plant or other cells are well known in the art and may be obtained such vendors as Invitrogen, Stratagene, New England Biolabs, Promega Corporation, and others. In addition, the construct may be joined to an amplifiable gene (e.g., DHFR) so that multiple copies of the gene may be made. For appropriate enhancer and other expression control sequences, see also Enhancers and Eukaryotic Gene Expression, Cold Spring Harbor Press, N.Y. (1983). While such expression vectors may replicate autonomously, they may less preferably replicate by being inserted into the genome of the host cell.
Expression and cloning vectors will likely contain a selectable marker, that is, a gene encoding a protein necessary for the survival or growth of a host cell transformed with the vector. Although such a marker gene may be carried on another polynucleotide sequence co-introduced into the host cell, it is most often contained on the cloning vector. Only those host cells into which the marker gene has been introduced will survive and/or grow under selective conditions. Typical selection genes encode proteins that confer resistance to antibiotics or other toxic substances, e.g., ampicillin, neomycin, methotrexate, etc.; complement auxotrophic deficiencies; or supply critical nutrients not available from complex media. The choice of the proper selectable marker will depend on the host cell; appropriate markers for different hosts are known in the art.
Recombinant host cells, in the present context, are those which have been genetically modified to contain an isolated DNA molecule of the instant invention. The DNA can be introduced by any means known to the art which is appropriate for the particular type of cell, including without limitation, transformation, lipofection or electroporation.
It is recognized by those skilled in the art that the DNA sequences may vary due to the degeneracy of the genetic code and codon usage. All DNA sequences which code for the polypeptide or protein of interest are included in this invention.
Additionally, it will be recognized by those skilled in the art that allelic variations may occur in the DNA sequences which will not significantly change activity of the amino acid sequences of the peptides which the DNA sequences encode. All such equivalent DNA sequences are included within the scope of this invention and the definition of the regulated promoter region. The skilled artisan will understand that the sequence of the exemplified sequence can be used to identify and isolate additional, nonexemplified nucleotide sequences which are functionally equivalent to the sequences given.
Hybridization procedures are useful for identifying polynucleotides with sufficient homology to the subject regulatory sequences to be useful as taught herein. The particular hybridization technique is not essential to the subject invention. As improvements are made in hybridization techniques, they can be readily applied by one of ordinary skill in the art.
A probe and sample are combined in a hybridization buffer solution and held at an appropriate temperature until annealing occurs. Thereafter, the membrane is washed free of extraneous materials, leaving the sample and bound probe molecules typically detected and quantified by autoradiography and/or liquid scintillation counting. As is well known in the art, if the probe molecule and nucleic acid sample hybridize by forming a strong non-covalent bond between the two molecules, it can be reasonably assumed that the probe and sample are essentially identical, or completely complementary if the annealing and washing steps are carried out under conditions of high stringency. The probe's detectable label provides a means for determining whether hybridization has occurred.
In the use of the oligonucleotides or polynucleotides as probes, the particular probe is labeled with any suitable label known to those skilled in the art, including radioactive and non-radioactive labels. Typical radioactive labels include 32P, 35S, and the like. Non-radioactive labels include, for example, ligands such as biotin or thyroxine, as well as enzymes such as hydrolases or peroxidases, or a chemiluminescer such as luciferin, or fluorescent compounds like fluorescein and its derivatives. Alternatively, the probes can be made inherently fluorescent as described in International Application No. WO 93/16094.
Various degrees of stringency of hybridization can be employed. The more stringent the conditions, the greater the complementarity that is required for duplex formation. Stringency can be controlled by temperature, probe concentration, probe length, ionic strength, time, and the like. Preferably, hybridization is conducted under moderate to high stringency conditions by techniques well know in the art, as described, for example in Keller, G. H., M. M. Manak (1987) DNA Probes, Stockton Press, New York, N.Y., pp. 169-170, hereby incorporated by reference.
As used herein, moderate to high stringency conditions for hybridization are conditions which achieve the same, or about the same, degree of specificity of hybridization as the conditions employed by the current inventors. An example of high stringency conditions are hybridizing at 68° C. in 5×SSC/5× Denhardt's solution/0.1% SDS, and washing in 0.2×SSC/0.1% SDS at room temperature. An example of conditions of moderate stringency are hybridizing at 68° C. in 5×SSC/5× Denhardt's solution/0.1% SDS and washing at 42° C. in 3×SSC. The parameters of temperature and salt concentration can be varied to achieve the desired level of sequence identity between probe and target nucleic acid. See, e.g., Sambrook et al. (1989) vide infra or Ausubel et al. (1995) Current Protocols in Molecular Biology, John Wiley & Sons, NY, N.Y., for further guidance on hybridization conditions.
Specifically, hybridization of immobilized DNA in Southern blots with 32P-labeled gene specific probes was performed by standard methods (Maniatis et al.) In general, hybridization and subsequent washes were carried out under moderate to high stringency conditions that allowed for detection of target sequences with homology to the exemplified sequences. For double-stranded DNA gene probes, hybridization can be carried out overnight at 20-25° C. below the melting temperature (Tm) of the DNA hybrid in 6×SSPE 5× Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. The melting temperature is described by the following formula (Beltz, G. A., Jacobe, T. H., Rickbush, P. T., Chorbas, and F. C. Kafatos (1983) Methods of Enzymology, R. Wu, L, Grossman and K. Moldave (eds) Academic Press, New York 100:266-285).
Tm=81.5° C.+16.6 Log[Na+]+0.41(+G+C)−0.61(% formamide)−600/length of duplex in base pairs.
Washes are typically carried out as follows: twice at room temperature for 15 minutes in 1×SSPE, 0.1% SDS (low stringency wash), and once at TM-20° C. for 15 minutes in 0.2×SSPE, 0.1% SDS (moderate stringency wash).
For oligonucleotide probes, hybridization was carried out overnight at 10-20° C. below the melting temperature (Tm) of the hybrid 6×SSPE, 5× Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. Tm for oligonucleotide probes was determined by the following formula: TM(° C.)=2(number T/A base pairs+4(number G/C base pairs) (Suggs, S. V., T. Miyake, E. H., Kawashime, M. J. Johnson, K. Itakura, and R. B. Wallace (1981) ICB-UCLA Symp. Dev. Biol. Using Purified Genes, D. D. Brown (ed.), Academic Press, New York, 23:683-693).
Washes were typically carried out as follows: twice at room temperature for 15 minutes 1×SSPE, 0.1% SDS (low stringency wash), and once at the hybridization temperature for 15 minutes in 1×SSPE, 0.1% SDS (moderate stringency wash).
In general, salt and/or temperature can be altered to change stringency. With a labeled DNA fragment of more than 70 or so bases in length, the following conditions can be used: Low, 1 or 2×SSPE, room temperature; Low, 1 or 2×SSPE, 42° C.; Moderate, 0.2× or 1×SSPE, 65° C.; and High, 0.1×SSPE, 65° C.
Duplex formation and stability depend on substantial complementarity between the two strands of a hybrid, and, as noted above, a certain degree of mismatch can be tolerated. Therefore, the probe sequences of the subject invention include mutations (both single and multiple), deletions, insertions of the described sequences, and combinations thereof, wherein said mutations, insertions and deletions permit formation of stable hybrids with the target polynucleotide of interest. Mutations, insertions, and deletions can be produced in a given polynucleotide sequence in many ways, and those methods are known to an ordinarily skilled artisan. Other methods may become known in the future.
Mutational, insertional, and deletional variants of the disclosed nucleotide sequences can be readily prepared by methods which are well known to those skilled in the art. These variants can be used in the same manner as the exemplified primer sequences so long as the variants have substantial sequence homology with the original sequence. As used herein, substantial sequence homology refers to homology which is sufficient to enable the variant polynucleotide to function in the same capacity as the polynucleotide from which the probe was derived. Preferably, this homology is greater than 80%, more preferably, this homology is greater than 85%, even more preferably this homology is greater than 90%, and most preferably, this homology is greater than 95%. The degree of homology or identity needed for the variant to function in its intended capacity depends upon the intended use of the sequence. It is well within the skill of a person trained in this art to make mutational, insertional, and deletional mutations which are equivalent in function or are designed to improve the function of the sequence or otherwise provide a methodological advantage.
Polymerase Chain Reaction (PCR) is a repetitive, enzymatic, primed synthesis of a nucleic acid sequence. This procedure is well known and commonly used by those skilled in this art (see Mullis, U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki et al. (1985) Science 230:1350-1354 and commercial suppliers of reagents for PCR, real time PCR and reverse transcriptase PCR). In the context of the present application, RT-PCR is reverse transcriptase PCR. PCR is based on the enzymatic amplification of a DNA fragment of interest that is flanked by two oligonucleotide primers that hybridize to opposite strands of the target sequence. The primers are oriented with the 3′ ends pointing towards each other. Repeated cycles of heat denaturation of the template, annealing of the primers to their complementary sequences, and extension of the annealed primers with a DNA polymerase result in the amplification of the segment defined by the 5′ ends of the PCR primers. Since the extension product of each primer can serve as a template for the other primer, each cycle essentially doubles the amount of DNA template produced in the previous cycle. This results in the exponential accumulation of the specific target fragment, up to several million-fold in a few hours. By using a thermostable DNA polymerase such as the Taq polymerase, which is isolated from the thermophilic bacterium Thermus aquaticus, the amplification process can be completely automated. Other enzymes which can be used are known to those skilled in the art.
It is well known in the art that the polynucleotide sequences of the present invention can be truncated and/or mutated such that certain of the resulting fragments and/or mutants of the original full-length sequence can retain the desired characteristics of the full-length sequence. A wide variety of restriction enzymes which are suitable for generating fragments from larger nucleic acid molecules are well known. In addition, it is well known that an exonuclease such as Bal31 can be conveniently used for time-controlled limited digestion of DNA. See, for example, Maniatis (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, pages 135-139, incorporated herein by reference. See also Wei et al. (1983 J. Biol. Chem. 258:13006-13512. By use of Bal31 exonuclease, the ordinarily skilled artisan can remove nucleotides from either or both ends of the subject nucleic acids to generate a wide spectrum of fragments which are functionally equivalent to the subject nucleotide sequences. One of ordinary skill in the art can, in this manner, generate hundreds of fragments of controlled, varying lengths from locations all along the original molecule. The ordinarily skilled artisan can routinely test or screen the generated fragments for their characteristics and determine the utility of the fragments as taught herein. It is also well known that the mutant sequences of the full length sequence, or fragments thereof, can be easily produced with site directed mutagenesis. See, for example, Larionov, O. A. and Nikiforov, V. G. (1982) Genetika 18(3):349-59; Shortle, D, DiMaio, D., and Nathans, D. (1981) Annu. Rev. Genet. 15:265-94; both incorporated herein by reference.
As used herein percent sequence identity of two nucleic acids is determined using the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:402-410. BLAST nucleotide searches are performed with the NBLAST program, score=100, word length=12, to obtain nucleotide sequences with the desired percent sequence identity. To obtain gapped alignments for comparison purposes, Gapped BLAST is used as described in Altschul et al. (1997) Nucl. Acids. Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (NBLAST and XBLAST) are used. See, for example, the National Center for Biotechnology Information website on the internet.
Monoclonal or polyclonal antibodies, preferably monoclonal, specifically reacting with an E4mtNOX-derived protein may be made by methods known in the art. See, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratories; Goding (1986) Monoclonal Antibodies: Principles and Practice, 2d ed., Academic Press, New York.
Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art. A number of standard techniques are described in Sambrook et al. (1989) Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, N. Y.; Maniatis et al. (1982) Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, N.Y.; Wu (ed.) (1993) Meth. Enzymol. 218, Part I; Wu (ed.) (1979) Meth Enzymol. 68; Wu et al. (eds.) (1983) Meth. Enzymol. 100 and 101; Grossman and Moldave (eds.) Meth. Enzymol. 65; Miller (ed.) (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Old and Primrose (1981) Principles of Gene Manipulation, University of California Press, Berkeley; Schleif and Wensink (1982) Practical Methods in Molecular Biology; Glover (ed.) (1985) DNA Cloning Vol. I and II, IRL Press, Oxford, UK; Hames and Higgins (eds.) (1985) Nucleic Acid Hybridization, IRL Press, Oxford, UK; and Setlow and Hollaender (1979) Genetic Engineering: Principles and Methods, Vols. 1-4, Plenum Press, New York. Abbreviations and nomenclature, where employed, are deemed standard in the field and commonly used in professional journals such as those cited herein.
All references cited herein are hereby incorporated by reference to the extent they are not inconsistent with the present disclosure. Those cited references are indicative of the state of the relevant arts.
Although the description herein contains certain specific examples and statements, these should not be construed as limiting the scope of the invention but as merely providing illustrations of some embodiments of the invention. For example, thus the scope of the invention should be determined by the appended claims and their equivalents, rather than by the examples given. Any variations in the exemplified articles which occur to the skilled artisan are intended to fall within the scope of the present invention.
Most chemicals and reagents were from Sigma-Aldrich, St. Louis, Mo. DNA markers, Taq polymerase, and Random Hexamer Priming Kit were from Promega Corporation Madison, Wis. SuperScript II Reverse Transcriptase was from GIBCO/Invitrogen, Carlsbad, Calif. Restriction enzymes were from New England Biolabs, Beverly, Mass. Human Multiple Tissue Northern Blot was from Clontech, Palo Alto, Calif. Fast-Link DNA Ligation Kit was from Epicentre, Madison, Wis. Cloned pfu DNA polymerase and E. coli strain XL1-blue were from Stratagene, La Jolla, Calif. Gel Purification kit, Plasmid DNA Mini and Midi Purification Kit were from Qiagen, Valencia, Calif. Mammalian expression vector pcDNA 3.1, and Calcium Phosphate Transfection Kit were from Invitrogen, Carlsbad, Calif. Alkaline phosphatase conjugated monoclonal anti-rabbit IgG was from Sigma Chemical Co., St. Louis, Mo. Peptide antibodies PU02 and PU04 were generated by Covance Research Products, Inc., Denver, Pa.
Approximately 106 cells of HeLa (human cervical carcinoma), BT-20 (human breast adenocarcinoma), and MCF-1OA (human breast epithelial cells) were collected and washed twice with cold PBS, pH 7.4. Each cell pellet were resuspended in 300 μl RNA Preparation Solution I (10 mM Tris-Cl, 0.15 M NaCl, 1.5 M MgCl2, 0.65% NP-40, DEPC treated). After centrifugation at 800 g for 10 min, the upper phase was transferred to a new tube and 200 μl RNA Preparation Solution 11 (7 M urea, 1% SDS, 0.35 M NaCl, 10 mM EDTA, 10 mM Tris-Cl, DEPC treated) were added. Then 400 μl phenol/chloroform/isoamyl alcohol (50:50:1) were added and the mixture was centrifuged at 15,000 g for 5 min. The clear phase that contained RNA was precipitated with 95% ethanol and dissolved in DEPC (diethyl pyrocarbonate) treated water.
A 20 μl reaction volume containing 1 μl Oligo (dT)12-18 (500 μg/ml), 1 μl 10 mM dNTP, 4 μl 5× First-Strand Buffer, 2 μl 0.1 M DTT, 5 μg of total RNA, and 1 μl Reverse Transcriptase was used. Reaction mixture was incubated at 42° C. for 1 hour, followed by heating at 70° C. for 15 min to inactivate. The synthesized first-strand cDNA was amplified by PCR using primers 5′-CAGCCGATAACAGTAGMCTCTGA-3′ (forward) (SEQ ID NO:1) and 5′-CTGATTCCTCAGTTTCTTTTGTTTCTG-3′ (reverse) (SEQ ID NO:2). PCR products were separated on a 1% agarose gel and recovered a using Gel Extraction Kit(Qiagen). Purified PCR products were sequenced to verify identities.
cDNA probes used in the northern blot experiments were generated from PCR using a pcDNA 3.1-tNOX plasmid. The probes were labeled with (α-32P) dCTP using Random Hexamer Priming Kit (Promega) prior to use. Two probes were used: 1.2 kb tNOX cDNA corresponding to tNOX sequence 680 bp to 1,830 bp; and exon 4 of tNOX. The human MTN (Multiple Tissue Northern) blot was incubated with prehybridization solution at 65° C. for 6 hours. Then hybridization solution with either of the probes was added and incubated at 65° C. overnight. The MTN Blot was washed and autoradiograms (X ray film) were exposed at −80° C.
Total RNA from BT-20 and MCF-10A cells were prepared by the guanidine isothiocyanate/acid-phenol method. Total RNA was quantified by Hitachi U-1100 UV spectrometry and stored at −20° C. in 30 μl DEPC treated water. A custom primer, 109L, is a 20 nt reverse primer matching the end of exon 2: 5′-GGTAAAATTGGTGTCCGGC-3′ (SEQ ID NO:3), was used in first strand synthesis and the later in the PCR process. 20 mM Tris-HCl (pH 8.4), 50 mM KC1, 2.5 mM MgCl2, 10 mM DTT, 100 nM cDNA custom primer 109L, 400 μM each dATP, dCTP, dGTP, dTTP, 5 μg RNA, and 200 units of Superscript II RT were used in the first strand cDNA synthesis. After incubation at 42° C. for one hour, the reaction was terminated by incubation at 70° C. for 15 min. Then RNAse H was added to the mix to digest the RNA strand in RNA/cDNA hetero-double strand. The digest was transferred to the GlassMAX spin cartridge and centrifuge at 13,000 g for 20 s. After three washes, the first strand cDNA was purified and concentrated for the next step of TdT tailing. TdT adds several Cs at the 3′ end of the first strand cDNA. Using anchoring primers and the customer primer 109L, the cDNA was specifically amplified and resolved on 1% agarose gel. It was further cloned into T-vector and sequenced.
Total RNA was prepared from 2×107 cells. The forward primer, a 20 mer oligonucleotide (TCCCTCCAAATCCAATACCG) (SEQ ID NO:4) was from the junction of exon 3 and exon 5 with 15 nucleotides from exon 3 and 5 nucleotides from exon 5. The reverse primer (CTGATTCCTCAGTTTCTTTTCTTTCTG) (SEQ ID NO:5) was from the end of exon 8. A product of 670 nucleotides was expected. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were used as controls (640 nucleotides).
Parts of the Exon 4 minus and Exon 5 minus forms of tNOX (E4mtNOX and E5m tNOX) cDNAs were first amplified by PCR using primers 5′-CCGATMCAGTAGMCTCTGMC-3′ (forward) (SEQ ID NO:6) and 5′-GGCTGATTCCTCAGTTTCTTTTGTTTC-3′ (reverse) (SEQ ID NO:7). The PCR product was then ligated to pGEM-T Easy Vector (Promega, Madison, Wis.) for sequencing. The construct was digested with NotI. The digested products were separated on an agarose gel and extracted using a DNA Extraction Kit (Qiagen, Valencia, Calif.). The DNA then was double digested with BanI and AseI. The digested products were also separated on an agarose gel and extracted (Fragment A). The pcDNA3.1-full length tNOX was double digested with HindIII and BamHI and a fragment containing pcDNA3.1 and part of tNOX (Fragment D) and the other fragment F was obtained. Fragment F was then double digested with BanI and AseI to get 3 fragments. There was a fragment between HindIII and BanI (Fragment B), a fragment between BanI and AseI (Fragment G) and a fragment between AseI and BamHI (Fragment C). All of these digested products were separated on an agarose gel and extracted. Finally, 4 fragments (Fragments A, B, C, and D) were ligated using a Fast-link kit (Epicenter). The resulting plasmid was used to transfect COS cells.
DNA sequences of the ligation products were confirmed by sequencing. Expression of the E4mtNOX and E5mtNOX protein was confirmed by SDS-PAGE with immunoblotting. Immunoblot analysis was with tNOX peptide antibody. Detection used alkaline phosphate-conjugated anti-rabbit antibody. The E4mtNOX and E5tNOX cDNA sequences are given in SEQ ID NO:10 and SEQ ID NO:11, respectively, while the full length tNOX cDNA sequence is depicted in SEQ ID NO:12.
COS cells (SV-40 transformed African monkey kidney cell line) were plated one day prior to transfection at 4×105 cells per 100 mm dish. 36 μl of 2 M CaCl2 and 30 μg of pcDNA3.1-E5m tNOX in 300 μl sterile H2O were slowly added dropwise into 300 μl, of 2× Hepes Buffered Saline (HBS) at room temperature for 30 min. The transfection mixtures then were added dropwise to the media to the cells and incubated overnight at 37° C. After overnight exposure to the DNA precipitate, the cells were washed twice with PBS and 3 ml of DMSO were added for 2.5 min. The DMSO then was removed and cells were fed with fresh media for 2-3 days. For selection of stable transfectants, 0.5 mg/ml of G418 sulfate (Invitrogen) was added into the media twice a week and the cultures were maintained until colonies 2-3 mm in diameter were formed. A total of 4 colonies were selected and trypsinized individually followed by transferring into wells of a 24-well plate and then into a 35 mm petri dish. Cells were harvested at 80% confluency.
Codons encoding methionines M220, M231, and M314 were replaced by alanine codons by site-directed mutagenesis according to Braman et al. (13).
Approximately 106 COS cells were transfected with pcDNA 3.1, pcDNA 3.1-tNOX, or pcDNA 3.1-exon 4 minus tNOX. Whole cell extracts were dissolved in 100 μl 10 mM Tris-Cl and immediately loaded into SDS-PAGE gels. After transfer, the nitrocellulose membrane was incubated with either PU02 or PU04 anti-tNOX antibody at 4° C. overnight. PU02 is polyclonal peptide antibody to the tNOX c-terminal containing the putative adenine nucleotide binding motif (T589GVGASLEKRWKFCGFE605) (SEQ ID NO:8) PU04 is polyclonal peptide antibody to the tNOX putative quinone binding site which begins at amino acid 394, with reference to SEQ ID NO:13 (EEMTECRREEEMEMSDDEIEEMTETK) (SEQ ID NO:9). The membrane was incubated with alkaline phosphatase conjugated monoclonal anti-rabbit IgG at room temperature for one h.
A pair of oligodeoxyribonucleotide primers (5′-GATCTCGAGCTCMGCTTGACCACACMTGCAA-3′, SEQ ID NO:16, forward; and 5′-ATCCCGGGCCCGCGGTACCGTCAGCTTCMGCC-3′, SEQ ID NO:17, reverse) were used to amplify the E4mtNOX coding region by PCR. The PCR products were digested with HindIII and KpnI restriction endonucleases and the DNA fragment was purified by agarose gel electrophoresis. The plasmid PEGFP-NI was digested with HindIII and KpnI, and the backbone of the expression plasmid was gel-isolated. The two purified fragments were then joined to give pE4m-EGFP.
The E4m-EGFP expression plasmid was constructed using pEGFP-N1, which allow expression of the enhanced green fluorescent protein fused at the C-terminus of E4m tNOX. E. coli cells were transformed with pE4m-EGFP. The plasmid was isolated from transformed E. coli cells. COS cells were transfected with pE4m-EGFP using calcium phosphate (Stratagene, Inc.) transfection and DMSO shock according to the instructions provided by the supplier.
Cells were grown in corner glass chambers and observed on a Bio-Rad MRC-1024 confocal microscope. Fluorescence of EGFP was excited using a 488 nm krypton/argon laser, and emitted fluorescence was detected with 515-540 band pass filter. For tetramethylrhodamine concanavalin A, a 568 nm krypton/argon laser was used for excitation and fluorescence was detected with a 590 nm band pass filter. Cells were rinsed with cold PBS and treated with tetramethylrhodamine concanavalin A (10 μg/ml in PBS) for 2 min at 4° C. After a final rinse with cold PBS, the cells were observed with a confocal microscope using the 60× oil immersion lens.
E4AS, E5AS and their corresponding scrambled controls E4C and E5C were synthesized. There were 3 phosphorothioate linkages at each end of the oligonucleotides. Each final product was desalted and purified by HPLC (IDT Inc). Their sequences are shown in Table 5.
Total RNA from HeLa cells treated with antisense oligonucleotides for 6 h was isolated using RNeasy mini kits (Qiagen). Specific tNOX mRNA was measured using a real-time quantitative PCR method. Sequences of primers and their nucleotide position on tNOX are shown in Table 6. Primer FLS and FLR was used to detect full length tNOX mRNA. Primer E3/5 S and E3/5 R was used to detect E4m tNOX mRNA. Primer E4/6 S and E4/6 R was used to detect E5m tNOX mRNA. The RNA was reverse-transcribed using M-MLV reverse transcriptase (Promega Corporation, Madison, Wis.). Real-time PCR was performed using the SYBR Green Supermix (BioRad Laboratories, Hercules, Calif.). Real-time PCR was 45 cycles. tNOX mRNA level of each sample was calculated relative to Lipofectamine 2000 (Invitrogen Corporation, Carlsbad, Calif.) transfection control cells. tNOX mRNA levels were determined by the cycle threshold method and were normalized for GAPDH mRNA content.
One day before in vitro transfection, HeLa cells were plated in 10 cm tissue culture plates. Antisense oligonucleotides were delivered into cells with the Lipofectamine 2000 transfection reagent. For 96-well plates, transfection complex was added then cells were seeded according to the manufacturer's protocol. On 24 h transfection, EGCg (1 mM) and Capsibiol-T (1.25 mg/ml) were added, and cells were incubated for an additional 48 h before analysis.
Cells were transfected with oligonucleotides using the Lipofectamine 2000 transfection reagent for 24 h, and then they were incubated for 48 h at 37° C. in the presence or absence of EGCg. At the end of the incubation period, the media were decanted, and the cells washed twice with cold PBS. The cells were then fixed in 2.5% (v/v) glutaraldehyde for 0.5 h and washed with distilled water. Viable cells were stained with 200 μl of 0.5% aqueous crystal violet solution for 0.5 h and then washed exhaustively with distilled water. Acetic acid (200 μl; 33% (v/v)) was added to solubilize the cells for 0.5 h, and the absorbance read at 580 nm using plate reader (Lin et al., 1996). The percentage of surviving cells treated with EGCg was normalized to untreated controls according to the following formula: [(b−c)×100]/(a−c), where a, b, and c are the absorbance values of cells in medium without EGCg, cells in medium with EGCg, and medium alone, as background, respectively.
The Student's t test was used to measure statistical significance between two treatment groups. Data were considered significant for a P<0.05.
The amino acid sequences of the approximately 46 kDa and approximately 44 kDa proteins, which are expression products of the E4mtNOX mRNA, are given in Tables 1 and 2, respectively. See also, SEQ ID NO:14 and SEQ ID NO:15.
*Phosphorothioated
13. Braman, J., Papworth, C. and Greener, A. (1996) Methods Mol. Biol. 57: 31-44
