P53-induced apoptosis

This application claims the benefit of co-pending provisional applications Ser. No. 60/059,153 filed Sep. 17, 1997 and Ser. No. 60/079,817 filed Mar. 30, 1998. These two applications are incorporated by reference herein.

This invention was made using grant funds from the U.S. National Institutes of Health (CA57345). Therefore the government retains some rights in the present 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.

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.

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.

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. p53-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 () 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.

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).

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52

10

Homo sapiens

52

ctgcacttac 10

53

10

Homo sapiens

53

gatcccaact 10

54

10

Homo sapiens

54

gggaagcaga 10

55

10

Homo sapiens

55

gctttctcac 10

56

10

Homo sapiens

56

ttcattataa 10

57

10

Homo sapiens

57

taaggagctg 10

58

10

Homo sapiens

58

tgagggaata 10

59

10

Homo sapiens

59

gggatggcag 10

60

9

Homo sapiens

60

tcttctctg 9

61

10

Homo sapiens

61

gcaccttatt 10

62

10

Homo sapiens

62

actttaaact 10

63

10

Homo sapiens

63

ccattccact 10

64

10

Homo sapiens

64

tcaaatgcat 10

65

10

Homo sapiens

65

gaaaaatggt 10

66

10

Homo sapiens

66

actaacaccc 10

67

10

Homo sapiens

67

ttggggtttc 10

68

498

Homo sapiens

68

ttaaagcaaa gaattccccg gtcccagcca tgtccaacgt cccccacaag tcctcgctgc 60

ccgagggcat ccgccctggc acggtgctga gaattcgcgg cttggttcct cccaatgcca 120

gcaggttcca tgtaaacctg ctgtgcgggg aggagcaggg ctccgatgcc gccctgcatt 180

tcaacccccg gctggacacg tcggaggtgg tcttcaacag caaggagcaa ggctcctggg 240

gccgcgagga gcgcgggccg ggcgttcctt tccagcgcgg gcagcccttc gaggtgctca 300

tcatcgcgtc agacgacggc ttcaaggccg tggttgggga cgcccagtac caccacttcc 360

gccaccgcct gccgctggcg cgcgtgcgcc tggtggaggt gggcggggac gtgcagctgg 420

actccgtgag gatcttctga gcagaagccc aggcggcccg gggccttggc tggcaaataa 480

agcgttagcc cgcagcgc 498

69

993

Homo sapiens

69

cggcggcgcg cgatcgaggt cgggtcgccg tccagcctgc agcatgagcg cccccagcgc 60

gacccccatc ttcgcgcccg gcgagaactg cagccccgcg tggggggcgg cgcccgcggc 120

ctacgacgca gcggacacgc acctgcgcat cctgggcaag ccggtgatgg agcgctggga 180

gaccccctat atgcacgcgc tggccgccgc cgcctcctcc aaagggggcc gggtcctgga 240

ggtgggcttt ggcatggcca tcgcagcgtc aaaggtgcag gaggcgccca ttgatgagca 300

ttggatcatc gagtgcaatg acggcgtctt ccagcggctc cgggactggg ccccacggca 360

gacacacaag gtcatcccct tgaaaggcct gtgggaggat gtggcaccca ccctgcctga 420

cggtcacttt gatgggatcc tgtacgacac gtacccactc tcggaggaga cctggcacac 480

acaccagttc aacttcatca agaaccacgc ctttcgcctg ctgaagccgg ggggcgtcct 540

cacctactgc aacctcacct cctgggggga gctgatgaag tccaagtact cagacatcac 600

catcatgttt gaggagacgc aggtgcccgc gctgctggag gccggcttcc ggagggagaa 660

catccgtacg gaggtgatgg cgctggtccc accggccgac tgccgctact acgccttccc 720

acagatgatc acgcccctgg tgaccaaagg ctgagccccc accccggccc ggccacaccc 780

atgccctccg ccgtgccttc ctggccggga gtccagggtg tcgcaccagc cctgggctga 840

tcccagctgt gtgtcaccag aagctttccc ggcttctctg tgaggggtcc caccagccca 900

gggctgatcc cagctgtgtg tcaccagcag ctttcccagc ttgctctgtg agggtcactg 960

ctgcccactg cagggtgccc tgaggtgaag ccg 993

70

1670

Homo sapiens

70

ccagccgtcc attccggtgg aggcagaggc agtcctgggg ctctggggct cgggctttgt 60

caccgggacc cgcagagcca gaaccactcg gcgccgctgg tgcatgggag gggagccggg 120

ccaggagtaa gtaactcata cgggcgccgg ggacccgggt cggctggggg cttccaactc 180

agagggagtg tgatttgcct gatcctcttc ggcgttgtcc tgctctgccg catccagccc 240

tgtaccgcca tcccacttcc cgccgttccc atctgtgttc cgggtgggat cggtctggag 300

gcggccgagg acttcccagg caggagctcg gggcggaggc gggtccgcgg cagaccaggg 360

cagcgaggcg ctggccggca gggggcgctg cggtgccagc ctgaggctgg ctgctccgcg 420

aggatacagc ggcccctgcc ctgtcctgtc ctgccctgcc ctgtcctgtc ctgccctgcc 480

ctgccctgtc ctgtcctgcc ctgccctgcc ctgtgtcctc agacaatatg ttagccgtgc 540

actttgacaa gccgggagga ccggaaaacc tctacgtgaa ggaggtggcc aagccgagcc 600

cgggggaggg tgaagtcctc ctgaaggtgg cggccagcgc cctgaaccgg gcggacttaa 660

tgcagagaca aggccagtat gacccacctc caggagccag caacattttg ggacttgagg 720

catctggaca tgtggcagag ctggggcctg gctgccaggg acactggaag atcggggaca 780

cagccatggc tctgctcccc ggtgggggcc aggctcagta cgtcactgtc cccgaagggc 840

tcctcatgcc tatcccagag ggattgaccc tgacccaggc tgcagccatc ccagaggcct 900

ggctcaccgc cttccagctg ttacatcttg tgggaaatgt tcaggctgga gactatgtgc 960

taatccatgc aggactgagt ggtgtgggca cagctgctat ccaactcacc cggatggctg 1020

gagctattcc tctggtcaca gctggctccc agaagaagct tcaaatggca gaaaagcttg 1080

gagcagctgc tggattcaat tacaaaaaag aggatttctc tgaagcaacg ctgaaattca 1140

ccaaaggtgc tggagttaat cttattctag actgcatagg cggatcctac tgggagaaga 1200

acgtcaactg cctggctctt gatggtcgat gggttctcta tggtctgatg ggaggaggtg 1260

acatcaatgg gcccctgttt tcaaagctac tttttaagcg aggaagtctg atcaccagtt 1320

tgctgaggtc tagggacaat aagtacaagc aaatgctggt gaatgctttc acggagcaaa 1380

ttctgcctca cttctccacg gagggccccc aacgtctgct gccggttctg gacagaatct 1440

acccagtgac cgaaatccag gaggcccata gtacatggag gccaacaaga acataggcaa 1500

gatcgtcctg gaactgcccc agtgaaggag gatgggggca ggacaggacg cggccacccc 1560

aggcctttcc agagcaaacc tggagaagat tcacaataga caggccaaga aacccggtgc 1620

ttcctccaga gccgtttaaa gctgatatga ggaaataaag agtgaactgg 1670

71

526

Homo sapiens

71

cagctacagc acagatcagc accatgaagc ttctcacggg cctggttttc tgctccttgg 60

tcctgagtgt cagcagccga agcttctttt cgttccttgg cgaggctttt gatggggctc 120

gggacatgtg gagagcctac tctgacatga gagaagccaa ttacatcggc tcagacaaat 180

acttccatgc tcgggggaac tatgatgctg ccaaaagggg acctgggggt gcctgggccg 240

cagaagtgat cagcaatgcc agagagaata tccagagact cacaggccat ggtgcggagg 300

actcgctggc cgatcaggct gccaataaat ggggcaggag tggcagagac cccaatcact 360

tccgacctgc tggcctgcct gagaaatact gagcttcctc ttcactctgc tctcaggaga 420

cctggctatg agccctcggg gcagggattc aaagttagtg aggtctatgt ccagagaagc 480

tgagatatgg catataatag gcatctaata aatgcttaag aggtgg 526

72

842

Homo sapiens

72

gcctcaaggg ctacgtcaac cacagcctgt ccgtcttcca caccaaggac ttccaggacc 60

ctgatgggat tgagggctca gaaaacgtga ctctgtgcag atacagggac taccgcaatc 120

ccccgattac aacttctccg agcagttctg gttcctcctg gccatccgcc tggccttcgt 180

catcctcttt gagcacgtgg ccttgtgcat caagctcatc gccgcctggt tcgtgcccga 240

catccctcag tcggtgaaga acaaggttct ggaggtgaag taccagaggc tgcgtgagaa 300

gatgtggatg gaaggcagag gctgggtggg gtgggggctg gctctcggcc cccaatgcct 360

gcccatccca ccccagcatc catcttcagt gccaggagca cagacgtgta gggccagagc 420

ccgtccagag gccaccagga gctgagacag tgccaccacc agcacctccc acaaacccac 480

cctgtgcgtg ttgaggggtg ctgtgagaag gctgtgccca tgtggggccg caggaatccc 540

ctgtatgttc agggctgtga gctgccaccc tattccgcct gctccgtctt tgtggggctc 600

tcaggcttgg cacagccctg acttgaactc tgggtgagcc tgggcaccca cagaactggg 660

agtgagggct cctcaggcag ccacaaggca ggaaaactgg cgcaaatttc ctgggcctcc 720

ctctgacttc tgggcgccag atcctgccgt gccccctacc tggctgttgg gggtgtcctg 780

agcccacctc gctggcctgt tcccttcagc caacccgttt ctgcagtaaa attaagcctg 840

tc 842

73

901

Homo sapiens

73

ggcgcatacc tggcccagga gcgagcccgt gcgcagatcg gctatgagga ccccatcaac 60

cccacgtacg aggccaccaa cgccatgtac cacaggtgcc tggactacgt gttggaggag 120

ctgaagcaca acgccaaggc caaggtgatg gtggcctccc acaatgagga cacagtgcgc 180

ttcgcactgc gcaggatgga ggagctgggc ctgcatcctg ctgaccacca ggtgtacttt 240

ggacagctgc taggcatgtg tgaccagatc agcttcccgc tgggccacgg ctggctaccc 300

cgtgtacaag tacgtgccct atggccccgt gatggaggtg ctgccctact tgtccccgcc 360

gtgccctgga agaacagcag cctcatgaag ggcacccatt cgggagcggc actggctgtg 420

gctggagctc ttgaagcggc tccgaactgg caacctcttc catcgccctg cctagcaccc 480

gccagcacac cctctagcct tccagcaccc cccgccccct gctccaggcc attcaaccaa 540

caagctgcaa gccaaacccc aatccttcaa cacagattca ccttttttca ccccaccact 600

ttgcagagct tgcttggagg tgaggtcagg tgcctcccag cccttgccca gagtatgggc 660

actcaggtgt gggccgaacc tgatacctgc ctgggacagc cactggaaac ttttgggaac 720

tctcctctga aatgtgtggg cccaaggccc ccacctctgt gacccccatg tccttggacc 780

tagaggattg tccaccttct gccaaggcca gcccacacag cccgagcccc ttggggagca 840

gtggccgggc tggggaggcc tgcctggtca ataaaccact gttcctgcaa aaaaaaaaaa 900

a 901

74

1677

Homo sapiens

74

cacgcgcagc atagcagagt cgacactaga ggcatccaaa gaataccggc acgagcaggc 60

ggcgcgggcg gcggttaaaa tgtcggttcc aggaccttac caggcggcca ctgggccttc 120

ctccgcacca tccgcacctc catcctatga agagacagtg gctgttaaca gttattaccc 180

cactcctcca gctcccatgc ctgggccaac tacggggctt gtgacggggc ctgatgggaa 240

gggcatgaat cctccttcgt attataccca gccagcgccc atccccaata acaatccaat 300

taccgtgcag acggtctacg tgcagcaccc catcaccttt ttggaccgcc ctatccaaat 360

gtgttgtcct tcctgcaaca agatgatcgt gagtcagctg tcctataacg ccggtgctct 420

gacctggctg tcctgcggga gcctgtgcct gctgggggtg catagcggcc tgctgcttca 480

tccccttctg cgtggatgcc ctgcaggacg tggaccatta ctgtcccaac tgcagagctc 540

tcctgggcac ctacaagcgt ttgtaggact cagccagacg tggagggagc cgggtgccgc 600

aggaagtcct ttccacctct catccagctt cacgcctggt ggaggttctg ccctggtggt 660

ctcacctctc cagggggccc accttcatgt cttcttttgg ggggaatacg tcgcaaaact 720

aacaaatctc caaaccccag aaattgctgc ttggagtcgt gcataggact tgcaaagaca 780

ttccccttga gtgtcagttc cacggtttcc tgcctccctg agaccctgag tcctgccatc 840

taactgttga tcattgccct atccgaatat tttcctgtcg accccgggcc accagtggct 900

cttttttcct gcttccatgg gcctttctgg tggcagtctc aaactgagga agccacagtt 960

gcctcatttt tgaggctgtt ctccccagga gcttcggctg gaaccaggcc tttaggtggc 1020

cttaccattt atctctatat ccggctcttt cccgttccct ggatggacaa aaatcttgcc 1080

cttgacagga ctttaacagg gcttgggctt tgagattctg ttaacccgca ggacttcatt 1140

aggcacacaa gattcacctt aatttctcta aatttttttt tttttaaaat accaagggaa 1200

gggggctaat taacaaccca gtacaggaca tatccacaag ggtcggtaaa tggcatgcta 1260

ggaaaaatag gggccttgga tcttattcac tggccctgtc ttccccttgg tttctcttgt 1320

ggccagatct ttcagttgcc ccttttccat aacaggggat tttttttctt cataggagtt 1380

aattattatg ggaacagttt tttatggacc tcccttttgg tctggaaata ccttttcgaa 1440

cagaatttct tttttttaaa aaaaaacaga gatggggtct tactatgttg cccaggctgg 1500

tgtcgaactc ctgggctcaa gcgatccttc tgccttggcc tcccgaagtg ctgggattgc 1560

aggcataagc ttaccatgct gggcctgaac ataatttcaa gaggaggatt tataaaacca 1620

ttttctgtaa tcaaatgatt ggtgtcattt tcccatttgc acaatgtagt ctcactt 1677

75

2608

Homo sapiens

75

agctcgccgg cctttggtct ccaggacttg tcccagcagc ccctcgaact gagaattaca 60

ccatcggacc cctggctctg aggccttcag acttggactg tgtcacactg ccaggcttcc 120

agggctccaa cttgcagacg gcctgttgtg ggacagtctc tgtaatcgcg aaagcaacca 180

tggaagacct gggggaaaac accatggttt tatccaccct gagatctttg aacaacttca 240

tctctcagcg tgtggaggga ggctctggac tggatatttc tacctcggcc ccaggttctc 300

tgcagatgca gtaccagcag agcatgcagc tggaggaaag agcagagcag atccgttcga 360

agtcccacct catccaggtg gagcgggaga aaatgcagat ggagctgagt cacaagaggg 420

ctcgagtgga gctggagaga gcagccagca ccagtgccag gaactacgag cgtgaggtcg 480

accgcaacca ggagctcctg acgcgcatcc ggcagcttca ggagcgggag gccggggcgg 540

aggagaagat gcaggagcag ctggagcgca acaggcagtg tcagcagaac ttggatgctg 600

ccagcaagag gctgcgtgag aaagaggaca gtctggccca ggctggcgag accatcaacg 660

cactgaaggg gaggatctcg gaactgcagt ggagcgtgat ggaccaggag atgcgggtga 720

agcgcctgga gtcggagaag caggacgtgc aggagcagct ggacctgcaa cacaaaaaat 780

gccaggaagc caatcagaaa atccaggaac tccaggccag ccaagaagca agagcagacc 840

acgagcagca gattaaggat ctggagcaga agctgtccct gcaagagcag gatgcagcga 900

ttgtgaagaa catgaagtct gagctggtac ggctccctag gctggaacgg gagctggagc 960

agctgcggga ggagagcgca ctgcgggaga tgagagagac caacgggctg ctccaggaag 1020

agctggaagg gctgcagagg aagctggggc gccaggagaa gatgcaggag acgctggttg 1080

gcttggagct ggagaacgag aggctgctgg ccaagctgca aagctgggag agactggacc 1140

agaccatggg cctgagcatc aggactccag aagacctttc cagattcgtg gttgagctgc 1200

agcagaggga gcttgccttg aaggacaaga acagcgccgt caccagcagc gcccgggggc 1260

tggagaaggc caggcagcag ctgcaggagg agctccggca ggtcagcggc cagctgttgg 1320

aggagaggaa gaagcgcgag acccacgagg cgctggcccg gaggctccag aaacgggtcc 1380

tgctgctcac caaggagcgg gacggtatgc gggccatcct ggggtcctac gacagcgagc 1440

tgaccccggc cgagtactca ccccagctga cgcggcgcat gcgggaggct gaggatatgg 1500

tgcagaaggt gcacagccac agcgccgaga tggaggctca gctgtcgcag gccctggagg 1560

agctgggagg ccagaaacaa agagcagaca tgctggagat ggagctgaag atgctgaagt 1620

ctcagtccag ctctgccgaa cagagcttcc tgttctccag ggaggaggcg gacacgctca 1680

ggttgaaggt cgaggagctg gaaggcgagc ggagtcggct ggaggaggaa aagaggatgc 1740

tggaggcaca gctggagcgg cgagctctgc agggtgacta tgaccagagc aggaccaaag 1800

tgctgcacat gagcctgaac cccaccagtg tggccaggca gcgcctgcgc gaggaccaca 1860

gccagctgca ggcggagtgc gagcgactgc gcgggctcct gcgcgccatg gagagaggag 1920

gcaccgtccc agccgacctt gaggctgccg ccgcgagtct gccatcgtcc aaggaggtgg 1980

cagagctgaa gaagcaggtg gagagtgccg agctgaagaa ccagcggctc aaggaggttt 2040

tccagaccaa gatccaggag ttccgcaagg cctgctacac gctcaccggc taccagatcg 2100

acatcaccac ggagaaccag taccggctga cctcgctgta cgccgagcac ccaggcgact 2160

gctcatcttc aaggccacca gcccctcggg ttccaagatg cagctactgg agacagagtt 2220

ctcacacacc gtgggcgagc tcatcgaggt gcacctgcgg cgccaggaca gcatccctgc 2280

cttcctcagc tcgctcaccc tcgagctctt cagccgccag accgtggcgt agcctgcagg 2340

ctcgggggca tagccggagc cactctgctt ggcctgacct gcaggtcccc tgccccgcca 2400

gccacaggct gggtgcacgt cctgcctctc cagccccaca gggcagcagc atgactgaca 2460

gacacgctgg gacctacgtc gggcttcctg ctggggcggc cagcaccctc tccacgtgca 2520

gaccccatgc gtcccggagc ctggtgtgtg ggcgtcggcc accagcctgg gttcctcacc 2580

ttgtgaaata aaatcttctc ccctaaaa 2608

76

2326

Homo sapiens

76

aggccggaga ggaggcggtg cggcggtggc cgtgcggaga cccggtccag acgcctggcg 60

gccgccggca cacaaggcgc tttctagctc cctcccccga gcgcacagcc cgcctccttc 120

cgcggcgcct gcagtggcac ggattgctct gccctaccgt gacgcgctcc ggagacgctc 180

tgcgggtcct ggacaccggg tccggcggcg tggggacgac agacggaggc gaacgcatcc 240

ggtagccggt ccgcgagcca tcgttcgggg cgcagtcctc tccccggctg gccctccttt 300

ctccggggca ttcgccaccg cttccctggg gctgagacga ccggttcgtc gcctccttgc 360

ccgtgaccgt cgctagaact cagttgtgcg ttgcggccag tcgccactgc tgagtggaag 420

caaaatgtca gtcagtgtgc atgagaaccg caagtccagg gccagcagcg gctccattaa 480

catctatctg tttcacaagt cctcctacgc tgacagcgtc ctcactcacc tgaatctttt 540

acgccagcag cgtctcttca ctgacgtcct tctccatgcc ggaaatagga ccttcccttg 600

ccaccgggca gtgctggctg catgcagtcg ctactttgag gccatgttca gtggtggcct 660

gaaagagagc caggacagtg aggtcaactt tgacaattcc atccacccag aagtcttgga 720

gctgctgctt gactatgcgt actcctcccg ggtcattcat caattggaag gaaaatgcag 780

aaattcgctc ctgggaagct tggtgacatg ctggagtttc aaggacatcc gggatgcatg 840

tgcagagttc ctggaaaaga acctgcatcc caccaactgc ctgggcatgc tgctgctgtc 900

tgatgcacac cagtgcacca agctgtacga actatcttgg agaatgtgtc tcagcaactt 960

ccaaaccatc aggaagaatg aagatttcct ccagctgccc caggacatgg tagtgcaact 1020

cttgtccagt gaagagctgg agacagagga tgaaaggctt gtgtacgagt ctgcaattaa 1080

ctggatcagc tatgacctga agaagcgcta ttgctacctc ccagaactgt tgcagacagt 1140

aacgcgggca cttctgccag ccatctatct catggagaat gtggccatgg aggaactcat 1200

caccaagcag agaaagagta aggaaattgt ggaagaggcc atcaggtgca aactaaaaat 1260

cctgcagaat gacggtgtgg taaccagcct ctgtgcccga cctcggaaaa ctggccatgc 1320

cctcttcctt ctgggaggac agactttcat gtgtgacaag ttgtatctgg tagaccagaa 1380

ggccaaagaa atcattccca aggctgacat tcccagccca agaaaagagt ttagtgcatg 1440

tgcgattggc tgcaaagtgt acattactgg ggggcggggg tctgaaaatg gggtctcgaa 1500

agatgtctgg gtttatgata ccctgcacga ggagtggtcc aaggctgccc ccatgctggt 1560

ggccaggttt ggccatggct ctgctgaact gaagcactgc ctgtatgtgg ttggggggca 1620

cacggccgca actggctgcc tcccggcctc cccctcagtc tctctaaagc aggtagaaca 1680

ttatgacccc acaatcaaca aatggaccat ggcggcccca cgtccgagaa ggcgttacaa 1740

ctgcgcacag gtagtgagtg ccaaacttaa gttatttgct ttcggaggta ccagtgtcag 1800

tcatgacaag ctccccaaag ttcagtgtta cgatcagtgt gaaaacaggt ggactgtacc 1860

ggccacctgt ccccagccct ggcgtataca cagccaagca agctgtcctg ggggaaccca 1920

ggatttttta ttatgggggg tgatacagaa tttctctgcc tgcttctgct tataaattcg 1980

caacagtgag acttaccagt ggaccaaagg tgggagatgt gacagcaaag cgcatgagct 2040

gccatgctgt tggcctctgg aaacaaactc ttacgtggtt ggaggatact ttgggcattc 2100

agcgatgcaa gactttggac tgctacgatc caacattaga cgtgtggaac agcatcacca 2160

ctgtcccgta ctcgctgatt cctactgcat tttgtcagca cctggaaaca tctgccttct 2220

taaatgcagt acattctaaa gagaagatga gcatgagctc actccatcac tcgatgagat 2280

aatatgagat ttctacttcg gagaggccaa gtctaatgaa gagaaa 2326

77

2302

Homo sapiens

77

ctaaatcaag ctggagtcat gagggtagtg ggctaagtcg agggtccagc ctcttctgcc 60

aggaagccct tcttgctttt gagagagggc tgtgaccacc ccccatcctt ctccctacac 120

tcccagccaa cctagtgccc aagcagctaa acttggcttc cttctaatcc tggaaaaccc 180

tgtacccctc ctcctcaatc tggccctctc cacatgcaca ccctgagaac acacacagac 240

acacaacaca cacacataca cacccctgaa cacacacaca gacacacata cacccatgat 300

gtgagcaaac acacacacgt gcgccttcat agcccagcca aggcatcgca ggcagggtgt 360

gctgcctgag atggcacctc cctttcagcc attcttcaag aatgggccac acacagctag 420

aagtcctctc ccagctagaa gtcctgtccc actctcctgg cctgacaaga tgagctctcc 480

tgggaccttg ctctagggca ctctgcctct accctaggac actggaatgc cctgggagcc 540

ccctccctgc aaccagcctg agttcagccc cacggacaaa gggacacaca gcccccaatg 600

gagaccattg taagtggtgg ggctgggaga ggaggaacag aaggaaagcc atagcgctct 660

cttgcccctt ggcatgtacc ccaaggcctg atggccactg ggctcagcct gtcccccact 720

cctgcctgct tcccggtgag ctgcccccga cacgtgcagc ccgggctgcc tccagggtct 780

ggctgagtgg gatcaggtgg ccctccaact cagcacagga aataagtaga aacatttcag 840

caggccacct cccctcatct tccccgccct gtccagcgcc ctggcaaagg ctgacaactg 900

gctgtcttgg ggccgaacag ccctgcctgc tctgagggcc acagcctgtg ctgcataccc 960

accgcccagc ttctccctga gggcccacca gcctgtgctg catacccacc acccagcttc 1020

tccctgaggg cccaccagcc tgtgctgtac accccgttag tccctgatcc caaccttctc 1080

cctcctgcca gcacaccgat gcacacaccg gaagtggcga gcccaagccc tggggacagg 1140

tgtagggaga aaagcagccc caggcctcag actcgctctc ccatcactgg catagagtgg 1200

gaggatggct ggagggtgtc tataggtaca gcccgctctg gctgctgcca ggtgggcccc 1260

tgccaggggt cctcacccct gtccaccctg tgcctggctg tccctgcacc cagatacagc 1320

aacatggcct gtacccagca gagtggtggc aaccaccatg gttacagcgg atgccccgag 1380

actctgcttg gtaaacgtgg cagagcagaa tgggaggctg ggaccctgag gaagggcccc 1440

tctcctggca tctgtctctt gctacctaag cctgtgcctc tccctaaaga gctgcctccc 1500

tgctgccgag ccctggtctg gccacgagcc actactgcct cccacaggca ccactgcctc 1560

ccgctgctgc ccacaggtgg tgccgccaat gggcagtgcc tccaggccga agccttcaat 1620

cccccatctt gagccagggc ctaaatcctc ttaatagtga tggttggttt tgtcctccca 1680

ttaactgcag gtgggatttc cacctggggg aatgaggctt gcgttgttcg ggcgtctgct 1740

ggccctgaga catccagtct tccacactca actgtgggat gggagggtgg cgtggcttta 1800

ccccatggag gctgttccag ggctctgggc acacagctgt gctcacacaa aatactgggt 1860

ggcttggttt agagctaatt gtagtggaag cctgcaaggt tgaggggtga aggggagggg 1920

gcttgcaagg tccaggtaaa gatctggaaa gacagaacgt acagcttgga gggcaagggg 1980

gactctaaag tgcaaggaga tttacagttg ggaaaggagg cagtggcaga ggggttgagg 2040

gacaggggcc cttaagtcca gcgaggaaag ctcggtgtgg ggcccgctct acgctccgtt 2100

tggggtgacc tggaacgcct cttctcccag ctccctccag ccatcagcag cctcttgtca 2160

agcttctgcc tcgccccagt ctatccccaa ccccaaatca agaccacctt tcttcaacgg 2220

tcactattta ttctttgttc ctttttcttt tgtgtaagaa acattcacaa aaaccagtgc 2280

caaaaccatc aaaaaaaaaa aa 2302

78

1729

Homo sapiens

78

tggccagaga tgcctgccca cagcctggtg atgagcagcc cggccctccc ggccttcctg 60

ctctgcagca cgctgctggt catcaagatg tacgtggtgg ccatcatcac gggccaagtg 120

aggctgcgga agaaggcctt tgccaacccc gaggatgccc tgagacacgg aggaggcccc 180

cagtattgca ggagcgaccc cgacgtggaa cgctgcctca gggcccaccg gaacgacatg 240

gagaccatct accccttcct tttcctgggc ttcgtctact cctttctggg tcctaaccct 300

tttgtcgcct ggatgcactt cctggtcttc ctcgtgggcc gtgtggcaca caccgtggcc 360

tacctgggga agctgcgggc acccatccgc tccgtgacct acaccctggc ccagctcccc 420

tgcgcctcca tggctctgca gatcctctgg gaagcggccc gccacctgtg accagcagct 480

gatgcctcct tggccaccag accatgggcc aagagccgcc gtggctatac ctggggactt 540

gatgttcctt ccagattgtg gtgtgggccc tgagtcctgg tttcctggca gcctgctgcg 600

cgtgtgggtc tctgggcaca gtgggcctgt gtgtgtgccc gtgtgtgtgt atgtgtgtgt 660

gtatgtttct tagccccttg gattcctgca cgaagtggct gatgggaacc atttcaagac 720

agattgtgaa gattgataga aaatccttca gctaaagtaa cagagcatca aaaacatcac 780

tccctctccc tccctaacag tgaaaagaga gaagggagac tctatttaag attcccaaac 840

ctaatgatca tctgaatccc gggctaagaa tgcagacttt tcagactgac cccagaaatt 900

ctggcccagc caatctagag gcaagcctgg ccatctgtat tttttttttc caagacagag 960

tcttgctctc gttgcccaag ctggagtgaa gtggtacaat ctggctcact gcagcctccg 1020

cctcccgggt tcaagcgatt ctcccgcctc agcctcctga gtagctggga ttacaggcgc 1080

gtatcaccat acccagctaa tttttgtatt tttagtagag acgggttcac catgttgccc 1140

aggagggtct cgaactcctg gcctcaagtg atccacgcct cggcctccca aagtgctggg 1200

atgacaggca tgaatcactg tgctcagcca ccatctggag tttaaaagga cctcccatgt 1260

gagtccctgt gtggccaggc cagggacccc tgccagttct atgtggaagc aaggctgggg 1320

tcttgggttc ctgtatggtg gaagctgggt gagccaagga cagggctggc tcctctgccc 1380

ccgctgacgc ttcccttgcc gttggctttg gatgtctttg ctgcagtctt ctctctggct 1440

caggtgtggg tgggaggggc ccacaggaag ctcagccttc tcctcccaag gtttgagtcc 1500

ctccaaaggg cagtgggtgg aggaccggga gctttgggtg accagccact caaaggaact 1560

ttctggtccc ttcagtatct tcaaggtttg gaaactgcaa atgtcccctg atggggaatc 1620

ctgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgttt tctcctagac 1680

ccgtgacctg agatgtgtga tttttagtca ttaaatggaa gtgtctgcc 1729

79

136

Homo sapiens

79

Met Ser Asn Val Pro His Lys Ser Ser Leu Pro Glu Gly Ile Arg Pro

1 5 10 15

Gly Thr Val Leu Arg Ile Arg Gly Leu Val Pro Pro Asn Ala Ser Arg

20 25 30

Phe His Val Asn Leu Leu Cys Gly Glu Glu Gln Gly Ser Asp Ala Ala

35 40 45

Leu His Phe Asn Pro Arg Leu Asp Thr Ser Glu Val Val Phe Asn Ser

50 55 60

Lys Glu Gln Gly Ser Trp Gly Arg Glu Glu Arg Gly Pro Gly Val Pro

65 70 75 80

Phe Gln Arg Gly Gln Pro Phe Glu Val Leu Ile Ile Ala Ser Asp Asp

85 90 95

Gly Phe Lys Ala Val Val Gly Asp Ala Gln Tyr His His Phe Arg His

100 105 110

Arg Leu Pro Leu Ala Arg Val Arg Leu Val Glu Val Gly Gly Asp Val

115 120 125

Gln Leu Asp Ser Val Arg Ile Phe

130 135

80

236

Homo sapiens

80

Met Ser Ala Pro Ser Ala Thr Pro Ile Phe Ala Pro Gly Glu Asn Cys

1 5 10 15

Ser Pro Ala Trp Gly Ala Ala Pro Ala Ala Tyr Asp Ala Ala Asp Thr

20 25 30

His Leu Arg Ile Leu Gly Lys Pro Val Met Glu Arg Trp Glu Thr Pro

35 40 45

Tyr Met His Ala Leu Ala Ala Ala Ala Ser Ser Lys Gly Gly Arg Val

50 55 60

Leu Glu Val Gly Phe Gly Met Ala Ile Ala Ala Ser Lys Val Gln Glu

65 70 75 80

Ala Pro Ile Asp Glu His Trp Ile Ile Glu Cys Asn Asp Gly Val Phe

85 90 95

Gln Arg Leu Arg Asp Trp Ala Pro Arg Gln Thr His Lys Val Ile Pro

100 105 110

Leu Lys Gly Leu Trp Glu Asp Val Ala Pro Thr Leu Pro Asp Gly His

115 120 125

Phe Asp Gly Ile Leu Tyr Asp Thr Tyr Pro Leu Ser Glu Glu Thr Trp

130 135 140

His Thr His Gln Phe Asn Phe Ile Lys Asn His Ala Phe Arg Leu Leu

145 150 155 160

Lys Pro Gly Gly Val Leu Thr Tyr Cys Asn Leu Thr Ser Trp Gly Glu

165 170 175

Leu Met Lys Ser Lys Tyr Ser Asp Ile Thr Ile Met Phe Glu Glu Thr

180 185 190

Gln Val Pro Ala Leu Leu Glu Ala Gly Phe Arg Arg Glu Asn Ile Arg

195 200 205

Thr Glu Val Met Ala Leu Val Pro Pro Ala Asp Cys Arg Tyr Tyr Ala

210 215 220

Phe Pro Gln Met Ile Thr Pro Leu Val Thr Lys Gly

225 230 235

81

322

Homo sapiens

81

Met Leu Ala Val His Phe Asp Lys Pro Gly Gly Pro Glu Asn Leu Tyr

1 5 10 15

Val Lys Glu Val Ala Lys Pro Ser Pro Gly Glu Gly Glu Val Leu Leu

20 25 30

Lys Val Ala Ala Ser Ala Leu Asn Arg Ala Asp Leu Met Gln Arg Gln

35 40 45

Gly Gln Tyr Asp Pro Pro Pro Gly Ala Ser Asn Ile Leu Gly Leu Glu

50 55 60

Ala Ser Gly His Val Ala Glu Leu Gly Pro Gly Cys Gln Gly His Trp

65 70 75 80

Lys Ile Gly Asp Thr Ala Met Ala Leu Leu Pro Gly Gly Gly Gln Ala

85 90 95

Gln Tyr Val Thr Val Pro Glu Gly Leu Leu Met Pro Ile Pro Glu Gly

100 105 110

Leu Thr Leu Thr Gln Ala Ala Ala Ile Pro Glu Ala Trp Leu Thr Ala

115 120 125

Phe Gln Leu Leu His Leu Val Gly Asn Val Gln Ala Gly Asp Tyr Val

130 135 140

Leu Ile His Ala Gly Leu Ser Gly Val Gly Thr Ala Ala Ile Gln Leu

145 150 155 160

Thr Arg Met Ala Gly Ala Ile Pro Leu Val Thr Ala Gly Ser Gln Lys

165 170 175

Lys Leu Gln Met Ala Glu Lys Leu Gly Ala Ala Ala Gly Phe Asn Tyr

180 185 190

Lys Lys Glu Asp Phe Ser Glu Ala Thr Leu Lys Phe Thr Lys Gly Ala

195 200 205

Gly Val Asn Leu Ile Leu Asp Cys Ile Gly Gly Ser Tyr Trp Glu Lys

210 215 220

Asn Val Asn Cys Leu Ala Leu Asp Gly Arg Trp Val Leu Tyr Gly Leu

225 230 235 240

Met Gly Gly Gly Asp Ile Asn Gly Pro Leu Phe Ser Lys Leu Leu Phe

245 250 255

Lys Arg Gly Ser Leu Ile Thr Ser Leu Leu Arg Ser Arg Asp Asn Lys

260 265 270

Tyr Lys Gln Met Leu Val Asn Ala Phe Thr Glu Gln Ile Leu Pro His

275 280 285

Phe Ser Thr Glu Gly Pro Gln Arg Leu Leu Pro Val Leu Asp Arg Ile

290 295 300

Tyr Pro Val Thr Glu Ile Gln Glu Ala His Ser Thr Trp Arg Pro Thr

305 310 315 320

Arg Thr

82

122

Homo sapiens

82

Met Lys Leu Leu Thr Gly Leu Val Phe Cys Ser Leu Val Leu Ser Val

1 5 10 15

Ser Ser Arg Ser Phe Phe Ser Phe Leu Gly Glu Ala Phe Asp Gly Ala

20 25 30

Arg Asp Met Trp Arg Ala Tyr Ser Asp Met Arg Glu Ala Asn Tyr Ile

35 40 45

Gly Ser Asp Lys Tyr Phe His Ala Arg Gly Asn Tyr Asp Ala Ala Lys

50 55 60

Arg Gly Pro Gly Gly Ala Trp Ala Ala Glu Val Ile Ser Asn Ala Arg

65 70 75 80

Glu Asn Ile Gln Arg Leu Thr Gly His Gly Ala Glu Asp Ser Leu Ala

85 90 95

Asp Gln Ala Ala Asn Lys Trp Gly Arg Ser Gly Arg Asp Pro Asn His

100 105 110

Phe Arg Pro Ala Gly Leu Pro Glu Lys Tyr

115 120

83

253

Homo sapiens

83

Gly Ala Tyr Leu Ala Gln Glu Arg Ala Arg Ala Gln Ile Gly Tyr Glu

1 5 10 15

Asp Pro Ile Asn Pro Thr Tyr Glu Ala Thr Asn Ala Met Tyr His Arg

20 25 30

Cys Leu Asp Tyr Val Leu Glu Glu Leu Lys His Asn Ala Lys Ala Lys

35 40 45

Val Met Val Ala Ser His Asn Glu Asp Thr Val Arg Phe Ala Leu Arg

50 55 60

Arg Met Glu Glu Leu Gly Leu His Pro Ala Asp His Gln Val Tyr Phe

65 70 75 80

Gly Gln Leu Leu Gly Met Cys Asp Gln Ile Ser Phe Pro Leu Gly His

85 90 95

Gly Trp Leu Pro Arg Val Gln Val Arg Ala Leu Trp Pro Arg Asp Gly

100 105 110

Gly Ala Ala Leu Leu Val Pro Ala Val Pro Trp Lys Asn Ser Ser Leu

115 120 125

Met Lys Gly Thr His Ser Gly Ala Ala Leu Ala Val Ala Gly Ala Leu

130 135 140

Glu Ala Ala Pro Asn Trp Gln Pro Leu Pro Ser Pro Cys Leu Ala Pro

145 150 155 160

Ala Ser Thr Pro Ser Ser Leu Pro Ala Pro Pro Ala Pro Cys Ser Arg

165 170 175

Pro Phe Asn Gln Gln Ala Ala Ser Gln Thr Pro Ile Leu Gln His Arg

180 185 190

Phe Thr Phe Phe His Pro Thr Thr Leu Gln Ser Leu Leu Gly Gly Glu

195 200 205

Val Arg Cys Leu Pro Ala Leu Ala Gln Ser Met Gly Thr Gln Val Trp

210 215 220

Ala Glu Pro Asp Thr Cys Leu Gly Gln Pro Leu Glu Thr Phe Gly Asn

225 230 235 240

Ser Pro Leu Lys Cys Val Gly Pro Arg Pro Pro Pro Leu

245 250

84

228

Homo sapiens

84

Met Ser Val Pro Gly Pro Tyr Gln Ala Ala Thr Gly Pro Ser Ser Ala

1 5 10 15

Pro Ser Ala Pro Pro Ser Tyr Glu Glu Thr Val Ala Val Asn Ser Tyr

20 25 30

Tyr Pro Thr Pro Pro Ala Pro Met Pro Gly Pro Thr Thr Gly Leu Val

35 40 45

Thr Gly Pro Asp Gly Lys Gly Met Asn Pro Pro Ser Tyr Tyr Thr Gln

50 55 60

Pro Ala Pro Ile Pro Asn Asn Asn Pro Ile Thr Val Gln Thr Val Tyr

65 70 75 80

Val Gln His Pro Ile Thr Phe Leu Asp Arg Pro Ile Gln Met Cys Cys

85 90 95

Pro Ser Cys Asn Lys Met Ile Val Ser Gln Leu Ser Tyr Asn Ala Gly

100 105 110

Ala Leu Thr Trp Leu Ser Cys Gly Ser Leu Cys Leu Leu Gly Val His

115 120 125

Ser Gly Leu Leu Leu His Pro Leu Leu Arg Gly Cys Pro Ala Gly Arg

130 135 140

Gly Pro Leu Leu Ser Gln Leu Gln Ser Ser Pro Gly His Leu Gln Ala

145 150 155 160

Phe Val Gly Leu Ser Gln Thr Trp Arg Glu Pro Gly Ala Ala Gly Ser

165 170 175

Pro Phe His Leu Ser Ser Ser Phe Thr Pro Gly Gly Gly Ser Ala Leu

180 185 190

Val Val Ser Pro Leu Gln Gly Ala His Leu His Val Phe Phe Trp Gly

195 200 205

Glu Tyr Val Ala Lys Leu Thr Asn Leu Gln Thr Pro Glu Ile Ala Ala

210 215 220

Trp Ser Arg Ala

225

85

803

Homo sapiens

85

Met Glu Asp Leu Gly Glu Asn Thr Met Val Leu Ser Thr Leu Arg Ser

1 5 10 15

Leu Asn Asn Phe Ile Ser Gln Arg Val Glu Gly Gly Ser Gly Leu Asp

20 25 30

Ile Ser Thr Ser Ala Pro Gly Ser Leu Gln Met Gln Tyr Gln Gln Ser

35 40 45

Met Gln Leu Glu Glu Arg Ala Glu Gln Ile Arg Ser Lys Ser His Leu

50 55 60

Ile Gln Val Glu Arg Glu Lys Met Gln Met Glu Leu Ser His Lys Arg

65 70 75 80

Ala Arg Val Glu Leu Glu Arg Ala Ala Ser Thr Ser Ala Arg Asn Tyr

85 90 95

Glu Arg Glu Val Asp Arg Asn Gln Glu Leu Leu Thr Arg Ile Arg Gln

100 105 110

Leu Gln Glu Arg Glu Ala Gly Ala Glu Glu Lys Met Gln Glu Gln Leu

115 120 125

Glu Arg Asn Arg Gln Cys Gln Gln Asn Leu Asp Ala Ala Ser Lys Arg

130 135 140

Leu Arg Glu Lys Glu Asp Ser Leu Ala Gln Ala Gly Glu Thr Ile Asn

145 150 155 160

Ala Leu Lys Gly Arg Ile Ser Glu Leu Gln Trp Ser Val Met Asp Gln

165 170 175

Glu Met Arg Val Lys Arg Leu Glu Ser Glu Lys Gln Asp Val Gln Glu

180 185 190

Gln Leu Asp Leu Gln His Lys Lys Cys Gln Glu Ala Asn Gln Lys Ile

195 200 205

Gln Glu Leu Gln Ala Ser Gln Glu Ala Arg Ala Asp His Glu Gln Gln

210 215 220

Ile Lys Asp Leu Glu Gln Lys Leu Ser Leu Gln Glu Gln Asp Ala Ala

225 230 235 240

Ile Val Lys Asn Met Lys Ser Glu Leu Val Arg Leu Pro Arg Leu Glu

245 250 255

Arg Glu Leu Glu Gln Leu Arg Glu Glu Ser Ala Leu Arg Glu Met Arg

260 265 270

Glu Thr Asn Gly Leu Leu Gln Glu Glu Leu Glu Gly Leu Gln Arg Lys

275 280 285

Leu Gly Arg Gln Glu Lys Met Gln Glu Thr Leu Val Gly Leu Glu Leu

290 295 300

Glu Asn Glu Arg Leu Leu Ala Lys Leu Gln Ser Trp Glu Arg Leu Asp

305 310 315 320

Gln Thr Met Gly Leu Ser Ile Arg Thr Pro Glu Asp Leu Ser Arg Phe

325 330 335

Val Val Glu Leu Gln Gln Arg Glu Leu Ala Leu Lys Asp Lys Asn Ser

340 345 350

Ala Val Thr Ser Ser Ala Arg Gly Leu Glu Lys Ala Arg Gln Gln Leu

355 360 365

Gln Glu Glu Leu Arg Gln Val Ser Gly Gln Leu Leu Glu Glu Arg Lys

370 375 380

Lys Arg Glu Thr His Glu Ala Leu Ala Arg Arg Leu Gln Lys Arg Val

385 390 395 400

Leu Leu Leu Thr Lys Glu Arg Asp Gly Met Arg Ala Ile Leu Gly Ser

405 410 415

Tyr Asp Ser Glu Leu Thr Pro Ala Glu Tyr Ser Pro Gln Leu Thr Arg

420 425 430

Arg Met Arg Glu Ala Glu Asp Met Val Gln Lys Val His Ser His Ser

435 440 445

Ala Glu Met Glu Ala Gln Leu Ser Gln Ala Leu Glu Glu Leu Gly Gly

450 455 460

Gln Lys Gln Arg Ala Asp Met Leu Glu Met Glu Leu Lys Met Leu Lys

465 470 475 480

Ser Gln Ser Ser Ser Ala Glu Gln Ser Phe Leu Phe Ser Arg Glu Glu

485 490 495

Ala Asp Thr Leu Arg Leu Lys Val Glu Glu Leu Glu Gly Glu Arg Ser

500 505 510

Arg Leu Glu Glu Glu Lys Arg Met Leu Glu Ala Gln Leu Glu Arg Arg

515 520 525

Ala Leu Gln Gly Asp Tyr Asp Gln Ser Arg Thr Lys Val Leu His Met

530 535 540

Ser Leu Asn Pro Thr Ser Val Ala Arg Gln Arg Leu Arg Glu Asp His

545 550 555 560

Ser Gln Leu Gln Ala Glu Cys Glu Arg Leu Arg Gly Leu Leu Arg Ala

565 570 575

Met Glu Arg Gly Gly Thr Val Pro Ala Asp Leu Glu Ala Ala Ala Ala

580 585 590

Ser Leu Pro Ser Ser Lys Glu Val Ala Glu Leu Lys Lys Gln Val Glu

595 600 605

Ser Ala Glu Leu Lys Asn Gln Arg Leu Lys Glu Val Phe Gln Thr Lys

610 615 620

Ile Gln Glu Phe Arg Lys Ala Cys Tyr Thr Leu Thr Gly Tyr Gln Ile

625 630 635 640

Asp Ile Thr Thr Glu Asn Gln Tyr Arg Leu Thr Ser Leu Tyr Ala Glu

645 650 655

His Pro Gly Asp Cys Ser Ser Ser Arg Pro Pro Ala Pro Arg Val Pro

660 665 670

Arg Cys Ser Tyr Trp Arg Gln Ser Ser His Thr Pro Trp Ala Ser Ser

675 680 685

Ser Arg Cys Thr Cys Gly Ala Arg Thr Ala Ser Leu Pro Ser Ser Ala

690 695 700

Arg Ser Pro Ser Ser Ser Ser Ala Ala Arg Pro Trp Arg Ser Leu Gln

705 710 715 720

Ala Arg Gly His Ser Arg Ser His Ser Ala Trp Pro Asp Leu Gln Val

725 730 735

Pro Cys Pro Ala Ser His Arg Leu Gly Ala Arg Pro Ala Ser Pro Ala

740 745 750

Pro Gln Gly Ser Ser Met Thr Asp Arg His Ala Gly Thr Tyr Val Gly

755 760 765

Leu Pro Ala Gly Ala Ala Ser Thr Leu Ser Thr Cys Arg Pro His Ala

770 775 780

Ser Arg Ser Leu Val Cys Gly Arg Arg Pro Pro Ala Trp Val Pro His

785 790 795 800

Leu Val Lys

86

516

Homo sapiens

86

Met Ser Val Ser Val His Glu Asn Arg Lys Ser Arg Ala Ser Ser Gly

1 5 10 15

Ser Ile Asn Ile Tyr Leu Phe His Lys Ser Ser Tyr Ala Asp Ser Val

20 25 30

Leu Thr His Leu Asn Leu Leu Arg Gln Gln Arg Leu Phe Thr Asp Val

35 40 45

Leu Leu His Ala Gly Asn Arg Thr Phe Pro Cys His Arg Ala Val Leu

50 55 60

Ala Ala Cys Ser Arg Tyr Phe Glu Ala Met Phe Ser Gly Gly Leu Lys

65 70 75 80

Glu Ser Gln Asp Ser Glu Val Asn Phe Asp Asn Ser Ile His Pro Glu

85 90 95

Val Leu Glu Leu Leu Leu Asp Tyr Ala Tyr Ser Ser Arg Val Ile His

100 105 110

Gln Leu Glu Gly Lys Cys Arg Asn Ser Leu Leu Gly Ser Leu Val Thr

115 120 125

Cys Trp Ser Phe Lys Asp Ile Arg Asp Ala Cys Ala Glu Phe Leu Glu

130 135 140

Lys Asn Leu His Pro Thr Asn Cys Leu Gly Met Leu Leu Leu Ser Asp

145 150 155 160

Ala His Gln Cys Thr Lys Leu Tyr Glu Leu Ser Trp Arg Met Cys Leu

165 170 175

Ser Asn Phe Gln Thr Ile Arg Lys Asn Glu Asp Phe Leu Gln Leu Pro

180 185 190

Gln Asp Met Val Val Gln Leu Leu Ser Ser Glu Glu Leu Glu Thr Glu

195 200 205

Asp Glu Arg Leu Val Tyr Glu Ser Ala Ile Asn Trp Ile Ser Tyr Asp

210 215 220

Leu Lys Lys Arg Tyr Cys Tyr Leu Pro Glu Leu Leu Gln Thr Val Thr

225 230 235 240

Arg Ala Leu Leu Pro Ala Ile Tyr Leu Met Glu Asn Val Ala Met Glu

245 250 255

Glu Leu Ile Thr Lys Gln Arg Lys Ser Lys Glu Ile Val Glu Glu Ala

260 265 270

Ile Arg Cys Lys Leu Lys Ile Leu Gln Asn Asp Gly Val Val Thr Ser

275 280 285

Leu Cys Ala Arg Pro Arg Lys Thr Gly His Ala Leu Phe Leu Leu Gly

290 295 300

Gly Gln Thr Phe Met Cys Asp Lys Leu Tyr Leu Val Asp Gln Lys Ala

305 310 315 320

Lys Glu Ile Ile Pro Lys Ala Asp Ile Pro Ser Pro Arg Lys Glu Phe

325 330 335

Ser Ala Cys Ala Ile Gly Cys Lys Val Tyr Ile Thr Gly Gly Arg Gly

340 345 350

Ser Glu Asn Gly Val Ser Lys Asp Val Trp Val Tyr Asp Thr Leu His

355 360 365

Glu Glu Trp Ser Lys Ala Ala Pro Met Leu Val Ala Arg Phe Gly His

370 375 380

Gly Ser Ala Glu Leu Lys His Cys Leu Tyr Val Val Gly Gly His Thr

385 390 395 400

Ala Ala Thr Gly Cys Leu Pro Ala Ser Pro Ser Val Ser Leu Lys Gln

405 410 415

Val Glu His Tyr Asp Pro Thr Ile Asn Lys Trp Thr Met Ala Ala Pro

420 425 430

Arg Pro Arg Arg Arg Tyr Asn Cys Ala Gln Val Val Ser Ala Lys Leu

435 440 445

Lys Leu Phe Ala Phe Gly Gly Thr Ser Val Ser His Asp Lys Leu Pro

450 455 460

Lys Val Gln Cys Tyr Asp Gln Cys Glu Asn Arg Trp Thr Val Pro Ala

465 470 475 480

Thr Cys Pro Gln Pro Trp Arg Ile His Ser Gln Ala Ser Cys Pro Gly

485 490 495

Gly Thr Gln Asp Phe Leu Leu Trp Gly Val Ile Gln Asn Phe Ser Ala

500 505 510

Cys Phe Cys Leu

515

87

153

Homo sapiens

87

Met Pro Ala His Ser Leu Val Met Ser Ser Pro Ala Leu Pro Ala Phe

1 5 10 15

Leu Leu Cys Ser Thr Leu Leu Val Ile Lys Met Tyr Val Val Ala Ile

20 25 30

Ile Thr Gly Gln Val Arg Leu Arg Lys Lys Ala Phe Ala Asn Pro Glu

35 40 45

Asp Ala Leu Arg His Gly Gly Gly Pro Gln Tyr Cys Arg Ser Asp Pro

50 55 60

Asp Val Glu Arg Cys Leu Arg Ala His Arg Asn Asp Met Glu Thr Ile

65 70 75 80

Tyr Pro Phe Leu Phe Leu Gly Phe Val Tyr Ser Phe Leu Gly Pro Asn

85 90 95

Pro Phe Val Ala Trp Met His Phe Leu Val Phe Leu Val Gly Arg Val

100 105 110

Ala His Thr Val Ala Tyr Leu Gly Lys Leu Arg Ala Pro Ile Arg Ser

115 120 125

Val Thr Tyr Thr Leu Ala Gln Leu Pro Cys Ala Ser Met Ala Leu Gln

130 135 140

Ile Leu Trp Glu Ala Ala Arg His Leu

145 150

88

155

Rat

88

Met Ala Asp Leu Lys Gln Leu Met Asp Asn Glu Val Leu Met Ala Phe

1 5 10 15

Thr Ser Tyr Ala Thr Ile Ile Leu Ala Lys Met Met Phe Leu Ser Ser

20 25 30

Ala Thr Ala Phe Gln Arg Leu Thr Asn Lys Val Phe Ala Asn Pro Glu

35 40 45

Asp Cys Ala Gly Phe Gly Lys Gly Glu Asn Ala Lys Lys Phe Leu Arg

50 55 60

Thr Asp Glu Lys Val Glu Arg Val Arg Arg Ala His Leu Asn Asp Leu

65 70 75 80

Glu Asn Ile Val Pro Phe Leu Gly Ile Gly Leu Leu Tyr Ser Leu Ser

85 90 95

Gly Pro Asp Leu Ser Thr Ala Leu Ile His Phe Arg Ile Phe Val Gly

100 105 110

Ala Arg Ile Tyr His Thr Ile Ala Tyr Leu Thr Pro Leu Pro Gln Pro

115 120 125

Asn Arg Gly Leu Ala Phe Phe Val Gly Tyr Gly Val Thr Leu Ser Met

130 135 140

Ala Tyr Arg Leu Leu Arg Ser Arg Leu Tyr Leu

145 150 155

89

324

Vigna

89

Met Val Lys Ala Ile Arg Val His Glu Gln Gly Gly Pro Gln Val Leu

1 5 10 15

Lys Trp Glu Asp Val Glu Ile Gly Glu Pro Lys Glu Gly Glu Val Arg

20 25 30

Val Arg Asn Lys Ala Val Gly Val Asn Phe Ile Asp Val Tyr Phe Arg

35 40 45

Lys Gly Val Tyr Lys Pro Pro Ser Phe Pro Phe Thr Pro Gly Met Glu

50 55 60

Ala Val Gly Val Val Thr Ala Val Gly Ala Gly Leu Thr Gly Arg Gln

65 70 75 80

Val Gly Asp Leu Val Ala Tyr Ala Gly Gln Pro Met Gly Ser Tyr Ala

85 90 95

Glu Glu Gln Ile Leu Pro Ala Asn Lys Val Val Pro Val Pro Ser Ser

100 105 110

Ile Asp Pro Pro Ile Ala Ala Ser Ile Met Leu Lys Gly Met Thr Thr

115 120 125

His Phe Leu Val Arg Arg Cys Phe Lys Val Glu Pro Gly His Thr Ile

130 135 140

Leu Val His Ala Ala Ala Gly Gly Val Gly Ser Leu Leu Cys Gln Trp

145 150 155 160

Ala Asn Ala Leu Gly Ala Thr Val Ile Gly Thr Val Ser Asn Lys Glu

165 170 175

Lys Ala Ala Gln Ala Lys Glu Asp Gly Cys His His Val Ile Ile Tyr

180 185 190

Lys Glu Glu Asp Phe Val Ala Arg Val Asn Glu Ile Thr Ser Gly Asn

195 200 205

Gly Val Glu Val Val Tyr Asp Ser Val Gly Lys Asp Thr Phe Glu Gly

210 215 220

Ser Leu Ala Cys Leu Lys Leu Arg Gly Tyr Met Val Ser Phe Gly Gln

225 230 235 240

Ser Ser Gly Ser Pro Asp Pro Val Pro Leu Ser Ser Leu Ala Ala Lys

245 250 255

Ser Leu Phe Leu Thr Arg Pro Ser Leu Met Gln Tyr Val Val Thr Arg

260 265 270

Asp Glu Leu Leu Glu Ala Ala Gly Glu Leu Phe Ala Asn Val Ala Ser

275 280 285

Gly Val Leu Lys Val Arg Val Asn His Thr Tyr Pro Leu Ser Glu Ala

290 295 300

Ala Lys Ala His Glu Asp Leu Glu Asn Arg Lys Thr Ser Gly Ser Ile

305 310 315 320

Val Leu Ile Pro

90

359

Homo sapiens

90

Met Ile Trp Gly His Phe Ser Leu Leu Cys Val Val Asp Ser Leu Gly

1 5 10 15

Gly Glu Glu Met Ala Asp Ser Val Lys Thr Phe Leu Gln Asp Leu Ala

20 25 30

Arg Gly Ile Lys Asp Ser Ile Trp Gly Ile Cys Thr Ile Ser Lys Leu

35 40 45

Asp Ala Arg Ile Gln Gln Lys Arg Glu Glu Gln Arg Arg Arg Arg Ala

50 55 60

Ser Ser Val Leu Ala Gln Arg Arg Pro Gln Ser Ile Glu Arg Lys Gln

65 70 75 80

Glu Ser Glu Pro Arg Ile Val Ser Arg Ile Phe Gln Cys Cys Ala Trp

85 90 95

Asn Gly Gly Val Phe Trp Phe Ser Leu Leu Leu Phe Tyr Arg Val Phe

100 105 110

Ile Pro Val Leu Gln Ser Val Thr Ala Arg Ile Ile Gly Asp Pro Ser

115 120 125

Leu His Gly Asp Val Trp Ser Trp Leu Gly Phe Phe Leu Thr Ser Ile

130 135 140

Phe Ser Ala Val Trp Val Leu Pro Leu Phe Val Leu Ser Lys Val Val

145 150 155 160

Asn Ala Ile Trp Phe Gln Asp Ile Ala Asp Leu Ala Phe Glu Val Ser

165 170 175

Gly Arg Lys Pro His Pro Phe Pro Ser Val Ser Lys Ile Ile Ala Asp

180 185 190

Met Leu Phe Asn Leu Leu Leu Gln Ala Leu Phe Leu Ile Gln Gly Met

195 200 205

Phe Val Ser Leu Phe Pro Ile His Leu Val Gly Gln Leu Val Ser Leu

210 215 220

Leu His Met Ser Leu Leu Tyr Ser Leu Tyr Cys Phe Glu Tyr Arg Trp

225 230 235 240

Phe Asn Lys Gly Ile Glu Met His Gln Arg Leu Ser Asn Ile Glu Arg

245 250 255

Asn Trp Pro Tyr Tyr Phe Gly Phe Gly Leu Pro Leu Ala Phe Leu Thr

260 265 270

Ala Met Gln Ser Ser Tyr Ile Ile Ser Gly Cys Leu Phe Ser Ile Leu

275 280 285

Phe Pro Leu Phe Ile Ile Ser Ala Asn Glu Ala Lys Thr Pro Gly Lys

290 295 300

Ala Tyr Leu Phe Gln Leu Arg Leu Phe Ser Leu Val Val Phe Leu Ser

305 310 315 320

Asn Arg Leu Phe His Lys Thr Val Tyr Leu Gln Ser Ala Leu Ser Ser

325 330 335

Ser Thr Ser Ala Glu Lys Phe Pro Ser Pro His Pro Ser Pro Ala Lys

340 345 350

Leu Lys Ala Thr Ala Gly His

355

91

315

C. elegans

91

Met Val Lys Phe Gln Ile Ile Ala Arg Asp Phe Tyr His Gly Phe Ile

1 5 10 15

Asp Ser Phe Lys Gly Ile Thr Phe Val Arg Arg Ile Arg Glu Glu Glu

20 25 30

Ala Lys Glu Val Lys Val Glu Pro Pro Lys Pro Val Glu Arg Thr Val

35 40 45

Leu Met Met Arg Arg Glu Lys Gln Gly Ile Phe Lys Arg Pro Pro Glu

50 55 60

Pro Pro Lys Lys Lys Asp Ser Phe Leu Lys Lys Leu Trp Gln Ile Tyr

65 70 75 80

Ala Met Asn Ile Gly Phe Leu Val Leu Trp Gln Val Cys Ile Leu Ile

85 90 95

Leu Gly Leu Phe Phe Ser Phe Phe Asp Arg Thr Asp Leu Gly His Asn

100 105 110

Ile Gly Tyr Ile Leu Ile Ile Pro Ile Phe Phe Ala Ser Arg Ile Ile

115 120 125

Gln Ala Leu Trp Phe Ser Asp Ile Ser Gly Ala Cys Met Arg Ala Leu

130 135 140

Lys Leu Pro Pro Pro Pro Val Val Pro Phe Ser Ser Met Leu Ala Gly

145 150 155 160

Thr Leu Ile Ser Ala Leu His Gln Ile Phe Phe Leu Ile Gln Gly Met

165 170 175

Leu Ser Gln Tyr Leu Pro Ile Pro Leu Ile Thr Pro Val Ile Val Tyr

180 185 190

Leu His Met Ala Leu Leu Asn Ser Met Tyr Cys Phe Asp Tyr Phe Phe

195 200 205

Asp Gly Tyr Asn Leu Ser Phe Leu Arg Arg Lys Asp Ile Phe Glu Ser

210 215 220

His Trp Pro Tyr Phe Leu Gly Phe Gly Thr Pro Leu Ala Leu Ala Cys

225 230 235 240

Ser Ile Ser Ser Asn Met Phe Val Asn Ser Val Ile Phe Ala Leu Leu

245 250 255

Phe Pro Phe Phe Ile Ile Thr Ser Tyr Pro Ala Asn Trp Asn Arg Lys

260 265 270

Tyr Glu Glu Glu Ile Pro Lys Ile Ala Phe Cys Arg Ile Ser Tyr Met

275 280 285

Phe Thr Glu Leu Val Gly Lys Phe Val Lys Ser Ile Thr Pro Thr Asn

290 295 300

Asn Pro Thr Ala Ala Arg Asn Asn Ala Gln Asn

305 310 315

92

83

Homo sapiens

92

Ile Gly Tyr Glu Asp Pro Ile Asn Pro Thr Tyr Glu Ala Thr Asn Ala

1 5 10 15

Met Tyr His Arg Cys Leu Asp Tyr Val Leu Glu Glu Leu Lys His Asn

20 25 30

Ala Lys Ala Lys Val Met Val Ala Ser His Asn Glu Asp Thr Val Arg

35 40 45

Phe Ala Leu Arg Arg Met Glu Glu Leu Gly Leu His Pro Ala Asp His

50 55 60

Gln Val Tyr Phe Gly Gln Leu Leu Gly Met Cys Asp Gln Ile Ser Phe

65 70 75 80

Pro Leu Gly

93

89

Drosophila

93

Ile Gly Tyr Glu Asp Pro Val Asn Pro Thr Phe Glu Ala Thr Thr Asp

1 5 10 15

Met Tyr His Arg Leu Ser Glu Cys Leu Arg Arg Ile Lys Leu Met Lys

20 25 30

Asp Cys Asp Asp Asp Ala Arg Lys Ile Gly Ile Met Val Ala Ser His

35 40 45

Asn Glu Asp Thr Val Arg Phe Ala Ile Gln Gln Met Lys Glu Ile Gly

50 55 60

Ile Ser Pro Glu Asp Lys Val Ile Cys Phe Gly Gln Leu Leu Gly Met

65 70 75 80

Cys Asp Tyr Ile Thr Phe Pro Leu Gly

85