Detection of altered expression of genes regulating cell proliferation

The present invention relates to polynucleotides comprising at least a fragment of genes that regulate cell proliferation and methods comprising the use of such polynucleotides.

Cellular proliferation in normal tissues is strictly regulated by multiple factors and mechanisms. Cells grow and differentiate, carry out their structural or metabolic roles, participate in organismal development, and respond to their environment by altering their gene expression. Cellular functions are controlled by the timing and the amount of expression attributable to thousands of individual genes.

Aberrant cellular proliferation can be the result of under- or overexpression of genes which regulate cell growth, differentiation, and metastasis. Overexpression of oncogenes, which include growth factors, growth factor receptors, transcriptional activators, proteases, cell matrix components and other proteins necessary for cell growth regulation, can result in aberrant cell proliferation. Conversely, underexpression of regulatory proteins, inhibitors or suppressor of oncogenes, cell cycle regulators, and apoptosis inducers may contribute to abnormal cell proliferation. In many cases of aberrant cellular proliferation changes in expression of multiple factors contribute to the development of cell growth disorders such as cancers.

The present invention identifies particular polynucleotides that can be used to detect the altered expression of genes that regulate cell proliferation and consequently, diagnose diseases involving aberrant cell proliferation, such as cancer. The polynucleotides may also be used to treat such diseases.

In one aspect, the present invention provides a composition comprising one or more polynucleotides, wherein each of said polynucleotides comprises at least a fragment of a gene implicated in the regulation of cell proliferation. The plurality of polynucleotides can be selected from I) first polynucleotides, wherein each of said first polynucleotides comprises at least a fragment of a gene differentially expressed in precancerous biological samples; or II) second polynucleotides, wherein each of said second polynucleotides comprises at least a fragment of a gene differentially expressed in cancerous biological samples. The composition is useful for the detection of the altered expression of genes that regulate cell proliferation.

Generally, first polynucleotide sequences are selected by a first method comprising a) preparing more than one first, second and third transcript profiles from noncancerous, precancerous and cancerous biological samples, respectively; b) comparing said first, second and third transcript profiles to detect a plurality of genes that are differentially expressed in either noncancerous, precancerous and cancerous biological samples, respectively; and c) identifying one of said detected genes that are differentially expressed in precancerous biological samples. Second polynucleotide sequences are selected by a second method comprising a) preparing more than one first, second and third transcript profiles from noncancerous, precancerous and cancerous biological samples, respectively; b) comparing said first, second and third transcript profiles to detect a plurality of genes that are differentially expressed in either noncancerous, precancerous and cancerous biological samples, respectively; and c) identifying one of said detected genes that are differentially expressed in cancerous biological samples. The polynucleotides may be complementary DNAs (cDNAs), genomic DNA fragments, oligonucleotides and the like.

In one preferred embodiment, the composition comprises a polynucleotide comprising a sequence selected from the group consisting of: a) a polynucleotide sequence selected from the group consisting of SEQ ID NOs:1-16; b) a polynucleotide sequence, comprising a polynucleotide sequence which encodes the polypeptide sequence selected from the group consisting of: of SEQ ID NOs:17-23; c) a polynucleotide sequence having at least 70% identity to the polynucleotide sequence of (a) or (b); (d) a polynucleotide sequence comprising at least 18 sequential nucleotides of the polynucleotide sequence of (a), (b) or (c); e) a polynucleotide sequence which is complementary to the polynucleotide sequence of (a), (b),(c) or (d); and f) a polynucleotide which hybridizes under stringent conditions to the polynucleotide of (a),(b), (c), (d) or (e).

The composition is particularly useful as hybridizable array elements in a microarray for monitoring the expression of a plurality of sample polynucleotides implicated in the regulation of cell proliferation. The microarray can be used, for example, in the prognosis, diagnosis and treatment of a cancer or other disease relating to the altered expression of genes involved in cell proliferation.

In yet another aspect, the present invention provides a method for identifying polynucleotides for diagnosing, prognosing, or monitoring the progression of cancer or other diseases relating to the altered expression of genes involved in the regulation of cell proliferation. The method comprises selecting first polynucleotide sequences that comprise at least a fragment of genes differentially expressed in precancerous biological samples and second polynucleotide sequences that comprise at least a fragment of genes differentially expressed in cancerous biological samples.

Further, the present invention provides a method for diagnosing, prognosing, or monitoring the treatment of a disease. The method comprises using the composition described above comprising polynucleotide sequences to detect hybridization complexes formed between the polynucleotide sequences and sample polynucleotides. The sample polynucleotides may be from diseased, asymptomatic or nondiseased samples. The polynucleotides are contacted with a sample containing sample polynucleotides under conditions effective to form hybridization complexes between said polynucleotide sequences and sample genes that regulate cell proliferation. Hybridization complexes are then detected. The levels of hybridization complexes in diseased or nonsymptomatic samples are then compared with those in nondiseased samples.

In another aspect, the invention is a polypeptide comprising a sequence selected from the group consisting of: a) a polypeptide sequence selected from the group consisting of SEQ ID NOs:17-23; b) a polypeptide sequence having at least 70% identity to the polypeptide sequence of (a); c) a polypeptide sequence comprising at least 10 sequential amino acids of the polypeptide sequence of (a) or (b).

In another aspect, the invention entails a pharmaceutical composition comprising a polynucleotide or polypeptide in conjunction with a suitable pharmaceutical carrier and a method for treating or preventing a disease or condition associated with the altered expression of genes that regulate cell proliferation comprising administering to a subject in need such a composition in an amount effective for treating or preventing said disease.

In a further aspect, the invention provides a ribozyme that cleaves a gene whose altered expression correlates with a disease associated with cell proliferation and a method for treating or preventing a disease or condition associated with the altered expression of genes that regulate cell proliferation. The method comprises administering to a subject in need the ribozyme in an amount effective for treating or preventing said disease.

In yet another aspect, the invention provides an antisense molecule that hybridizes to a gene whose altered expression correlates with a disease associated with cell proliferation and a method for treating or preventing a disease or condition associated with the altered expression of genes that regulate cell proliferation. The method comprises administering to a subject in need the antisense molecule that hybridizes to the gene.

The Sequence Listing is a compilation of exemplary polynucleotide sequences obtained by sequencing clone inserts of different cDNA libraries and the amino acid sequences which are coded for by selected nucleotide sequences. Each polynucleotide sequence is identified by a sequence identification number (SEQ ID NO:), by the Incyte Clone number from which the polynucleotide sequence was first identified and by the cDNA library from which the polynucleotide sequence was obtained.

A “polynucleotide sequence” refers to a chain of nucleotides. Preferably, the chain has from about 50 to 10,000 nucleotides, more preferably from about 150 to 3,500 nucleotides. In some instances, the sequences will be fully complementary (no mismatches) when aligned. In other instances, there may be up to a 30% mismatch in the sequences.

A “fragment” refers to a nucleic acid sequence that is preferably at least 10 nucleic acids in length, more preferably 40 nucleic acids, and most preferably 100 nucleic acids in length and encompasses, for example, fragments consisting of nucleic acids 1-100, 300-400, 500-600, 800-900 of SEQ ID NOs: 1-16 or fragments of similar length at the 3′ end of SEQ ID NOs: 1-16. A “fragment” can also mean a stretch of at least 100 consecutive nucleotides that contains one or more deletions, insertions or substitutions. A “fragment” can also mean the whole coding sequence of a gene and may include 5′ and 3′ untranslated regions.

Preferred fragments are those that lack secondary structure as identified by using computer software programs such as OLIGO 4.06 Primer Analysis Software (National Biosciences), Lasergene (DNASTAR), MacDNAsis (Hitachi Software Engineering Co., Ltd.) and the like. A “fragment” can also refer to polypeptide sequences which are preferably at least 5 to about 15 amino acids in length, most preferably at least 10 amino acids long, and which retain some biological activity or immunological activity of a sequence, such as SEQ ID NOs:17-23.

The term “gene” or “genes” refers to the partial or complete coding sequence of a gene. The term also refers to 5′ or 3′ untranslated regions of a transcript. The phrase “gene differentially expressed in precancerous biological samples” refers to a gene whose abundance in a transcript profile derived from precancerous sample differs preferably by least about 1.5 fold, more preferably at least about 2 fold, than that in a transcript profile comprising a noncancerous sample. The phrase also refers to genes that are not detectable in the noncancerous transcript profile but are preferably at levels of at least about 2 copies per cell, more preferably at least about 3 copies per cell, in the precancerous tissue transcript profile. “Precancerous biological sample” means a sample derived from tissue adjacent a cancerous focus. Also, a precancerous sample entails the tissue condition that typically predates the appearance of cancer.

The phrase “gene differentially expressed in a cancerous biological sample” refers to a gene whose abundance in a transcript profile derived from a one or more cancerous samples differs preferably by least about 1.5 fold, more preferably at least about 2 fold, than that in a transcript profile derived from one or more noncancerous biological sample. The phrase also refers to genes that are not detectable in the noncancerous sample transcript profiles but are preferably at levels of at least about 2 copies per cell, more preferably at least about 3 copies per cell, in the cancerous sample transcript profile. “A cancerous biological sample” refers to tissue undergoing uncontrolled cell growth.

The phrase “gene differentially expressed in noncancerous biological sample” refers to a gene whose abundance in a transcript profile derived from one or more non cancerous tissues differs preferably by least about 1.5 fold, more preferably by at least about 2 fold, than that in a transcript profile comprising one or more precancerous or cancerous biological samples. The phrase also refers to genes that are not detectable in precancerous or cancerous transcript profile but are preferably at levels of at least about 2 copies per cell, more preferably at least about 3 copies per cell, in the transcript profile derived from noncancerous biological samples.

The phrase “genes that regulate cell proliferation” refers to genes whose altered expression results in a cancerous or a precancerous stage in a biological sample.

The Invention

The present invention provides polynucleotide sequences comprising at least a fragment of one or more genes that regulate cell proliferation. The polynucleotide sequences are useful individually or as a group of two or more polynucleotide sequences or fragments of a sequence selected from the group consisting of SEQ ID NOs:1-16 for diagnosis and prognosis of diseases of aberrant cell proliferation. Preferably, the plurality of polynucleotide sequences comprise at least a fragment of one or more of the polynucleotide sequences (SEQ ID NOs:1-16) presented in the Sequence Listing. In one preferred embodiment, the polynucleotide sequences comprise a plurality of polynucleotides, wherein each polynucleotide comprises at least a fragment of a sequence selected from the group consisting of SEQ ID NOs:1-16. In another embodiment, the polynucleotides comprise a plurality of polynucleotides wherein said polynucleotides comprise at least a fragment of substantially all the sequences of SEQ ID NOs:1-16. In an additional embodiment, the polynucleotide sequences comprise at least a fragment of two, five, ten, fourteen or more sequences selected from the group consisting of SEQ ID NOs:1-16.

The polynucleotide sequences are particularly useful when they are hybridizable array elements in a microarray. Such a microarray can be employed to monitor the expression of genes of unknown function, but which are differentially expressed in precancerous or cancerous tissue. In addition, the microarray can be used to monitor the expression of genes with a known function in the control of cell proliferation.

The microarray can be used for large scale genetic or gene expression analysis of a large number of polynucleotide sequences. The microarray can be used in the diagnosis of diseases, such as in the diagnosis of early stages of ductal carcinoma before other definitive symptoms are evident, and in the differential diagnosis of diseases with similar symptoms. The microarray can also be used in the monitoring and evaluation of treatments where altered expression of genes coding for polypeptides implicated in the control of cell proliferation cause disease, such as cancer. Additionally, the microarray can be used to investigate an individual's predisposition to a disease, such as cancer. Furthermore, the microarray can be employed to investigate cellular responses, such as cell proliferation and the like.

When the polynucleotide sequences of the invention are employed as hybridizable array elements in a microarray, the array elements are organized in an ordered fashion so that each element is present at a specified location on the substrate. Because the array elements are at specified locations on the substrate, the hybridization patterns and intensities (which together create a unique expression profile) can be interpreted in terms of expression levels of particular genes and can be correlated with a particular disease or condition or treatment.

The invention also entails a pharmaceutical composition comprising a polynucleotide sequence of the invention in conjunction with a suitable pharmaceutical carrier and a method for treating or preventing a disease or condition associated with the altered expression of genes that regulate cell proliferation comprising administering to a subject in need such a composition in an amount effective for treating or preventing a disease or condition associated with cell proliferation.

The polynucleotide sequences may be selected by identifying genes that are differentially expressed in cancerous or precancerous tissue, but not in their noncancerous counterparts. Since some of the polynucleotide sequences are identified solely based on expression levels, it is not essential to know a priori the function of a particular gene in the control of cell proliferation.

Where the selection method is based on differential expression, expression sequence tag (EST) cluster analysis is employed. EST cluster analysis entails sequencing, in whole or in part, isolated clone inserts from a cDNA library, clustering overlapping sequences and determining the clustered sequences' frequency in the cDNA library. ESTs are sequenced by methods well known in the art. The methods can employ such enzymes as the Klenow fragment of DNA polymerase I, Taq polymerase, thermostable T7 polymerase, or combinations of polymerases and proofreading exonucleases. Preferably, the process is automated. ESTs can be combined to form a cluster of ESTs. Clusters are formed by identifying overlapping EST sequences and assembling the ESTs. A nucleic acid fragment assembly tool, such as the Phrap tool (WashU-Merck), the GELVIEW Fragment Assembly system (Genetics Computer Group) and the like, can be used for this purpose. The minimum number of clones necessary to constitute a cluster is two.

After assembling EST clusters, a transcript profile for a particular biological sample is generated and the frequency or abundance of a given EST cluster can be determined. The frequency of an EST cluster in a clone population is correlated to the level of expression of a particular gene. By this process those genes that are abundantly expressed in a biological sample can be identified.

Furthermore, EST analysis can be employed to identify genes that are differentially expressed in one biological sample but not in another biological sample. For this purpose, transcript profiles from both biological samples are generated and compared. By comparing transcript profiles those genes that are differentially expressed in a biological sample can be identified.

With a large enough number of transcript profiles derived from different biological samples, a statistically significant correlation can emerge between cell and tissue source information, such as disease states, treatment outcomes, exposure to various environmental factors or genotypes, and the expression levels of particular genes or groups of genes. Comparisons between transcript profiles of different cells or tissues or of the same cells or tissues under different conditions can be used to discern differences in transcriptional activities. For example, a transcript profile can show differences occurring between two different tissues, such as liver and prostate; between normal and diseased tissue, such as normal and breast tumor or between untreated and treated tissues, such as prostate tumor and irradiated prostate tumor.

The biological samples from which transcript profiles are derived can be selected from a variety of sources. For purposes of this invention, since the intent is to select polynucleotides useful for investigating gene expression as it relates to the control of cell proliferation or cancer, biological samples include those derived from noncancerous, precancerous and cancerous biological samples.

In particular, where polynucleotide sequences are sought that are derived from genes differentially expressed in precancerous tissue, the transcript profiles of precancerous tissue are compared to those of noncancerous biological samples. Examples of precancerous tissues include dysplastic bladder, breast, colon, lung and prostate tissues. Where polynucleotide sequences are sought that are derived from genes differentially expressed in cancerous tissue, the transcript profiles of cancerous tissue are compared to those of noncancerous biological samples. Examples of cancerous tissues include bladder, breast, colon, lung and prostate tumors.

Transcript profile comparisions can be obtained by methods well known to those skilled in the art. Transcript levels and profiles can be obtained and compared, for example, by a differential gene expression assay based on a quantitative hybridization of arrayed DNA clones (Nguyen, et al. (1995) Genomics 29: 207-216), based on the serial analysis of gene expression (SAGE) technology (Velculescu et al. (1995) Science 270: 484-487), based on the polymerase chain reaction (Liang et al. (1992) Science 257: 967-971, Prashar et al. (1996) Proc. Natl. Acad. Sci. USA 93: 659-663), by a differential amplification protocol (Van Gelder et al. 5,545,522)or based on electronic analysis, such as the Transcript Imaging tool or the GEMTOOLS gene expression analysis program (Incyte Pharmaceuticals) or the GeneCalling and Quantitative Expression Analysis technology (Curagen). Preferably, comparisons (subtractions) between two or more transcript profiles are performed electronically.

For purposes of this invention, a number of cDNA libraries are prepared from noncancerous, precancerous and cancerous biological samples, for example from different cDNA libraries derived from bladder, breast, colon, lung and prostate tissues which can be matched to normal and diseased conditions including precancerous and cancerous conditions. ESTs, or sequences derived from ESTs, are assembled and then analyzed to determine whether expression of the different EST clusters can be correlated in a statistical significant manner to the noncancerous, precancerous or cancerous state.

Specifically, transcript profiles can be compared to identify polynucleotide sequences whose transcripts (1) are present only in noncancerous tissue, (2) are present in noncancerous and precancerous tissue only, (3) are present in precancerous tissue only, (4) are present in precancerous and cancerous tissue only, (5) are present in cancerous tissue only, (5) are expressed at higher levels in noncancerous tissue in comparison with precancerous tissue, (6) are expressed at higher levels in noncancerous and precancerous tissue in comparison with cancerous tissue, (7) are expressed at higher levels in noncancerous and precancerous tissue in comparison with cancerous tissue, (8) are expressed at higher levels in tumor tissue in comparison with noncancerous and precancerous tissue, (9) are expressed at higher levels in precancerous tissue in comparison with noncancerous tissue, (10) are expressed at higher levels in precancerous in comparison with tumor tissue, (11) are expressed at higher levels in tumor tissue in comparison with precancerous tissue, (12) are expressed at higher levels in noncancerous in comparison with cancerous tissue, (13) are expressed at higher levels in tumor tissue in comparison with noncancerous tissue and (14) show no statistically significant differences in expression when comparing the tissues.

To identify polynucleotide sequences particularly useful in the detection of the altered expression of genes that regulate cell proliferation seven ductal carcinoma primary tumor cDNA samples and four non-diseased breast tissue cDNA samples were prepared for hybridization to diseased and non diseased sequences. Scan sensitivity, probe labeling, and cDNA quantitation controls were included in the hybridization experiments. The GEMTOOLS gene expression analysis program was used to identify gene transcripts whose expression levels in the diseased samples were at least 2-fold higher or 2-fold lower than their expression level in the non-diseased samples. These diseased sample gene transcripts were further compared in order to specifically identify those whose expression levels were consistently at least 2-fold higher or 2-fold lower in at least six of the seven diseased tissue samples than in the non-diseased samples. These specifically identified polynucleotide sequences are useful in the detection and monitoring of altered expression of genes that regulate cell proliferation. The polynucleotide sequences, SEQ ID NOs 1-16, and the amino acid sequences corresponding to some of the polynucleotide sequences, SEQ ID NOs:17-23, are provided in the Sequence Listing.

The selected polynucleotide sequences may be manipulated further to optimize the performance of the polynucleotide sequences as hybridization targets. Some sequences may not hybridize effectively under hybridization conditions due to secondary structure. To optimize polynucleotide sequence hybridization, the sequences are examined using a computer algorithm to identify fragments of genes without potential secondary structure. Such computer algorithms are well known in the art, such as OLIGO 4.06 Primer Analysis Software (National Biosciences) or Lasergene (DNASTAR, Madison Wis.). These programs can search nucleotide sequences to identify stem loop structures and tandem repeats and to analyze G+C content of the sequence (those sequences with a G+C content greater than 60% are excluded). Alternatively, the sequences can be optimized by trial and error. Experiments can be performed to determine whether sequences and complementary polynucleotide sequences hybridize optimally under experimental conditions.

Where the number of different polynucleotide sequences is desired to be greatest, the sequences are extended to assure that different polynucleotide sequences are not derived from the same gene, i.e., the polynucleotide sequences are not redundant. The sequences may be extended utilizing the partial nucleotide sequences derived from EST sequencing by employing various methods known in the art. For example, one method which may be employed, “restriction-site” PCR, uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, G. (1993) PCR Methods Applic. 2: 318-322).

The polynucleotides can be DNA or RNA, or any RNA-like or DNA-like material, such as peptide nucleic acids, branched DNAs and the like. The polynucleotide can be sense or antisense polynucleotides. In one embodiment, the polynucleotides are cDNAs. The size of the DNA sequence of interest may vary, and is preferably from 50 to 10,000 nucleotides, more preferably from 150 to 3,500 nucleotides. In a second embodiment, the polynucleotides are synthetic polynucleotides. The polynucleotides can be synthesized, in whole or in part, using chemical methods well known in the art (Caruthers et al. (1980) Nucl. Acids Res. Symp. Ser. 215-233).

The polynucleotides may be immobilized on a substrate. Preferred substrates are any suitable rigid or semirigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. The substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which the polynucleotides are bound.

In order to conduct sample analysis, a sample containing polynucleotidcs is provided. The samples can be any sample containing polynucleotides and obtained from any bodily fluid, cultured cells, tissue biopsies, or other tissue preparations.

DNA or RNA can be isolated from the sample according to any of a number of methods well known to those of skill in the art. For example, methods of purification of nucleic acids are described in Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization With Nucleic Acid Targets, Part I. Theory and Nucleic Acid Preparation , P. Tijssen, ed. Elsevier (1993). When sample polynucleotides are amplified it is desirable to amplify the nucleic acid sample and maintain the relative abundances of the original sample, including low abundance transcripts. Total mRNA can be amplified by reverse transcription using a reverse transcriptase, a primer consisting of oligo d(T), and a sequence encoding the phage T7 promoter to provide a single stranded DNA template. The second DNA strand is polymerized using a DNA polymerase and a RNAse which assists in breaking up the DNA/RNA hybrid. After synthesis of the double stranded DNA, T7 RNA polymerase can be added and RNA transcribed from the second DNA strand template (Van Gelder et al. U.S. Pat. No. 5,545,522). RNA can be amplified in vitro, in situ or in vivo (See Eberwine U.S. Pat. No. 5,514,545).

The polynucleotides may be labeled with one or more labeling moieties to allow for detection of hybridized polynucleotide complexes. The labeling moieties can include compositions that can be detected by spectroscopic, photochemical, biochemical, bioelectronic, immunochemical, electrical, optical or chemical means. The labeling moieties include radioisotopes, chemiluminescent compounds, labeled binding proteins, heavy metal atoms, spectroscopic markers, such as fluorescent markers and dyes, magnetic labels, linked enzymes, mass spectrometry tags, spin labels, electron transfer donors and acceptors, and the like.

Hybridization causes a denatured polynucleotide and a denatured sample polynucleotide to form a stable duplex through base pairing. Hybridization methods are well known to those skilled in the art (See, for example, Laboratory Techniques in Biochemistry and Molecular Biology , Vol. 24: Hybridization With Nucleic Acid Targets , P. Tijssen, ed. Elsevier, N.Y. (1993)) Hybridization conditions can be defined by salt concentration, temperature, and other chemicals and conditions well known in the art. In particular, stringency can be increased by reducing the concentration of salt, or raising the hybridization temperature.

For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and most preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C., more preferably of at least about 37° C., and most preferably of at least about 60° C. Varying additional parameters, such as hybridization time, the concentration of detergent or solvent, and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Additional variations on these conditions will be readily apparent to those skilled in the art (Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A. R. (1987) Methods Enzymol. 152:507-511; Ausubel, F. M. et al. (1997) Short Protocols in Molecular Biology , John Wiley & Sons, New York, N.Y.; and Sambrook, J. et al. (1989) Molecular Cloning A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).

Hybridization reactions can be performed in absolute or differential hybridization formats. In the absolute hybridization format, polynucleotides from one sample are hybridized to the polynucleotides in a microarray format and signals detected after hybridization complex formation correlate to polynucleotide levels in a sample. In the differential hybridization format, the differential expression of a set of genes in two biological samples is analyzed. For differential hybridization, polynucleotides from both biological samples are prepared and labeled with different labeling moieties. A mixture of the two labeled polynucleotides is added to a microarray. The microarray is then examined under conditions in which the emissions from the two different labels are individually detectable. Polynucleotides in the microarray that are hybridized to substantially equal numbers of polynucleotides derived from both biological samples give a distinct combined fluorescence (Shalon et al. PCT publication WO95/35505). In a preferred embodiment, the fluorophores Cy3 and Cy5 (Amersham Pharmacia Biotech, Piscataway N.J.) are employed as labels.

After hybridization, the microarray is washed to remove nonhybridized nucleic acids and complex formation between the hybridizable array elements and the polynucleotides is detected. Methods for detecting complex formation are well known to those skilled in the art.

In a differential hybridization experiment, polynucleotides from two or more different biological samples are labeled with two or more different fluorescent labels with different emission wavelengths. Fluorescent signals are detected separately with different photomultipliers set to detect specific wavelengths. The relative abundances/expression levels of the polynucleotides in two or more samples is obtained.

Typically, microarray fluorescence intensities can be normalized to take into account variations in hybridization intensities when more than one microarray is used under similar test conditions. In a preferred embodiment, individual polynucleotide complex hybridization intensities are normalized using the intensities derived from internal normalization controls contained on each microarray.

Diagnostic and Therapeutics

The sequences of the invention may be used in diagnosis, prognosis, treatment, prevention, and evaluation of therapies for diseases associated with cell proliferation.

In one preferred embodiment, the polynucleotides of SEQ ID NOs:1-16 or the polynucleotides encoding SEQ ID NOs:17-23 are used for diagnostic purposes to determine the absence, presence, and expression levels of genes regulating cell proliferation. The polynucleotides may be at least 10, preferably 18 nucleotides long, complementary RNA and DNA molecules, branched nucleic acids, or peptide nucleic acids (PNAs). In particular, the polynucleotides may be used to detect and quantitate gene expression in samples in which altered expression of the polynucleotides SEQ ID NOs:1-16, the polypeptides SEQ ID NOs:17-23 or the polypeptides encoded by SEQ ID NOs:1-16 are correlated with disease. Alternatively, the polynucleotides may be used to monitor the levels of such genes during therapeutic intervention. Additionally, SEQ ID NOs:1-16 can be used to detect genetic polymorphisms associated with a disease. These polymorphisms may be detected at the transcript cDNA or genomic level from mapping experiments.

The specificity of the probe, whether it is made from a highly specific region, e.g., the 5′ regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification (maximal, high, intermediate, or low), will determine whether the probe identifies only naturally occurring instances of SEQ ID NOs 1-16, sequences encoding SEQ ID NOs:17-23, allelic variants, or other related sequences.

Probes may also be used for the detection of related sequences, and should preferably have at least 70% sequence identity to any of the SEQ ID NOs:1-16.

Means for producing specific hybridization probes for DNAs encoding SEQ ID NOs:1-16 include the cloning of SEQ ID NOs:1-16 into vectors for the production of mRNA probes. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides. Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as 32 P or 35S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, by fluorescent labels and the like. The polynucleotide sequences may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; and in microarrays utilizing fluids or tissues from patients to detect altered expression of SEQ ID NOs:1-16. Such qualitative or quantitative methods are well known in the art.

SEQ ID NOs:1-16 can be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantitated and compared with a standard value, typically, derived from a non-diseased sample. If the amount of signal in the patient sample is altered in comparison to the standard value then the presence of altered levels of nucleotide sequences of SEQ ID NOs:1-16 in the sample indicates the presence of the associated disease. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.

Once the presence of a disease is established and a treatment protocol is initiated, hybridization or amplification assays can be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in a healthy subject. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.

The polynucleotides may be used for the diagnosis of diseases associated with altered expression of genes that regulate cell proliferation such as cancer.

Alternatively, the polynucleotides may be used as targets in a microarray. The microarray can be used to monitor the expression level of large numbers of genes simultaneously and to identify splice variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disease, to diagnose a disease, and to develop and monitor the activities of therapeutic agents.

In yet another alternative, polynucleotides may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence and detecting genetic diversity. Fluorescent in situ hybridization (FISH) may be correlated with other physical chromosome mapping techniques and genetic map data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, R. A. (ed.) Molecular Biology and Biotechnology, VCH Publishers New York, N.Y., pp. 965-968). Microarrays may be used to detect genetic diversity at the genome level.

In another embodiment, antibodies which specifically bind SEQ ID NOs:17-23 may be used for the diagnosis of diseases characterized by the over-or-underexpression of SEQ ID NOs:17-23 or polypeptides encoded by SEQ ID NOs:1-16. A variety of protocols for measuring SEQ ID NOs:17-23 or the polypeptides encoded by SEQ ID NOs:1-16, including ELISAs, RIAs, and FACS, are well known in the art and provide a basis for diagnosing altered or abnormal levels of the expression of SEQ ID NOs:17-23 or the polypeptides encoded by SEQ ID NOs:1-16. Standard values for expression levels of SEQ ID NOs:17-23 are established by combining body fluids or cell extracts taken from healthy subjects, preferably human, with antibody to SEQ ID NOs:17-23 or a polypeptide encoded by SEQ ID NOs:1-16 under conditions suitable for complex formation The amount of complex formation may be quantitated by various methods, preferably by photometric means. Quantities of SEQ ID NOs:17-23 or the polypeptides encoded by SEQ ID NOs:1-16 expressed in disease samples from, for example, biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing or monitoring disease. Alternatively, one may use competitive drug screening assays in which neutralizing antibodies capable of binding SEQ ID NOs:17-23 or the polypeptides encoded by SEQ ID NOs:1-16 specifically compete with a test compound for binding the polypeptides. Antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with SEQ ID NOs:17-23 or the polypeptides encoded by SEQ ID NOs:1-16.

In another aspect, the polynucleotides and polypeptides of the present invention can be employed for treatment of diseases associated with the altered expression of genes associated with cell proliferation. The polynucleotides of SEQ ID NOs:1-16 or those encoding SEQ ID NOs:17-23, or any fragment or complement thereof, may be used for therapeutic purposes. In one aspect, the complement of the polynucleotides of SEQ ID NOs:1-16 or those encoding SEQ ID NOs:17-23 may be used in situations in which it would be desirable to block the transcription or translation of the mRNA, ie using antisense technologies.

Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. Methods which are well known to those skilled in the art can be used to construct vectors to express nucleic acid sequences complementary to the polynucleotides. (See, e.g., Sambrook, supra; and Ausubel, supra.) Genes having polynucleotide sequences of SEQ ID NOs:1-16 or those encoding SEQ ID NOs:17-23 can be turned off by transforming a cell or tissue with expression vectors which express high levels of a polynucleotide, or fragment thereof. Such constructs may also be used to introduce untranslatable sense or antisense sequences into a cell. Oligonucleotides derived from the transcription initiation site, e.g., between about positions −10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature. (See, e.g., Gee, J. E. et al. (1994) in Huber, B. E. and B. I. Carr, Molecular and Immunologic Approaches , Futura Publishing Co., Mt. Kisco, N.Y., pp. 163-177.)

Ribozymes, enzymatic RNA molecules, may also be used to catalyze the cleavage of mRNA and decrease the levels of particular mRNAs, such as those comprising the polynucleotide sequences of the invention. (See, e.g. Rossi, 1994, Current Biology 4: 469-471). Ribozymes may cleave mRNA at specific cleavage sites. Alternatively, ribozymes may cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The construction and production of ribozymes is well known in the art and is described in Myers, (1995) Molecular Biology and Biotechnology: A Comprehensive Desk Reference, VCH Publishers, New York, N.Y.)

Alternatively, the polynucleotides of the invention may be integrated into a genome by somatic or germ cell gene therapy. Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art. (See, e.g., Goldman, C. K. et al. (1997) Nature Biotechnology 15:462-466.)

Additionally, endogeous polynucleotide expression may be inactivated using homologous recombination methods which insert inactive gene sequence at the target sequence location. (See, e.g., Thomas and Capecchi (1987) Cell 51: 503-512).

Further, an antagonist or antibody of a polypeptide of SEQ ID NOs:17-23 or a polypeptide encoded by SEQ ID NOs:1-16 may be administered to a subject to treat or prevent a cancer associated with increased expression or activity of SEQ ID NOs:17-23 or polypeptides encoded by SEQ ID NOs:1-16. An antibody which specifically binds the polypeptide may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express the polypeptide.

Antibodies to SEQ ID NOs:17-23 or polypeptides encoded by SEQ ID NOs:1-16 may also be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies (i.e., those which inhibit dimer formation) are especially preferred for therapeutic use. Monoclonal antibodies to SEQ ID NOs:17-23 or to polypeptides encoded by SEQ ID NOs:1-16 may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique. In addition, techniques developed for the production of chimeric antibodies can be used. (See, for example, Molecular Biology and Biotechnology, R. A. Myers, ed.,(1995)John Wiley & Sons, Inc., New York, N.Y.). Alternatively, techniques described for the production of single chain antibodies may be employed. Antibody fragments which contain specific binding sites for SEQ ID NOs:17-23 or the polypeptide sequences encoded by SEQ ID NOs:1-16 may also be generated.

Various immunoassays may be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art.

Yet further, an agonist of a polypeptide of SEQ ID NOs:17-23 or that encoded by SEQ ID NOs:1-16 may be administered to a subject to treat or prevent a cancer associated with altered expression or activity of the polypeptide.

An additional aspect of the invention relates to the administration of a pharmaceutical composition in conjunction with a pharmaceutically acceptable carrier. Such pharmaceutical compositions may consist of polynucleotides of SEQ ID Nos:1-16, polypeptides of SEQ ID NOs:17-23 or those encoded by SEQ ID NOs:1-16, antibodies to the polypeptides, and mimetics, agonists, antagonists, or inhibitors of the polypeptides. The compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water. The compositions may be administered to a patient alone, or in combination with other agents, drugs, or hormones.

The pharmaceutical compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.

In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).

For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells or in animal models such as mice, rats, rabbits, dogs, or pigs. An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of active ingredient, for example, polypeptides of SEQ ID NOs:17-23 or those encoded by SEQ ID NOs:1-16, or fragments thereof, antibodies of the polypeptides, and agonists, antagonists or inhibitors of the polypeptides, which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the ED 50 (the dose therapeutically effective in 50% of the population) or LD 50 (the dose lethal to 50% of the population) statistics.

Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

For purposes of example, the preparation of the BRSTNOT04 cDNA library, from which Incyte Clones 914930 and 1962202 were isolated, is described.

I cDNA Library Construction

The BRSTNOT04 cDNA library was constructed from microscopically noncancerous breast tissue removed from a 62-year-old female during unilateral extended simple mastectomy following diagnosis of invasive grade 3 (of 4), nuclear grade 2 (of 3) mammary ductal carcinoma. The surgical margins were found negative for tumor. Also, a 0.4 cm focus of in-situ carcinoma was identified in the lower quadrant of the breast. Prior to surgery, the patient was diagnosed with benign hypertension, cerebrovascular disease, atherosclerosis, hyperlipidemia, and hematuria. The patient family history included liver cancer in a sibling.

The frozen tissue was homogenized and lysed using a Brinkmann Homogenizer Polytron PT-3000 (Brinkmann Instruments, Westbury, N.J.) in guanidinium isothiocyanate solution. The lysate was centrifuged over a 5.7 M CsCl cushion using a Beckman SW28 rotor in a Beckman L8-70M Ultracentrifuge (Beckman Instruments) for 18 hours at 25,000 rpm at ambient temperature. The RNA was extracted with acid phenol pH 4.0, precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol, resuspended in RNAse-free water, and DNase treated at 37° C. The RNA extraction and precipitation were repeated as before. The mRNA was then isolated with the OLIGOTEX kit (Qiagen, Chatsworth, Calif.) and used to construct the cDNA library.

The mRNA was handled according to the recommended protocols in the SuperScript Plasmid System (Life Technologies). BRSTNOT04 cDNAs were fractionated on a Sepharose CL4B column (Pharmacia Upjohn), and those cDNAs exceeding 400 bp were ligated into a pSPORT I plasmid and subsequently transformed into DH5a TM competent cells (Life Technologies).

The cDNA library was initiated using oligo d(T) priming. The cDNAs were treated with T4 polymerase and synthetic adapter oligonucleotides were ligated onto the cDNAs enabling them to be inserted directionally into the pINCY vector (Incyte) using Eco RI and NotI.

Plasmid DNA was released from the cells and purified using the REAL Prep 96 Plasmid Kit (Qiagen). The recommended protocol was employed except for the following changes: 1) the bacteria were cultured in 1 ml of sterile Terrific Broth (Life Technologies) with carbenicillin at 25 mg/L and glycerol at 0.4%; 2) after inoculation, the cultures were incubated for 19 hours and at the end of incubation, the cells were lysed with 0.3 ml of lysis buffer; and 3) following isopropanol precipitation, the plasmid DNA pellet was resuspended in 0.1 ml of distilled water. After the last step in the protocol, samples were transferred to a 96-well block for storage at 4° C.

II Isolation and Sequencing of cDNA Clones

Plasmids were recovered from host cells by in vivo excision, using the UNIZAP vector system (Stratagene) or cell lysis. Plasmids were purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg Md.); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the REAL Prep 96 plasmid kit from QIAGEN. Following precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4° C.

Alternatively, plasmid DNA was amplified from host cell lysates using direct link PCR in a high-throughput format (Rao, V. B. (1994) Anal. Biochem. 216:1-14). Host cell lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384-well plates, and the concentration of amplified plasmid DNA was quantified fluorometrically using PICOGREEN dye (Molecular Probes, Eugene Oreg.) and a Fluoroskan II fluorescence scanner (Labsystems Oy, Helsinki, Finland).

The cDNAs were prepared for sequencing using the ABI CATALYST 800 (Perkin-Elmer) or the HYDRA microdispenser (Robbins Scientific) or MICROLAB 2200 (Hamilton) systems in combination with the PTC-200 thermal cyclers (MJ Research). The cDNAs were sequenced using the ABI PRISM 373 or 377 sequencing systems (Perkin-Elmer) and standard ABI protocols, base calling software, and kits. In one alternative, cDNAs were sequenced using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics). In another alternative, the cDNAs were amplified and sequenced using the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-Elmer). In yet another alternative, cDNAs were sequenced using solutions and dyes from Amersham Pharmacia Biotech. Reading frames for the ESTs were determined using standard methods (reviewed in Ausubel, F. M. (1997) Short Protocols in Molecular Biology , John Wiley & Sons, New York N.Y., unit 7.7).

III Homology Searching of cDNA Clones and Their Deduced Proteins

As used herein, “homology” refers to sequence similarity between a reference sequence and at least a fragment of a newly sequenced clone insert, and can refer to either a nucleic acid or amino acid sequence. The Genbank databases which contain previously identified and annotated sequences, were searched for regions of homology using BLAST (Basic Local Alignment Search Tool). (See, e.g., Altschul, S. F. (1993) J. Mol. Evol. 36: 290-300; and Altschul et al. (1990) J. Mol. Biol. 215: 403-410.)

BLAST involves first finding similar segments between the query sequence and a database sequence, then evaluating the statistical significance of any matches that are found and finally reporting only those matches that satisfy a user-selectable threshold of significance. BLAST produces alignments of both nucleotide and amino acid sequences to determine sequence similarity. The fundamental unit of the BLAST algorithm output is the High scoring Segment Pair (HSP). An HSP consists of two sequence fragments of arbitrary, but equal lengths, whose alignment is locally maximal and for which the alignment score meets or exceeds a threshold or cutoff score set by the user.

The basis of the search is the product score, which is defined as: %     sequence     identity    ×    %     maximum     BLAST     score 100

The product score takes into account both the degree of similarity (identity) between two sequences and the length of the sequence match as reflected in the BLAST score. The BLAST score is calculated by scoring +5 for every base that matches in an HSP and −4 for every mismatch. For example, with a product score of 40, the match will be exact within a 1% to 2% error, and, with a product score of 70, the match will be exact. Homologous molecules are usually identified by selecting those which show product scores between 15 and 40, although lower scores may identify related molecules. The P-value for any given HSP is a function of its expected frequency of occurrence and the number of HSPs observed against the same database sequence with scores at least as high. Percent sequence identity refers to the percentage of sequence similarity found in a comparison of two or more amino acid or nucleic acid sequences. Percent identity can be determined electronically, e.g., by using the MegAlign program (DNASTAR, Inc., Madison Wis.). The percentage similarity between two amino acid sequences, e.g., sequence A and sequence B, is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no homology between the two amino acid sequences are not included in determining percentage similarity.

IV Transcript Imaging

To discover transcripts that were differentially expressed in noncancerous, precancerous and cancerous biological samples, the following cDNA libraries were employed: cDNA libraries derived from noncancerous bladder (BLADNOT01, BLADNOT04), precancerous bladder (BLADNOT03, BLADNOT05, BLADNOT06), cancerous bladder (BLADTUT02, BLADTUT04, BLADTUT05, BLADTUT06, BLADTUT07); noncancerous breast (BRSTNOM01, BRSTNOM02, BRSTNOT01, BRSTNOT02), precancerous breast (BRSTNOT03, BRSTNOT04, BRSTNOT05, BRSTNOT07, BRSTNOT09, BRSTNOT13, BRSTNOT14),cancerous breast (BRSTTUT01, BRSTTUT02, BRSTTUT03, BRSTTUT08, BRSTTUT13); noncancerous colon (COLNNOT13, COLNOT19), precancerous colon (COLNNOT01, COLNNOT05, COLNNOT07, COLNNOT08, COLNNOT09, COLNNOT11, COLNNOT16), cancerous colon (COLNTUM01, COLNTUT02, COLNTUT03, COLNTUT06, COLNTUT15, COLNTUT16, COLNPOT01); noncancerous lung (LUNGNOM01, LUNGNOTO01, LUNGNOT02, LUNGNOT04), precancerous lung (LUNGNOT03, LUNGNOT04, LUNGNOT12, LUNGNOT14, LUNGNOT15, LUNGNOT18), cancerous lung (LUNGTUM01, LUNGTUT01, LUNGTUT03, LUNGTUT06, LUNGTUT07, LUNGTUT08, LUNGTUT09, LUNGTUT10, LUNGTUT11); noncancerous prostate (PROSNOT01, PROSNOT11, PROSNON01), precancerous prostate (PROSNOT02, PROSNOT05, PROSNOT06, PROSNOT07, PROSNOT14, PROSNOT15, PROSNOT16, PROSNOT18, PROSNOT19, PROSNOT20, PROSNOT26, PROSNON08), cancerous prostates (PROSTUT01, PROSTUT03, PROSTUT04, PROSTUT05, PROSTUT08, PROSTUT09, PROSTUT10, and PROSTUT12).

Transcript expression was analyzed by using a program that (a) assembled the sequences into clusters and (b) classified the sequences according to where the sequences were expressed. Also, the statistical significance of the expression patterns was analyzed. The categories were as follows: (1) are present only in noncancerous tissue, (2) are present in noncancerous and precancerous tissue only, (3) are present in precancerous tissue only, (4) are present in precancerous and tumor tissue only, (5) are present in tumor only, (5) are expressed at higher levels in noncancerous tissue in comparison with precancerous tissue, (6) are expressed at higher levels in noncancerous and precancerous tissue in comparison with cancerous tissue, (7) are expressed at higher levels in noncancerous and precancerous tissue in comparison with cancerous tissue, (8) are expressed at higher levels in cancerous tissue in comparison with noncancerous and precancerous tissue, (9) are expressed at higher levels in precancerous rather in comparison with noncancerous tissue, (10) are expressed at higher levels in precancerous in comparison with cancerous tissue, (11) are expressed at higher levels in cancerous tissue in comparison with precancerous tissue, (12) are expressed at higher levels in noncancerous tissue rather than in cancerous tissue, (13) are expressed at higher levels in tumor tissue rather than in noncancerous tissue and (14) no statistically significant differences in expression. For categories (1) through (5), the statistically significant cutoff point was selected whereby the transcript should be expressed in at least 50% of the libraries, if the number of libraries was equal or greater than 4 but less than 10. If the number of libraries was greater than 10, then the transcript only needed to be expressed in 33% of the libraries. When fewer than 4 libraries were available, a transcript would typically have to be expressed in all tissue samples for selection. For categories (6) through (13), selected transcripts were expressed at levels greater than 2.5×, more preferably at levels of about 3× and 4× in the tissue where overexpression is observed. Also at least 50% of the cDNA libraries of a given biological sample type have to show overexpression.

V Sample Preparation

PolyA + RNA was purchased from BioChain Institute (San Leandro, Calif., USA) where total RNA was isolated from tissue samples using the guanidinium thiocyanate method and poly + RNA was purified using the oligo (dT)cellulose method. The four non-diseased breast tissue polyA + RNA samples were prepared from tissue from three female patients, ages 32-42 and a pooled tissue sample from two donors, ages 43 and 58. The seven ductal carcinoma primary tumor tissue polyA + RNA samples were prepared from tissue from six different female patients, ages 46-56 and a pool of 18 donors (ages 40-72), all undergoing no chemotherapeutic treatment. Each polyA + RNA sample was reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/ul oligo-dT primer (21mer), 1×first strand buffer, 0.03 units/ul RNase inhibitor, 500 uM dATP, 500 uM dGTP, 500 uM dTTP, 40 uM dCTP, 40 uM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The reverse transcription reaction was performed in a 25 ml volume containing 200 ng polyA + RNA with GEMBRIGHT kits (Incyte). Specific control polyA + RNAs (YCFR06, YCFR45, YCFR67, YCFR85, YCFR43, YCFR22, YCFR23, YCFR25, YCFR44, YCFR26) were synthesized by in vitro transcription from non-coding yeast genomic DNA (W. Lei, unpublished). As quantitative controls, the control mRNAs (YCFR06, YCFR45, YCFR67, YCFR85) at 0.002 ng, 0.02 ng, 0.2 ng, and 2 ng were diluted into reverse transcription reaction at ratios of 1:100,000, 1:10,000, 1:1000, 1:100 (w/w) to sample mRNA respectively. The control mRNAs (YCFR43, YCFR22, YCFR23, YCFR25, YCFR44, YCFR26) were diluted into reverse transcription reaction at ratios of 1:3, 3:1, 1:10, 10:1, 1:25, 25:1 (w/w) to sample mRNA differential expression patterns. After incubation at 37° C. for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labeling) was treated with 2.5 m 1 of 0.5M sodium hydroxide and incubated for 20 minutes at 85° C. to the stop the reaction and degrade the RNA. Probes were purified using two successive CHROMA SPIN 30 gel filtration spin columns (Clontech, Palo Alto, Calif. USA) and after combining, both reaction samples were ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100% ethanol. The probe was then dried to completion using a SpeedVAC (Savant) and resuspended in 14 ul 5×SSC/0.2% SDS.

VI Microarray Preparation

Purified array elements were immobilized on polymer-coated glass slides. Glass microscope slides (Corning, Coming, N.Y.) cleaned by ultrasound in 0.1% SDS and acetone, with extensive distilled water washes between and after treatments. Glass slides were etched in 4% hydrofluoric acid (VWR, West Chester, Pa.), washed extensively in distilled water, and coated with 0.05% aminopropyl silane (Sigma Aldrich, St. Louis, Mo.) in 95% ethanol. Coated slides were cured in a 110° C. oven.

Array elements were applied to the coated glass substrate using a procedure described in U.S. Pat. No. 5,807,522 and incorporated herein by reference. In brief, 1 μl of the array element DNA, at an average concentration of 100 ng/ul, was loaded into the open capillary printing element by a high-speed robotic apparatus. The apparatus then deposited about 5 nl of array element sample per slide.

Microarrays were UV-crosslinked using a STRATALINKER UV-crosslinker (Stratagene, La Jolla, Calif.). Microarrays were washed at room temperature once in 0.2% SDS and three times in distilled water. Non-specific binding sites were blocked by incubation of microarrays in 0.2% casein in phosphate buffered saline (PBS) (Tropix Inc., Bedford, Mass.) for 30 minutes at 60° C. followed by washes in 0.2% SDS and distilled water as before.

VII Hybridization

Hybridization reactions contained 9 μl of probe mixture consisting of 0.2 μg each of both Cy3 and Cy5 labeled cDNA synthesis products in 5×SSC, 0.2% SDS hybridization buffer. The probe mixture was heated to 65° C. for 5 minutes and was aliquoted onto the microarray surface and covered with an 1.8 cm 2 coverslip. The arrays were transferred to a waterproof chamber having a cavity just slightly larger than a microscope slide. The chamber was kept at 100% humidity intemally by the addition of 140 μof 5×SSC in a corner of the chamber. The chamber containing the arrays was incubated for about 6.5 hours at 60° C. The arrays were washed for 10 min at 45° C. in high stringency wash buffer (1×SSC, 0.1% SDS), three times for 10 minutes each at 45° C. in low stringency wash buffer (0.1×SSC), and then dried.

VIII Detection

The microscope used to detect the reporter-labeled hybridization complexes was equipped with an Innova 70 mixed gas 10 W laser (Coherent Lasers, Santa Clara, Calif.) capable of generating spectral lines at 488 nm for excitation of Cy3, and 632 nm for excitation of Cy5. The excitation laser light was focused on the array using a 20×microscope objective (Nikon). The slide containing the array was placed on a computer-controlled X-Y stage on the microscope and raster-scanned past the objective. The 1.8 cm×1.8 cm array used in the present example was scanned with a resolution of 20 micrometers.

In two separate scans, a mixed gas multiline laser excited the two fluorophores sequentially. Emitted light was split, based on wavelength, into two photomultiplier tube detectors (PMT R1477, Hamamatsu Photonics, San Jose, Calif.) corresponding to the two fluorophores. Appropriate filters positioned between the array and the photomultiplier tubes were used to filter the signals. The emission maxima of the fluorophores used were 565 nm for Cy3 and 650 nm for Cy5. Each array was typically scanned twice, one scan per fluorophore using the appropriate filters at the laser source, although the apparatus was capable of recording the spectra from both fluorophores simultaneously.

The sensitivity of the scans was typically calibrated using the signal intensity generated by a cDNA control species added to the probe mix at a known concentration. A specific location on the array contained a complementary DNA sequence, allowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1:100,000. When two probes from different sources (e.g., representing test and control cells), each labeled with a different fluorophore, are hybridized to a single array for the purpose of identifying genes that are differentially expressed, the calibration was done by labeling samples of the calibrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture.

The output of the photomultiplier tube was digitized using a 12-bit RTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Norwood, Mass.) installed in an IBM-compatible PC computer. The digitized data were displayed as an image where the signal intensity was mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal). The data was also analyzed quantitatively. Where two different fluorophores were excited and measured simultaneously, the data were first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore's emission spectrum.

A grid was superimposed over the fluorescence signal image such that the signal from each spot was centered in each element of the grid. The fluorescence signal within each element was then integrated to obtain a numerical value corresponding to the average intensity of the signal. The software used for signal analysis was the GEMTOOLS gene expression analysis program (Incyte).

IX Results

The GEMTOOLS (Incyte) gene expression analysis program was used to identify gene transcripts whose expression levels in the diseased samples was at least 2-fold higher or 2-fold lower than their expression level in the non-diseased samples. These diseased sample gene transcripts were further compared in order to specifically identify those whose expression levels were consistently at least 2-fold higher or 2-fold lower in six of the seven diseased tissue samples than in the non-diseased samples. These specifically identified gene transcripts are useful in the detection and monitoring of altered expression of genes that regulate cell proliferation. The polynucleotide sequences, SEQ ID NOs 1-16, and the amino acid sequences, SEQ ID NOs 17-23, are provided in the Sequence Listing. Table 1 is a list of the exemplary polynucleotide sequences according to their SEQ ID NOs. Column 1 shows the sequence identification numbers (SEQ ID NO:), column 2 lists the Incyte Clone ID numbers, and column 3, the cDNA libraries from which these clones were isolated. The fourth column describes a relevant Genbank identification number match, if any. The fifth column contains the annotation associated with the referenced GenBank identification number. For polynucleotide sequences that are not exact matches or homologous to GenBank nucleotide sequences the fourth and fifth columns contain the words INCYTE.

SEQ ID NO: 17 corresponds to a translation of polynucleotide sequence SEQ ID NO:1. SEQ ID NO: 18 corresponds to a translation of polynucleotide sequence SEQ ID NO:4. SEQ ID NO: 19 corresponds to a translation of polynucleotide sequence SEQ ID NO:9. SEQ ID NO: 20 corresponds to a translation of polynucleotide sequence SEQ ID NO:11. SEQ ID NO: 21 corresponds to a translation of polynucleotide sequence SEQ ID NO:13. SEQ ID NO: 22 corresponds to a translation of polynucleotide sequence SEQ ID NO:14. SEQ ID NO: 23 corresponds to a translation of polynucleotide sequence SEQ ID NO:16.

<160> NUMBER OF SEQ ID NOS: 23

<210> SEQ ID NO 1

<211> LENGTH: 2728

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE: -

<223> OTHER INFORMATION: 159452

<400> SEQUENCE: 1

cgagacatag cttttctcat tcaccctcac acttggggct aatgcacaga catgaacatc 60

tattgaggaa aaccacaaaa aacttcaaaa cagctacaac gggaaaaaga gagttttgtc 120

ccacagtcag caggccacta gtttattaac ttccagtcac cttgattttt gctaaaatga 180

agactctgca gtctacactt ctcctgttac tgcttgtgcc tctgataaag ccagcaccac 240

caacccagca ggactcacgc attatctatg attatggaac agataatttt gaagaatcca 300

tatttagcca agattatgag gataaatacc tggatggaaa aaatattaag gaaaaagaaa 360

ctgtgataat acccaatgag aaaagtcttc aattacaaaa agatgaggca ataacaccat 420

tacctcccaa gaaagaaaat gatgaaatgc ccacgtgtct gctgtgtgtt tgtttaagtg 480

gctctgtata ctgtgaagaa gttgacattg atgctgtacc acccttacca aaggaatcag 540

cctatcttta cgcacgattc aacaaaatta aaaagctgac tgccaaagat tttgcagaca 600

tacctaactt aagaagactc gattttacag gaaatttgat agaagatata gaagatggta 660

ctttttcaaa actttctctg ttagaagaac tttcacttgc tgaaaatcaa ctactaaaac 720

ttccagttct tcctcccaag ctcactttat ttaatgcaaa atacaacaaa atcaagagta 780

ggggaatcaa agcaaatgca ttcaaaaaac tgaataacct caccttcctc tacttggacc 840

ataatgccct ggaatccgtg cctcttaatt taccagaaag tctacgtgta attcatcttc 900

agttcaacaa catagcttca attacagatg acacattctg caaggctaat gacaccagtt 960

acatccggga ccgcattgaa gagatacgcc tggagggcaa tccaatcgtc ctgggaaagc 1020

atccaaacag ttttatttgc ttaaaaagat taccgatagg gtcatacttt taacctctat 1080

tggtacaaca tataaatgaa agtacaccta cactaatagt ctgtctcaac aatgagtaaa 1140

ggaacttaag tattggttta atattaacct tgtatctcat tttgaaggaa tttaatattt 1200

taagcaagga tgttcaaaat cttacatata ataagtaaaa agtaagactg aatgtctacg 1260

ttcgaaacaa agtaatatga aaatatttaa acagcattac aaaatcctag tttatactag 1320

actaccattt aaaaatcatg tttttatata aatgcccaaa tttgagatgc attattccta 1380

ttactaatga tgtaagtacg aggataaatc caagaaactt tcaactcttt gcctttcctg 1440

gcctttactg gatcccaaaa gcatttaagg tacatgttcc aaaaactttg aaaagctaaa 1500

tgtttcccat gatcgctcat tcttctttta tgattcatac gttattcctt ataaagtaag 1560

aactttgttt tcctcctatc aaggcagcta ttttattaaa tttttcactt agtctgagaa 1620

atagcagata gtctcatatt taggaaaact ttccaaataa aataaatgtt attctctgat 1680

aaagagctaa tacagaaatg ttcaagttat tttactttct ggtaatgtct tcagtaaaat 1740

attttcttta tctaaatatt aacattctaa gtctaccaaa aaaagtttta aactcaagca 1800

ggccaaaacc aatatgctta taagaaataa tgaaaagttc atccatttct gataaagttc 1860

tctatggcaa agtctttcaa atacgagata actgcaaaat attttccttt tatactacag 1920

aaatgagaat ctcatcaata aattagttca agcataagat gaaaacagaa tattctgtgg 1980

tgccagtgca cactaccttc ccacccatac acatccatgt tcactgtaac aaactgaata 2040

ttcacaataa agcttctgag taacactttc tgattactca tgataaactg acatggctaa 2100

ctgcaagaat taaatcttct atctgagagt aataatttat gatgactcag tggtgccaga 2160

gtaaagtttc taaaataaca ttcctctcac ttgtacccca ctaaaagtat tagtctacac 2220

attacattga agttaaacac aaaattatca gtgttttaga aacatgagtc cggactgtgt 2280

aagtaaaagt acaaacatta tttccaccat aaagtatgta ttgaaatcaa gttgtctctg 2340

tgtacagaat acatacttat tcccattttt aagcatttgc ttctgttttc cctacctaga 2400

atgtcagatg tttttcagtt atctccccat ttgtcaaagt tgacctcaag ataacatttt 2460

tcattaaagc atctgagatc taagaacaca attattattc taacaatgat tattagctca 2520

ttcacttatt ttgataacta atgatcacag ctattatact actttctcgt tattttgtgt 2580

gcatgcctca tttccctgac ttaaacctca ctgagagcgc aaaatgcagc tttatacttt 2640

ttactttcaa ttgcctagca caatagtgag tacatttgaa ttgaatatat aataaatatt 2700

gcaaaataaa atccatctaa ataaaaaa 2728

<210> SEQ ID NO 2

<211> LENGTH: 2125

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<221> NAME/KEY: unsure

<222> LOCATION: 52

<223> OTHER INFORMATION: a or g or c or t, unknown, or other

<220> FEATURE: -

<223> OTHER INFORMATION: 914930

<400> SEQUENCE: 2

ccccccaaac ggaaggaagc ccccatgccc ccaaaagcag ccaaacctat tnttggttta 60

tttaatggtg tagcttttta actttattaa agaatttacc agcgaaaccc ttacatgagt 120

aattgaaatg aaattaaatg agattacagc ataatgaaga agaaaactag aatctaacag 180

gtatgacaca ttcagttatt ctaacagggc acagtactgt gctatatgta gccgccttta 240

cagatgttat taacctatag tagtttacta ttaactagga agactggtgt atgaggaggg 300

agacattctg taataagatt aaaacctaaa gtcagctgtg taacattcca tgaaagatgc 360

attcatttat tgaagatgac aaaaaaggga gtcagatata ttctggaagc tattttaatt 420

ttaggcacac aatctcatac attttgagac ccttgggtca tttattactt tttacaaact 480

agttcctctc tttttttctg ccaagtcctg aattgaaaac tgtaggcttc cttgcacaga 540

tgttgcagcc agcctcagga gtaaagtgcc cgctgactgc tgccgccacc tctgtctcgc 600

tccctgtcag tgctgctggc acgtgggacg cggcaggtga cagccgttct cagcatgttt 660

tagaagcttg cctcacagac ttccatgcct ctccattcag ccgatgactt caaggtgtca 720

aactgtttta atttttcaaa caaatggaac agaagccatt gtggttcatc ctgattactt 780

gaacgttgca cttggtggac cgtgcctggg agcgctcgca tgccccctgg cttcagaagt 840

catgtcagtg tctctgtaga caaactccac tgtacatctt ggattaatct tctgattcat 900

tgttcatttt ctcaagactt tttgtggaat tctctgataa aggaagcttt taggatggta 960

tctatcaggc caccagcagg aattgaaaat gttttcacaa aaatcctttt ccttagaaat 1020

aaaagctggt gacagagatg gtttccttgt accgataaaa acaaaaccaa atccatatta 1080

tacatcaaaa ccttgtgaga cattcacttg ctcttttgcc atatttagat gtgttagtgg 1140

aatcagaaac ctgttttgat atgtgttctc catgagttaa gtctgatttg tctttttatt 1200

tcatgatgca tgtctttttt tttcttttgt caggataacg tcatatagca tcttgtttgt 1260

ttttccttat ctctatgtac atatctatct acttctgact gtagatgggt atatagatag 1320

atgccaagct tcttatgttc tgggggtagt atgcatcatt attgggtctc tgccttaaaa 1380

cacatcaaaa ttcattttag acaaaaaaac ttctgctttg tctttggtca ttagggagct 1440

ctaatgtgtg tttgtggctc caagttacat tttgtgtttc attgatctat atgtatatat 1500

gtgatgtttt catatatata tgtgtgtgtg tttaaatttt gtatcatcag gactgacacc 1560

caatttgaca ctttttgtat ctagaagacc ctccaaaaaa ggaaccacat aagcacacaa 1620

gaaaagagtg ctatgatgtt cttagcattt gctatcatgc ctatttttat ctagattttt 1680

aaatgtagct tgtcataaca aaattttaat tacaattggc ttgtatgaga agaaaaagta 1740

tttttattgt tttgagtgat gacgcagaga ctcaatgaac ttgaaaatag cattgcttcg 1800

tgcactttga ataccaatca ggtgttttct gtgctactag ttgtcacgtt gcattcatgt 1860

tcacctcctg atttaagtat ctcgggtgtg cccagccact aaagcactct ggactaatcg 1920

ctaaagagaa gcaacgtggg gggtggggtt gcgagggatg tgttcacatg tacccatcat 1980

ttgatcatag cactgtgatt gcttttgatg tgtgtctcta gtggtgtgtt gtctgttggc 2040

atgcttaaag cacatgtcca ttaaaattca ttttgttcct tttaaaaaaa aaaaaaaagg 2100

gcggccccga ctagtgagcc cgtcg 2125

<210> SEQ ID NO 3

<211> LENGTH: 1853

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE: -

<223> OTHER INFORMATION: 1283330

<400> SEQUENCE: 3

tgttttctcc cccatcacaa aaaaaaaaat tcttattttt agtagacatg tatttaccaa 60

aaatatgtac tcaattattg tattttggat tttatcaatt taaaaattgt ggaaatttgt 120

ttgctcttac gccaacataa tattgatttt gcctcttggc tctgaaagcc caaaatattt 180

accgtctagc ccgttacaga aaaagtctgc tgactactga gccagacctc cattacctcc 240

atccctgttg gattatttaa agaaagcctc agacagtaag ggctttttta aaagaataaa 300

atgacttggt ttgcgcttgg aagcagggga agcattcaga tgagcggttt ctgcattaac 360

cctgcctatc acgcatctcg tgtcctgtgt ggctggcgag ccccccttgg aaggttctgg 420

tgcttcagct ggctcctgca gagtccaccc cgcctcgtgg tgggaatgca gagccctttg 480

ctttccttct tgccgcctgc ttcctgttcc tggggacccg ctgggccttt ggtctgcatc 540

ccctggccag gtccctcagg gttgatgcgt ggagaaggac tttgagcagt ggtgggcagc 600

agtggcctcc tggccagctc acactcttgt cctgggaggg gcagcctgat ctcacctcca 660

cctagtacct tggggactga ggaccttttg gcttctctgg agcctgcaag cctcttccca 720

tgtgtccagc tgctcttcct gctacaaagg ggactgctca cagtggcctc agcttggtgg 780

ttttgagggg ccgccccccg gccctccata agggtatcct gggcctgaga attctgcatc 840

tgccattgga ggatggacag cctcaaatgg aaggagtccc acgggagatg ggtccgaggt 900

ccggctgtgg ccatccagcc ccctgtggct tgtccagcct ctgtgcaccc ctggtgtctt 960

cactccaggg gcagacagca gccactgcag ttcctttctt cgtgagtaac agtagtgata 1020

gcagctgggg ctaacaggct aggctttgtg ttctgcgcat ttggtcagct tctcactcga 1080

tcctccctaa agcaatgggg aggcccccac tagcccagtt ttcaggaagt caactgggag 1140

gttagatggg ggccagggtc ccacagctac tgatggcccg agccaggttg agcttcctgg 1200

tgtccagtcc ggatcccact tgcagatctc atgctctcag ataggtggga caagttcttt 1260

tgtcacagtg ctggctctgt cctgaggcct cattgctggc tgggtgtgct ctgctgggaa 1320

aagctttgcg gggcttgctt ggttaaccac agaagagaag gggactgttt ggggtgcctc 1380

tctgcagcct ccccgtgctg ggtggaagca cggttactgt gttctctaat gttcatgtat 1440

ttaaaatgat ttctttctaa agatgtaacc tccacacctt tctccagatt gggtgactct 1500

tttctaaagg tggtgggagt atctgtcggg gtggtgtggc ccttggatgg gtcaggtggg 1560

tgtgagaggt cctggggagg tgggcgttga gctcaaagtt gtcctactgc catgtttttg 1620

tacctgaaat aaagcatatt ttgcacttgt tactgtacca tagtgcggac gagaagtctg 1680

tatgtgggat ctgtgcttgg gttagaatgc aaataaaact cacatttgta agacaaaaaa 1740

aaaaacaaaa gagagaccca gcccaaggag acatacagta aacgagggga gtaaaagagg 1800

tcgaagaaac tcgacaagag acgaagtaaa aaaaggggcg gcccccccct ttt 1853

<210> SEQ ID NO 4

<211> LENGTH: 1741

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE: -

<223> OTHER INFORMATION: 1299627

<400> SEQUENCE: 4

ttcgctccaa gcctcaggcc accggcttgg atggacgctc cgaggctacc cgtgcgtcca 60

ggggtcttgc ttccgaagtt ggtcctgctc tttgtctacg cagatgattg ccttgctcag 120

tgtggcaaag attgcaaatc ttactgctgt gatggaacca cgccctactg ttgctcctac 180

tacgcttata ttgggaatat cctctcgggc actgcaattg cgggcattgt ttttggaata 240

gtatttatca tgggggtcat tgctgggatt gccatatgca tctgcatgtg catgaagaac 300

cacagggcga cccgcgtggg catcctcagg acgactcaca tcaacaccgt ctcctcctat 360

cctggaccac caccctacgg tcacgaccac gagatggaat actgtgcaga cttgcctcct 420

ccatactccc ccaccccaca gggtccagca cagcgttctc caccccctcc ttatcctgga 480

aacgcaagga aataatctat ctcccagaac agaacatgtg ccaatgggcg atcttgcctg 540

gaataaaatg cctctactca gaaacaggca ggaaagaatt gctccaagga atactttttg 600

gggtcagata atgtgtcagg tggaatatcc ctgctaggag atataggatt tctactctgc 660

tcaaagctga ccccatctgg agtattaatg tttggttcta tggaaccaca ttttaagaga 720

tctgctgatc cacctaagca cattcaggga agagtaatgt aattgacaaa atatctgata 780

atcatgttgt ttaagggcta ggtgaagaaa gtttcagtat tgatcctgga aaaaaagaag 840

atctaagtag gatgggagaa tgatttggcc cacacaagga agcaacttta ttctatatag 900

ctttaaaagt cagaactaga attgttcatt ctttcattca tcaataaatg tattttgagt 960

gcctaagagt ttactatgtg cctagcactg tttgaggtcc tgatggaagt tacaggatgg 1020

gtactctggt tttagtacaa gaaagagcaa tgactagatt gctttgtgaa gctcttggta 1080

gagacacgct ccagaaggga taacaaaatc aaatagtaga tgggttcatt gggcctcaga 1140

agttctgctc gtattttagg tgggtgtgaa gtgaatttct atatgtccag gagtgaatac 1200

aacagaaaga gttggatctt atttatttaa ttagggagtt aaaacaagac caaaaagact 1260

caacagccgc ttgaagccaa gaactcttca atgccagcta ctgccaccta aaaatcatct 1320

ggctttatag tggatcagaa taaaggttat tctaactgtg gggagaaaaa aaaaattgta 1380

tcaagttcca caggtagcag acacttcact tccaagtaaa agatgagaaa tcaattattc 1440

ccacaggatt ttaggtcagg gagcaaaaat ctcagaactt gaccatgaag atacacaaca 1500

gactcgcaaa aataaagtgg gaaatgaagt tcagattccc ttctgtagat ttccttaaaa 1560

ctattatttt tttcttcttc gtaaaatttt gataatctgt tctcttaaaa aagttaatga 1620

cacaattaag atactgacat caaattgttg ccttttacca aaatgcaaat tttatgaagt 1680

gcctaccttt atatgtataa agcatttaat aaataattct aatgtgccat aaaaaaaaaa 1740

a 1741

<210> SEQ ID NO 5

<211> LENGTH: 2678

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE: -

<223> OTHER INFORMATION: 1319129

<400> SEQUENCE: 5

tcggaattcg gctcgagata gactgtgagt tctgtggtga cagaaaccaa gtgtaacctg 60

tttaccattt gattcccagc acctggcata gtgcctgaaa tgtactgttc gggggtcttg 120

tctggatttt ggttgcctcc tccaatgttc ctctacctct actacaagga tgggtcatgt 180

ttgtgtccgt gacagcgttt ttcttttcgc tcctctttct gggcatgttc ctctctggca 240

tggtggctca aattgatgct aactggaact tcctggattt tgcctaccat tttacagtat 300

ttgtcttcta ttttggagcc tttttattgg aagcagcagc cacatccctg catgatttgc 360

attgcaatac aaccataacc gggcagccac tcctgagtga taaccagtat aacataaacg 420

tagcagcctc aatttttgcc tttatgacga cagcttgtta tggttgcagt ttgggtctgg 480

ctttacgaag atggcgaccg taacactcct tagaaactgg cagtcgtatg ttagtttcac 540

ttgtctactt tatatgtctg atcaatttgg ataccatttt gtccagatgc aaaaacattc 600

caaaagtaat gtgtttagta gagagagact ctaagctcaa gttctggttt atttcatgga 660

tggaatgtta attttattat gatattaaag aaatggcctt ttattttaca tctctcccct 720

ttttcccttt ccccctttat tttcctcctt ttctttctga aagtttcctt ttatgtccat 780

aaaatacaaa tatattgttc ataaaaaatt agtatccctt ttgtttggtt gctgagtcac 840

ctgaacctta attttaattg gtaattacag cccctaaaaa aaacacattt caaataggct 900

tcccactaaa ctctatattt tagtgtaaac caggaattgg cacacttttt ttagaatggg 960

ccagatggta aatatttatg cttcacggtc catacagtct ctgtcacaac tattcagttc 1020

tgctagtata gcgtgaaagc agctatacac aatacagaaa tgaatgagtg tggttatgtt 1080

ctaataaaac ttatttataa aaacaagggg aggctgggtt tagcctgtgg gccatagttt 1140

gtcaaccact ggtgtaaaac cttagttata tatgatctgc attttcttga actgatcatt 1200

gaaaacttat aaacctaaca gaaaagccac ataatattta gtgtcattat gcaataatca 1260

cattgccttt gtgttaatag tcaaatactt acctttggag aatacttacc tttggaggaa 1320

tgtataaaat ttctcaggca gagtcctgga tataggaaaa agtaatttat gaagtaaact 1380

tcagttgctt aatcaaacta atgatagtct aacaactgag caagatcctc atctgagagt 1440

gcttaaaatg ggatccccag agaccattaa ccaatactgg aactggtatc tagctactga 1500

tgtcttactt tgagtttatt tatgcttcag aatacagttg tttgccctgt gcatgaatat 1560

acccatattt gtgtgtggat atgtgaagct tttccaaata gagctctcag aagaattaag 1620

tttttacttc taattatttt gcattacttt gagttaaatt tgaatagagt attaaatata 1680

aagttgtaga ttcttatgtg tttttgtatt agcccagaca tctgtaatgt ttttgcactg 1740

gtgacagaca aaatctgttt taaaatcata tccagcacaa aaactatttc tggctgaata 1800

gcacagaaaa gtattttaac ctacctgtag agatcctcgt catggaaagg tgccaaactg 1860

ttttgaatgg aaggacaagt aagagtgagg ccacagttcc caccacacga gggcttttgt 1920

attgttctac tttttcagtc ctttactttc tggctgaagc atccccttgg agtgccatgt 1980

ataagttggg ctattagagt tcatggaaca tagaacaacc atgaatgagt ggcatgatcc 2040

gtgcttaatg atcaagtgtt acttatctaa taatcctcta gaaagaaccc tgttagatct 2100

tggtttgtga taaaaatata aagacagaag acatgaggaa aaacaaaagg tttgaggaaa 2160

tcaggcatat gactttatac ttaacatcag atcttttcta taatatccta ctactttggt 2220

tttcctagct ccataccaca cacctaaacc tgtattatga attacatatt acaaagtcat 2280

aaatgtgcca tatggatata cagtacattc tagttggaat cgtttactct gctagaattt 2340

aggtgtgaga ttttttgttt cccaggtata gcaggcttat gtttggtggc attaaattgg 2400

tttctttaaa atgctttggt ggcacttttg taaacagatt gcttctagat tgttacaaac 2460

caagcctaag acacatctgt gaatacttag atttgtagct taatcacatt ctagacttgt 2520

gagttgaatg acaaagcagt tgaacaaaaa ttatggcatt taagaattta acatgtctta 2580

gctgtaaaaa tgagaaagtg ttggttggtt ttaaaatctg gtaactccat gatgaaaaga 2640

aatttatttt atacgtgtta tgtctctaat aaagtata 2678

<210> SEQ ID NO 6

<211> LENGTH: 1585

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE: -

<223> OTHER INFORMATION: 1698542

<400> SEQUENCE: 6

tgggctcggg tgtgggggcc ctagctcgcc ctctctgggt gggtgggtga gagaggctgc 60

aggggaggct ggcaccgggc ttgggcctcc agtccctgcc ttgctgactt tggccgttgc 120

ggtatcacgt gacaatcaac tctcccttaa cgacgactgc ggctgttggg ctgcgctgcc 180

ctccctggcc tgcaggtggg gccgtggagc ccatgcccgc cggggttgca ggctcttccg 240

ggcctgggct gcagcctggc ccggccctgc ttccctggcc tcccctcctt ctcctgggtg 300

cttggaagag gtgggtcctg gcccgtggtt gaggtcttgc ctcgtggact ggaagcagag 360

ggctggctgt gcagcctcct ggggtagctc gttttttttg caccgggtag aatatttttt 420

caggcacgga ttcctttttt tctgggcccc catgggtggt ccggcatcct cagggggtgt 480

gagtgtgtgt ggggggtctc tgagctgaac ttgggtgggg tggggacttg ttcctcgggg 540

gccacctttg tgtccttgtc agcggtcgtc ctgctgtggc ctgggttgca tttcctcttg 600

gggggggtat tgaggacccc cagcctggaa tgagaagggt ccccggttcc atgtcagacc 660

cagaaaggtg gatcccccca ctgttggctg caggaggttt ttggtacccc cttttgttcc 720

agaaccgtcc tgcctctcgc ttggggacag gggggctttg gatggcactg gtgtgcacct 780

ggacccagcc ccggcctggc aggatccagg gatgggtgcc tgggatcctg gggggaggga 840

ggcaggggac gcccttctgg agctgggctc ggagggtcct gccccatcca gccctcggct 900

ctctgtgttc tctgtcccta gcctcaaacc ctcttctggc agcgctagtg agatgcctta 960

gtctgtgggg gtgggtgggg gactggggcc ccgttttcct ttgtgagtct tggttggccc 1020

cagccctggc aggggcctgt ctggagcagg ggtaggtagg gctgtgggtt ccagaactct 1080

ataactgggc ccctctccag tgtccagggg cttggagaga cctgcgccca cccctccact 1140

ctcccaggag tcgctgatca ctgggacctg gttccagccg tttgcagggg aggcggctcc 1200

gagaggcgtg tgctttccgt gggtggcgcc cctcccccgc cctgcctggc ctcatccttg 1260

tatttaatta attaaacaag ccccttttta aaccctaaaa aaaaaaggca gaagcaacat 1320

aacaaacagg aaacctataa gaaagaggaa gcacgtgaga aaaaaaaaaa gggggggccc 1380

ctctgggggt tccgggctta ggtgcgcttg catggggggt caaagctctt ctaaaggtgc 1440

tccctaattt tggattcagt gggcgggggt tttagaggct ccgtgacctg ggaaaaaccc 1500

ctggggttta gccgcatctt aaatcggctt ttggggggga attcccccct tttggggggg 1560

ttgggggtta aatggcgcag ggggg 1585

<210> SEQ ID NO 7

<211> LENGTH: 828

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE: -

<223> OTHER INFORMATION: 1962202

<400> SEQUENCE: 7

aatttctaac ctttggttgc ctttagtcaa agaaagagct tagatttaca tgcacatgtt 60

taaaaaattc agcaaatcta tgactgccat aaactctgca tgacatacac agtgtcagca 120

agttactgct atgttaggct gtaaattatt cattccatac cttgcacaag aaaatatatt 180

ccccttttta agaaagaaaa gttcacaact taggtgactg gaaatcaaag tattggcttc 240

taacctattt taaactaaga aatagtgatt tattaaatgt taaagttttc acaatttgat 300

gactagtccc ttttaaatat cccctataaa ttattagcaa tgatatcacc attctaaaat 360

gttataaatg taggaaacat tttgcctttt gtagaaattc atgaaaatct tcatttcatg 420

gtggagtaat ttgagaaaga agcacagaaa agtagacttg gcaacaaatg ctatgaaatg 480

tgacttttgt atactcaaaa cagacgaacc tttaactaaa taactctaca ttaaccccca 540

aaattctgtt gaaaattctg tctttgtgcc ttcttaaatg taatattgag tatctaagat 600

ggaatggtca agtagcctca agtattttat taagagagaa aaataacagt ggctaacatt 660

ttaaatatat atcttcccaa ccacttaacc tttatacata tttttgttga aaataaacaa 720

tgggtctcct gagaagttgt atccagacca gagaaaggga agtttaaaga ataggcctac 780

acctattgtt tctcagaggt tgtgtttttg ttttctttgt gggttttc 828

<210> SEQ ID NO 8

<211> LENGTH: 1892

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE: -

<223> OTHER INFORMATION: 2083433

<400> SEQUENCE: 8

cttttttgtg gtttcctgtg aagtgagcgt ttcccttgca catggctgct ttggtgcttt 60

ggcggctgtt ccaggggccg ttgcaaaacg ctcgtgcaag gagcacagct gcagccttgt 120

cctctgcagt aactcctccc agcacctctc tcacaccctt gttcccaaca gaacgtgttt 180

gtgcaactgt ccttggcctt tagaaatgac agctacactc tggaatctag aattaaccag 240

gctgaaaggg aacgcaacct gacagaggag aacactgaga aagaactgga aaacttcaaa 300

gcttccatta cgtcctcagc ttcactctgg caccactgtg agcaccggga aacctaccag 360

aagttgctgg aggacatcgc tgtcctgcac cgcctggctg cccgcctctc cagccgagct 420

gaggtggtag gcgccgtcca ccaggaaaag cgcatgtcga aagcaacgga agtgatgatg 480

cagtatgtgg agaatctaaa gaggacgtat gagaaggacc atgcggagct catggagttt 540

aaaaagcttg caaatcagaa ttcaagccgc agctgtggcc cctctgaaga tggggtccct 600

cgcacggcac ggtccatgtc cctcacgctg ggaaagaata tgcctcgccg gaggtcagcg 660

ttgctgtggt tcctaagttt aatgccctga atctgcctgg ccaaactccc agctcatcat 720

ccattccctc cttaccagcc ttgtcggaat cacccaatgg gaaaggcagc ctacctgtca 780

cttcagcact gcctgcactt ttggaaaatg gaaagacaaa tggggaccca gattgtgaag 840

cctctgctcc tgcgctgacc ctgagctgcc tggaggagct tagtcaggag accaaggcca 900

ggatggagga agaagcctac agcaagggat tccaagaagg tctaaagaag accaaagaac 960

ttcaagacct gaaggaggag gaggaagaac agaagagtga gagtcctgag gaacctgaag 1020

aggtagaaga aactgaggaa gaggaaaagg gcccaagaag cagcaaactt gaagaattgg 1080

tccatttctt acaagtcatg tatcccaaac tgtgtcagca ctggcaagtg atctggatga 1140

tggctgcagt gatgctggtc ttgactgttg tgctggggct ctacaattcc tataactctt 1200

gtgcagagca ggctgatggg ccccttggaa gatccacttg ctcggcagcc cagagggact 1260

cctggtggag ctcaggactc cagcatgagc agcctacaga gcagtaggaa acctcacacc 1320

tagccagtgc cctgctctga gacactcaga ctaccaccct ttccccaagt ataacgtcag 1380

gcccaagtgt ggacacactg ccgcccatcc catcaggtca tgaggaaggg ttcttttaac 1440

actcggcact tctgtgggag ctattcatac acagtgactt gatgttcttg gaggatcaac 1500

aaaactgccc tgggaaagca tccagtggat gaagaagtca ccttcaccaa ggaactctat 1560

tggaagggaa ggtctcctgc ccctagctca ggtggctggg gagaactaaa acaccttcac 1620

tggtggttgg gggtaaggag cggggcacgg gggaggagga ggtagggggc agtaaaaaac 1680

ttactctctt ttttcctctc tgtaattggt tatcaggaag aatttgctta atgactaaca 1740

ccctaagcat cagacctgga atttggagtt gcaaagtgac tatcttccca tttcccatct 1800

cattttcaat aacttcagcc tcccattctt tcctttggaa tgagagtttc tttttacaga 1860

agtaggaaag gcttctcaaa aaaaaaaaaa aa 1892

<210> SEQ ID NO 9

<211> LENGTH: 2055

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE: -

<223> OTHER INFORMATION: 2227688

<400> SEQUENCE: 9

agagcaggcg tcgggacgca gcaaagagag gagagacccc agagtcagaa ggagtgagaa 60

ccctgacccc taatcccact gcatccagcc aataggagcc cagccaccat ggcggagctg 120

caggaggtgc agatcacaga ggagaagcca ctgttgccag gacagacgcc tgaggcggcc 180

aaggaggctg agttagctgc ccgaatcctc ctggaccagg gacagactca ctctgtggag 240

acaccatacg gctctgtcac tttcactgtc tatggcaccc ccaaacccaa acgcccagcg 300

atccttacct accacgatgt gggactcaac tataaatctt gcttccagcc actgtttcag 360

ttcgaggaca tgcaggaaat cattcagaac tttgtgcggg ttcatgtgga tgcccctgga 420

atggaagagg gagcccctgt gttccctttg ggatatcagt acccatctct ggaccagctt 480

gcagacatga tcccttgcgt cctgcagtac ctaaatttct ctacaataat tggagttggt 540

gttggagctg gagcctacat cctggcgaga tatgctctta accacccgga cactgttgaa 600

ggtcttgtcc tcatcaacat tgatcccaat gccaagggtt ggatggattg ggcagcccac 660

aagctaacag gcctcacctc ttccattccg gagatgatcc ttggacatct tttcagccag 720

gaagagctct ctggaaattc tgagttgata caaaagtaca gaaatatcat tacacatgca 780

cccaacctgg ataacattga attgtactgg aacagctaca acaaccgccg agacctgaac 840

tttgagcgtg gaggtgatat caccctcagg tgtcctgtga tgctggtggt aggagaccaa 900

gcacctcatg aagatgcagt ggtggaatgt aactcaaaac tggaccccac ccagacctcg 960

ttcctcaaga tggctgactc cggaggtcag ccccagctga ctcagccagg caagctgacc 1020

gaggccttca agtacttcct gcaaggcatg ggctacatgg cctcatcctg catgactcgc 1080

ctgtcccggt ctcgtacagc ctctctgacc agtgcagcat ccgttgatgg caaccggtcc 1140

cgctctcgca ccctgtccca gagcagcgag tctggaactc tttcttcggg gcccccgggg 1200

cacaccatgg aggtctcctg ttgaatggcc cttgttgccc tagagtggga cccagccctc 1260

acctccccca gagctaacct gggaggtgct gaaggggcat tgggccaccg taagcaaggg 1320

aaaaagggca gatcatgcgg ggagatgacc ttgatctttg attgctaccc taaccttgac 1380

ctttaacccg tgattccccc cagctcctgg aagagatgtc ctaatatctc ttagggaccc 1440

agacccctaa attctcctcc tcccccattt tgatgttaag gtggagaggg catatgcatc 1500

ctctgtcctg atctaggtgt ctatagctga ggggtaagag gttgttgtag ttgtcctggt 1560

gcctccatca gactctccct acttgtccca tatttgcaag gggaggggat ttggggctgg 1620

ggctccattc accaaagctg aggtggcttc tcattaaccc tttaggactc tgaagggtat 1680

ggacctacgt gaatgtgtgt cagggggaga cttgctggtg ggttagtggt cctcaggatg 1740

tgatagaaac atccagtgta aaaaggaagt tggaatggga gttggcgggc agtgaacgag 1800

tgtggggaag gattggtgct ggggcaacag gaaggggcct ggggccgttt ggctgcacta 1860

actttggtag ctcagtgtgc atctagagtg ggactgggga gggagctaag cttgggctgg 1920

gctgcttggg gcttggcata gggtggaaag ggctaccctg gggctctgac cacactgtag 1980

tatgtgtgga gggtgccctc ccgtctccca caacttctgc tataacaata aactgtagag 2040

gaatctgaaa aaaaa 2055

<210> SEQ ID NO 10

<211> LENGTH: 2319

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<221> NAME/KEY: unsure

<222> LOCATION: 20, 21, 23, 25, 26, 28, 32, 34

<223> OTHER INFORMATION: a or g or c or t, unknown, or other

<220> FEATURE: -

<223> OTHER INFORMATION: 2313925

<400> SEQUENCE: 10

caggtcgact ctagagcaan ngncnncnac cncnaccacc ccctccacca ccacgacagc 60

caggagctgc cccagccccc ttacagttct cctatcagac ttgtgagctg ccaagcgctg 120

cttcccctgc gccagactat cccactccct gtcagtatcc tgtggatgga gcccagcaga 180

gcgacctaac ggggccagac tgtcccagaa gcccaggact gcaagaggcc ccctccagct 240

acgacccact agccctctct gagctacctg gactctttga ttgtgaaatg ctagacgctg 300

tggatccaca acacaacggg tatgtcctgg tgaattagtc tcagcacagg aattgaggtg 360

ggtcaggtga aggaagagtg tatgttccta tttttattcc agccttttaa atttaaagct 420

tattttcttg ccctctccct aacggggaga aatcgagcca cccaactgga atcagagggt 480

ctggctgggg tggatgttgc ttcctcctgg ttctgcccca ccacaaagtt ttctgtggca 540

agtgctggaa catagttgta ggctgaggct cctgcccttc ggtcgagtgg agcaagctct 600

cgagggcagc actgacaaat gtgttcctaa gaagacattc agacccaggt cttatgcagg 660

attacatccg tttattatca agggcaacct tggtgaaagc agaaagggtg tgtgctattg 720

catatatatg ggggaaaagg caatatattt ttcactgaag ctgagcaacc acatattgct 780

acaaggcaaa tcaagaagac atcaggaaat cagatgcaca ggaaataaag gaaagctgtg 840

ctttgtcatt gaatcctaag ttcttagctg ctgatgcaag ttgtccccca aggccatcac 900

aaagcagtgg ggcatgagct gtgtttcagg ggccactaaa taacagctgg tactgacccc 960

agaaaccgcc ttcatctcca ttcggaagca ggtgacacac cccttcagaa ggtgccctgg 1020

gttgccgagt gtcagaatat actcaggact ccagaggtgt cacacgtgga actgacagga 1080

gacccgccac cgtggaggca gggggcaaga aactcaagaa cgcatcaaga gcaccagccc 1140

tgggccaggg aagacaggct cttcctgcag tttctcgtgg acactgctgg cttgcgggca 1200

gtcggtctcc agggtacctg ttgtctcttt tccgatgtaa taactacttt gaccttacac 1260

tatatgttgc tagtagttta ttgagctttg tatatttgga cagtttcata tagggcttag 1320

agattttaag gacatgataa atgaactttt ctgtcccatg tgaagtggta gtgcggtgcc 1380

tttcccccag atcatgcttt aattctttct tttctgtaga aaccaacagt ttccatttat 1440

gtcaatgcta aatccaaagt cacttcagag tttgttttcc accatgtggg aatcagcatt 1500

cttaatttcg ttaaagtttt gacttgtaat gaaatgttca agtattacag caatattcaa 1560

agaaagaacc acagatgtgt taaccattta agcagatcat ctgccaaaca ttatattact 1620

aataaaactt aaccaacact tacaattcag tcatcaaagt aagtaaaaat tagatgctac 1680

agctagctaa ctgtatccct agaaatgatg aataatttgc catttggaca gttaacatcc 1740

aggtgttaca aagtcagtgt taattctaaa gatgatcatt tctgcccttt agaatggctt 1800

gtcccatcag cagatgaatg tgttaagcac aaagcatctt ccttaaagca caaagagagg 1860

gactaactga tgctgcatct agaaaacacc tttaagttgc ctttcctctt tgtagttagc 1920

gttcaggcag gtgacgtgtg gaaagtctag ggggttccat tctggccatg cgagcccagc 1980

tcctaccaac gtcggtaact tgagcagtcc ctgttgctgg ccagagactg cctggtcgcc 2040

agcgctcacc atgggtgcca ggatgcttcg cagaggcact gtgctcacgg ttggacttgg 2100

tgtcagtggg aaagggcagt gtggggactg tcatttttgt gatttaataa cacacagtga 2160

aaatccagga agaatgaatt aagcttcttc tgggagttgt ttattcctgc tcgtgcttaa 2220

gattgatgat ttcgtgaaat aaagaacaat catttcattt atgagatcat ttcattaaga 2280

tctctaatct gttttgagtc tttacaaaat agccagtta 2319

<210> SEQ ID NO 11

<211> LENGTH: 668

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE: -

<223> OTHER INFORMATION: 2507107

<400> SEQUENCE: 11

gcttcttctt tcttctgctc ataaaaggag gaacacttta gatagagggc aaatatatct 60

gaaaacctaa tttctttctt tttttgataa ggaaatcttt tccatctcca tcctaacatg 120

cacaacctgt gaagagaatt gtttctatag taactggtct gtgatctttt gtggccaaga 180

gaatagcagg caagaattag ggccttgaca gaatttccac gaagctctga gaacatgttt 240

gtttcgaatg tctgattcct ctttgtcatc aatgtgtatg ctctgtcccc atccttcact 300

cctcctcaag ctcacaccaa ttggtttggc acaggcacag agctggtccc tagttaagtg 360

gcatttatgt taaaaaaaaa tagttcagaa tctcagcctt ttctttgtgt catcaaaaca 420

gcttaagaag gggactactg ccaatgtcct ctagtctgac ctccacccag ggaggaccca 480

tggcaggtct tttcaacttt ctgattcatg agaacaacct tgtgaagctt ttcccacctc 540

ctaaagtgtt ttctgcatct gttccttcct ttggacctca caacaaatcc tgtgaagtaa 600

ctgagacatc tgttgttaga tacatttttg tgatgagtaa actgaggctt cgtgattcaa 660

aaaaaaaa 668

<210> SEQ ID NO 12

<211> LENGTH: 2112

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<221> NAME/KEY: unsure

<222> LOCATION: 4, 9, 13, 27, 35, 37, 62, 88, 95, 102, 120, 173

<221> NAME/KEY: unsure

<222> LOCATION:

<223> OTHER INFORMATION: a or g or c or t, unknown, or other

<220> FEATURE: -

<223> OTHER INFORMATION: 2544503

<400> SEQUENCE: 12

aacnaattng ggncccggtt ttcgggnatt tggtngnggt ttaggagtgg gcctttttca 60

ancttccctg gtaggagact tcccttgntg gaaancaact cntaaacttc caggcctccn 120

cccaagctca ctttatttaa tgcaaattcc accaaatcaa gagtggggaa tcnaagcaaa 180

tgcattcaaa aaactggata acctcacctt cctctacttg gaccataatg ccctggaatc 240

cgtgcctctt aatttaccag aaagtctacg tgtaattcat cttcagttca acaacatagc 300

ttcaattaca gatgacacat tctgcaaggc taatgacacc agttacatcc gggaccgcat 360

tgaagagata cgcctggagg gcaatccaat cgtcctggga aagcatccaa acagttttat 420

ttgcttaaaa agattaccga tagggtcata cttttaacct ctattggtac aacatataaa 480

tgaaagtaca cctacactaa tagtctgtct caacaatgtg taaaggaact taagtattgg 540

tttaatatta accttgtatc tcattttgaa ggaatttaat attttaagca aggatgttca 600

aaatcttaca tataataagt aaaaagtaag actgaatgtc tacgttcgaa acaaagtaat 660

atgaaaatat ttaaacagca ttacaaaatc ctagtttata ctagactacc atttaaaaat 720

catgttttta tataaatgcc caaatttgag atgcattatt cctattacta atgatgtaag 780

tacgaggata aatccaagaa actttcaact ctttgccttt cctggccttt actggatccc 840

aaaagcattt aaggtacatg ttccaaaaac tttgaaaagc taaatgtttc ccatgatcgc 900

tcattcttct tttatgattc atacgttatt ccttataaag taagaacttt gttttcctcc 960

tatcaaggca gctattttat taaatttttc acttagtctg agaaatagca gatagtctca 1020

tatttaggaa aactttccaa ataaaataaa tgttattctc tgataaagag ctaatacaga 1080

aatgttcaag ttattttact ttctggtaat gtcttcagta aaatattttc tttatctaaa 1140

tattaacatt ctaagtctac caaaaaaagt tttaaactca agcaggccaa aaccaatatg 1200

cttataagaa ataatgaaaa gttcatccat ttctgataaa gttctctatg gcaaagtctt 1260

tcaaatacga gataactgca aaatattttc cttttatact acagaaatga gaatctcatc 1320

aataaattag ttcaagcata agatgaaaac agaatattct gtggtgccag tgcacactac 1380

cttcccaccc atacacatcc atgttcactg taacaaactg aatattcaca ataaagcttc 1440

tgagtaacac tttctgatta ctcatgataa actgacatgg ctaactgcaa gaattaaatc 1500

ttctatctga gagtaataat ttatgatgac tcagtggtgc cagagtaaag tttctaaaat 1560

aacattcctc tcacttgtac cccactaaaa gtattagtct acacattaca ttgaagttaa 1620

acacaaaatt atcagtgttt tagaaacatg agtccggact gtgtaagtaa aagtacaaac 1680

attatttcca ccataaagta tgtattgaaa tcaagttgtc tctgtgtaca gaatacatac 1740

ttattcccat ttttaagcat ttgcttctgt tttccctacc tagaatgtca gatgtttttc 1800

agttatctcc ccatttgtca aagttgacct caagataaca tttttcatta aagcatctga 1860

gatctaagaa cacaattatt attctaacaa tgattattag ctcattcact tattttgata 1920

actaatgatc acagctatta tactactttc tcgttatttt gtgtgcatgc ctcatttccc 1980

tgacttaaac ctcactgaga gcgcaaaatg cagctttata ctttttactt tcaattgcct 2040

agcacaatag tgagtacatt tgaattgaat atataataaa tattgcaaaa taaaatccat 2100

ctaaataaaa aa 2112

<210> SEQ ID NO 13

<211> LENGTH: 2029

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE: -

<223> OTHER INFORMATION: 3044710

<400> SEQUENCE: 13

ccttgacaag tcagaagctt gaaagcaggg aaatccggat gtctcggtta tgaagtggag 60

cagtgagtgt gagcctcaac atagttccag aactctccat ccggactagt tattgagcat 120

ctgcctctca tatcaccagt ggccatctga ggtgtttccc tggctctgaa ggggtaggca 180

cgatggccag gtgcttcagc ctggtgttgc ttctcacttc catctggacc acgaggctcc 240

tggtccaagg ctctttgcgt gcagaagagc tttccatcca ggtgtcatgc agaattatgg 300

ggatcaccct tgtgagcaaa aaggcgaacc agcagctgaa tttcacagaa gctaaggagg 360

cctgtaggct gctgggacta agtttggccg gcaaggacca agttgaaaca gccttgaaag 420

ctagctttga aacttgcagc tatggctggg ttggagatgg attcgtggtc atctctagga 480

ttagcccaaa ccccaagtgt gggaaaaatg gggtgggtgt cctgatttgg aaggttccag 540

tgagccgaca gtttgcagcc tattgttaca actcatctga tacttggact aactcgtgca 600

ttccagaaat tatcaccacc aaagatccca tattcaacac tcaaactgca acacaaacaa 660

cagaatttat tgtcagtgac agtacctact cggtggcatc cccttactct acaatacctg 720

cccctactac tactcctcct gctccagctt ccacttctat tccacggaga aaaaaattga 780

tttgtgtcac agaagttttt atggaaacta gcaccatgtc tacagaaact gaaccatttg 840

ttgaaaataa agcagcattc aagaatgaag ctgctgggtt tggaggtgtc cccacggctc 900

tgctagtgct tgctctcctc ttctttggtg ctgcagctgg tcttggattt tgctatgtca 960

aaaggtatgt gaaggccttc ccttttacaa acaagaatca gcagaaggaa atgatcgaaa 1020

ccaaagtagt aaaggaggag aaggccaatg atagcaaccc taatgaggaa tcaaagaaaa 1080

ctgataaaaa cccagaagag tccaagagtc caagcaaaac taccgtgcga tgcctggaag 1140

ctgaagttta gatgagacag aaatgaggag acacacctga ggctggtttc tttcatgctc 1200

cttaccctgc cccagctggg gaaatcaaaa gggccaaaga accaaagaag aaagtccacc 1260

cttggttcct aactggaatc agctcaggac tgccattgga ctatggagtg caccaaagag 1320

aatgcccttc tccttattgt aaccctgtct ggatcctatc ctcctacctc caaagcttcc 1380

cacggccttt ctagcctggc tatgtcctaa taatatccca ctgggagaaa ggagttttgc 1440

aaagtgcaag gacctaaaac atctcatcag tatccagtgg taaaaaggcc tcctggctgt 1500

ctgaggctag gtgggttgaa agccaaggag tcactgagac caaggctttc tctactgatt 1560

ccgcagctca gaccctttct tcagctctga aagagaaaca cgtatcccac ctgacatgtc 1620

cttctgagcc cggtaagagc aaaagaatgg cagaaaagtt tagcccctga aagccatgga 1680

gattctcata acttgagacc taatctctgt aaagctaaaa taaagaaata gaacaaggct 1740

gaggatacga cagtacactg tcagcaggga ctgtaaacac agacagggtc aaagtgtttt 1800

ctctgaacac attgagttgg aatcactgtt tagaacacac acacttactt tttctggtct 1860

ctaccactgc tgatattttc tctaggaaat atacttttac aagtaacaaa aataaaaact 1920

cttataaatt tctattttta tctgagttac agaaatgatt actaaggaag attactcagt 1980

aatttgttta aaaagtaata aaattcaaca aacatttaaa aaaaaaaaa 2029

<210> SEQ ID NO 14

<211> LENGTH: 1726

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<221> NAME/KEY: unsure

<222> LOCATION: 1213, 1353, 1366, 1459, 1460, 1463, 1469, 1482, 1512,

1524,

<221> NAME/KEY: unsure

<222> LOCATION: 1557, 1567, 1583, 1599, 1619, 1638, 1649, 1662, 1696,

1708,

<221> NAME/KEY: unsure

<222> LOCATION: 1715

<223> OTHER INFORMATION: a or g or c or t, unknown, or other

<220> FEATURE: -

<223> OTHER INFORMATION: 3507515

<400> SEQUENCE: 14

aggcgccgta ggctggaagc gccagcgctg ccggcgggcg gtgtgattga tccgcgtccc 60

ctggagctgg aggctcgggg gaaagggcca gcacggagcg ggcgctcggt tgctgcgcac 120

aaaggctgag gctccaagag ctgcagggcg tgtttgggac cccagagtca gaaggagtga 180

gaaccctgac ccctaatccc actgcatcca gccaatagga gcccagccac catggcggag 240

ctgcaggagg tgcagatcac agaggagaag ccactgttgc caggacagac gcctgaggcg 300

gccaagactc actctgtgga gacaccatac ggctctgtca ctttcactgt ctatggcacc 360

cccaaaccca aacgcccagc gatccttacc taccacgatg tgggactcaa ctataaatct 420

tgcttccagc cactgtttca gttcgaggac atgcaggaaa tcattcagaa ctttgtgcgg 480

gttcatgtgg atgcccctgg aatggaagag ggagcccctg tgttcccttt gggatatcag 540

tacccatctc tggaccagct tgcagacatg atcccttgcg tcctgcagta cctaaatttc 600

tctacaataa ttggagttgg tgttggagct ggagcctaca tcctggcgag atatgctctt 660

aaccacccgg acactgttga aggtcttgtc ctcatcaaca ttgatcccaa tgccaagggt 720

tggatggatt gggcagccca caagctaaca ggcctcacct cttccattcc ggagatgatc 780

cttggacatc ttttcagcca ggaagagctc tctggaaatt ctgagttgat acaaaagtac 840

agaaatatca ttacacatgc acccaacctg gataacattg aattgtactg gaacagctac 900

aacaaccgcc gagacctgaa ctttgagcgt ggaggtgata tcaccctcag gtgtcctgtg 960

atgctggtgg taggagacca agcacctcat gaagatgcag tggtggaatg taactcaaaa 1020

ctggacccca cccagacctc gttcctcaag atggctgact ccggaggtca gccccagctg 1080

actcagccag gcaagctgac cgaggccttc aagtacttcc tgcaaggcat gggctacatg 1140

gcctcatcct gcatgactcg cctgtcccgg tctcgtacag cctctctgac cagtgcagca 1200

tccgttgatg gcnaccggtc ccgctctcgc accctgtccc agagcagcga gtctggaact 1260

cttttcttcg ggggcccccg ggggcacacc atgggaggtc tcctgttgaa tggcccttgt 1320

tgccctagag tgggacccag ccctcagctc ccncagagta acctgngagg tgctgaaagg 1380

gggcattggg gccaccgtaa gcaaagggga aaaagggcag attcatggcg ggggagatga 1440

ccttgattct ttgaattgnn aancctaanc ttgaacttta anccgtgatt cccccccagc 1500

tcctgggaag angaggtcct aatnatctct taagggaccc cagaacccct aaaattnctc 1560

cgtcttnccc cattttgaag gtnaaagggg aaaaggggna tatggaatcc tctgttccng 1620

gatttaaggg gtccaaangt tgagggggna aaaggttgtg gnaattggtc cctggtggct 1680

ccatcaagaa tttccnaaat tgtcccanat tttgnaaggg gggggt 1726

<210> SEQ ID NO 15

<211> LENGTH: 757

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<221> NAME/KEY: unsure

<222> LOCATION: 596, 606, 613, 636, 639, 655, 659, 671, 676, 677, 682, 700,

<221> NAME/KEY: unsure

<222> LOCATION: 708, 710, 728, 731, 742, 755

<223> OTHER INFORMATION: a or g or c or t, unknown, or other

<220> FEATURE: -

<223> OTHER INFORMATION: 3540909

<400> SEQUENCE: 15

ggaggcgctt cggctccgga ctacgctcct gctgtgcgct cgcggggcca gcagtgctgg 60

cttctgcagt aggaggcgcg ggggcatggc gcagaggctg ggcgagtggg cccgggggcc 120

ctccgatgcc accgggctct accgggctgt gctgctccgg tcggccgcca tgtacttcgg 180

agatccagag ggagcgggca gacattgggg gcctgatggc ccggccagaa tacagagagt 240

ggaatccgga gctcatcaag cccaagaagc tgctgaaccc cgtgaaggcc tctcggagtc 300

accaggagct ccaccgggag ctgctcatga accacagaag gggccttggt gtggacagca 360

agccagagct gcagcgtgtc ctagagcacc gccggcggaa ccagctcatc aagaagaaga 420

aggaggagct ggaagccaag cggctgcagt gcccctttga gcaggagctg ctgagacggc 480

agcagaggct gaaccagctg gaaaaaccac cagagaagga agaggatcac gcccccgagt 540

ttattaaagt cagggaaaac ctgcggagaa ttgccacact gaaccagcga agaganagag 600

ttttanggcc agntgccggg ctcaaggcca ttgccnacnt tgggcttgaa aatcnttcnt 660

taagcctttc ngtacnngga anccttgggg ccccaggccn tgggaacntn tgagattttc 720

ccaactgntt ntgtagaaat gngcaccccc cgttntt 757

<210> SEQ ID NO 16

<211> LENGTH: 2430

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE: -

<223> OTHER INFORMATION: 3688209

<400> SEQUENCE: 16

gcgctcctct agctgggagg tggaagcagc attgcccaag cctcccagga gtgacaggaa 60

ttgtttctgc ctgaggagac actctgcagc ctgggctctg tgagactgag gtggcggtca 120

gccggagtga gtgttggggt cctggggcac ctgccttaca tggcttgttt atgaacatta 180

aagggaagaa gttgaagctt gaggagcgag gatggcagtc aacaaaggcc tcaccttgct 240

ggatggagac ctccctgagc aggagaatgt gctgcagcgg gtcctgcagc tgccggtggt 300

gagtggcacc tgcgaatgct tccagaagac ctacaccagc actaaggaag cccaccccct 360

ggtggcctct gtgtgcaatg cctatgagaa gggcgtgcag agcgccagta gcttggctgc 420

ctggagcatg gagccggtgg tccgcaggct gtccacccag ttcacagctg ccaatgagct 480

ggcctgccga ggcttggacc acctggagga aaagatcccc gccctccagt acccccctga 540

aaagattgct tctgagctga aggacaccat ctccacccgc ctccgcagtg ccagaaacag 600

catcagcgtt cccatcgcga gcacttcaga caaggtcctg ggggccgctt tggccgggtg 660

cgagcttgcc tggggggtgg ccagagacac tgcggaattt gctgccaaca ctcgagctgg 720

ccgactggct tctggagggg ccgacttggc cttgggcagc attgagaagg tggtggagta 780

cctcctccct gcagacaagg aagagtcagc ccctgctcct ggacaccagc aagcccagaa 840

gtctcccaag gccaagccaa gcctcttgag cagggttggg gctctgacca acaccctctc 900

tcgatacacc gtgcagacca tggcccgggc cctggagcag ggccacaccg tggccatgtg 960

gatcccaggc gtggtgcccc tgagcagcct ggcccagtgg ggtgcctcag tggccatgca 1020

ggcggtgtcc cggcggagga gcgaagtgcg ggtaccctgg ctgcacagcc tcgcagccgc 1080

ccaggaggag gatcatgagg accagacaga cacggaggga gaggacacgg aggaggagga 1140

agaattggag actgaggaga acaagttcag tgaggtagca gccctgccag gccctcgagg 1200

cctcctgggt ggtgtggcac ataccctgca gaagaccctc cagaccacca tctcggctgt 1260

gacatgggca cctgcagctg tgctgggcat ggcagggagg gtgctgcacc tcacaccagc 1320

ccccgctgtc tcctcaacca aggggagggc catgtcccta tcagatgccc tgaagggcgt 1380

tactgacaac gtggtggaca cagtggtgca ttacgtgccg ctccccaggc tgtcgctgat 1440

ggagcccgag agcgaattcc gggacatcga caacccacca gccgaggtcg agcgccggga 1500

ggcggagcgc agagcgtctg gggcgccgtc cgccggcccg gagcccgccc cgcgtctcgc 1560

acagccccgc cgcagcctgc gcagcgcgca gagccccggc gcgccccccg gcccgggcct 1620

ggaggacgaa gtcgccacgc ccgcagcgcc gcgcccgggc ttcccggccg tgccccgcga 1680

gaagccaaag cgcagggtca gcgacagctt cttccggccc agcgtcatgg agcccatcct 1740

gggccgcacg cattacagcc agctgcgcaa gaagagctga gtcgccgcac cagccgccgc 1800

gccccgggcc ggcgggtttc tctaacaaat aaacagaacc cgcactgccc aggcgagcgt 1860

tgccactttc aaagtggtcc cctggggagc tcagcctcat cctgatgatg ctgccaaggc 1920

gcacttttta tttttatttt atttttattt tttttttagc atccttttgg ggcttcactc 1980

tcagagccag tttttaaggg acaccagagc cgcagcctgc tctgattcta tggcttggtt 2040

gttactataa gagtaattgc ctaacttgat ttttcatctc tttaaccaaa cttgtggcca 2100

aaagatattt gaccgtttcc aaaattcaga ttctgcctct gcggataaat atttgccacg 2160

aatgagtaac tcctgtcacc actctgaagg tccagacaga aggttttgac acattcttag 2220

cactgaactc ctctgtgatc taggatgatc tgttccccct ctgatgaaca tcctctgatg 2280

atctaggctc ccagcaggct actttgaagg gaacaatcag atggcaaaag ctcttgggtg 2340

tttatttaaa atactagtgt cactatctga gtacccgccg cttcacaggc tgagtccagc 2400

ctgtgtgcta tgtagagcag ctgcttgctc 2430

<210> SEQ ID NO 17

<211> LENGTH: 298

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE: -

<223> OTHER INFORMATION: 159452

<400> SEQUENCE: 17

Met Lys Thr Leu Gln Ser Thr Leu Leu Leu Leu Leu Leu Val Pro

1 5 10 15

Leu Ile Lys Pro Ala Pro Pro Thr Gln Gln Asp Ser Arg Ile Ile

20 25 30

Tyr Asp Tyr Gly Thr Asp Asn Phe Glu Glu Ser Ile Phe Ser Gln

35 40 45

Asp Tyr Glu Asp Lys Tyr Leu Asp Gly Lys Asn Ile Lys Glu Lys

50 55 60

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

65 70 75

Asp Glu Ala Ile Thr Pro Leu Pro Pro Lys Lys Glu Asn Asp Glu

80 85 90

Met Pro Thr Cys Leu Leu Cys Val Cys Leu Ser Gly Ser Val Tyr

95 100 105

Cys Glu Glu Val Asp Ile Asp Ala Val Pro Pro Leu Pro Lys Glu

110 115 120

Ser Ala Tyr Leu Tyr Ala Arg Phe Asn Lys Ile Lys Lys Leu Thr

125 130 135

Ala Lys Asp Phe Ala Asp Ile Pro Asn Leu Arg Arg Leu Asp Phe

140 145 150

Thr Gly Asn Leu Ile Glu Asp Ile Glu Asp Gly Thr Phe Ser Lys

155 160 165

Leu Ser Leu Leu Glu Glu Leu Ser Leu Ala Glu Asn Gln Leu Leu

170 175 180

Lys Leu Pro Val Leu Pro Pro Lys Leu Thr Leu Phe Asn Ala Lys

185 190 195

Tyr Asn Lys Ile Lys Ser Arg Gly Ile Lys Ala Asn Ala Phe Lys

200 205 210

Lys Leu Asn Asn Leu Thr Phe Leu Tyr Leu Asp His Asn Ala Leu

215 220 225

Glu Ser Val Pro Leu Asn Leu Pro Glu Ser Leu Arg Val Ile His

230 235 240

Leu Gln Phe Asn Asn Ile Ala Ser Ile Thr Asp Asp Thr Phe Cys

245 250 255

Lys Ala Asn Asp Thr Ser Tyr Ile Arg Asp Arg Ile Glu Glu Ile

260 265 270

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

275 280 285

Phe Ile Cys Leu Lys Arg Leu Pro Ile Gly Ser Tyr Phe

290 295

<210> SEQ ID NO 18

<211> LENGTH: 154

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE: -

<223> OTHER INFORMATION: 1299627

<400> SEQUENCE: 18

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

1 5 10 15

Lys Leu Val Leu Leu Phe Val Tyr Ala Asp Asp Cys Leu Ala Gln

20 25 30

Cys Gly Lys Asp Cys Lys Ser Tyr Cys Cys Asp Gly Thr Thr Pro

35 40 45

Tyr Cys Cys Ser Tyr Tyr Ala Tyr Ile Gly Asn Ile Leu Ser Gly

50 55 60

Thr Ala Ile Ala Gly Ile Val Phe Gly Ile Val Phe Ile Met Gly

65 70 75

Val Ile Ala Gly Ile Ala Ile Cys Ile Cys Met Cys Met Lys Asn

80 85 90

His Arg Ala Thr Arg Val Gly Ile Leu Arg Thr Thr His Ile Asn

95 100 105

Thr Val Ser Ser Tyr Pro Gly Pro Pro Pro Tyr Gly His Asp His

110 115 120

Glu Met Glu Tyr Cys Ala Asp Leu Pro Pro Pro Tyr Ser Pro Thr

125 130 135

Pro Gln Gly Pro Ala Gln Arg Ser Pro Pro Pro Pro Tyr Pro Gly

140 145 150

Asn Ala Arg Lys

<210> SEQ ID NO 19

<211> LENGTH: 371

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE: -

<223> OTHER INFORMATION: 2227688

<400> SEQUENCE: 19

Met Ala Glu Leu Gln Glu Val Gln Ile Thr Glu Glu Lys Pro Leu

1 5 10 15

Leu Pro Gly Gln Thr Pro Glu Ala Ala Lys Glu Ala Glu Leu Ala

20 25 30

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

35 40 45

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

50 55 60

Lys Arg Pro Ala Ile Leu Thr Tyr His Asp Val Gly Leu Asn Tyr

65 70 75

Lys Ser Cys Phe Gln Pro Leu Phe Gln Phe Glu Asp Met Gln Glu

80 85 90

Ile Ile Gln Asn Phe Val Arg Val His Val Asp Ala Pro Gly Met

95 100 105

Glu Glu Gly Ala Pro Val Phe Pro Leu Gly Tyr Gln Tyr Pro Ser

110 115 120

Leu Asp Gln Leu Ala Asp Met Ile Pro Cys Val Leu Gln Tyr Leu

125 130 135

Asn Phe Ser Thr Ile Ile Gly Val Gly Val Gly Ala Gly Ala Tyr

140 145 150

Ile Leu Ala Arg Tyr Ala Leu Asn His Pro Asp Thr Val Glu Gly

155 160 165

Leu Val Leu Ile Asn Ile Asp Pro Asn Ala Lys Gly Trp Met Asp

170 175 180

Trp Ala Ala His Lys Leu Thr Gly Leu Thr Ser Ser Ile Pro Glu

185 190 195

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

200 205 210

Ser Glu Leu Ile Gln Lys Tyr Arg Asn Ile Ile Thr His Ala Pro

215 220 225

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

230 235 240

Arg Asp Leu Asn Phe Glu Arg Gly Gly Asp Ile Thr Leu Arg Cys

245 250 255

Pro Val Met Leu Val Val Gly Asp Gln Ala Pro His Glu Asp Ala

260 265 270

Val Val Glu Cys Asn Ser Lys Leu Asp Pro Thr Gln Thr Ser Phe

275 280 285

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

290 295 300

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

305 310 315

Tyr Met Ala Ser Ser Cys Met Thr Arg Leu Ser Arg Ser Arg Thr

320 325 330

Ala Ser Leu Thr Ser Ala Ala Ser Val Asp Gly Asn Arg Ser Arg

335 340 345

Ser Arg Thr Leu Ser Gln Ser Ser Glu Ser Gly Thr Leu Ser Ser

350 355 360

Gly Pro Pro Gly His Thr Met Glu Val Ser Cys

365 370

<210> SEQ ID NO 20

<211> LENGTH: 75

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE: -

<223> OTHER INFORMATION: 2507107

<400> SEQUENCE: 20

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

1 5 10 15

Leu Phe Asn Phe Leu Ile His Glu Asn Asn Leu Val Lys Leu Phe

20 25 30

Pro Pro Pro Lys Val Phe Ser Ala Ser Val Pro Ser Phe Gly Pro

35 40 45

His Asn Lys Ser Cys Glu Val Thr Glu Thr Ser Val Val Arg Tyr

50 55 60

Ile Phe Val Met Ser Lys Leu Arg Leu Arg Asp Ser Lys Lys Lys

65 70 75

<210> SEQ ID NO 21

<211> LENGTH: 322

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE: -

<223> OTHER INFORMATION: 3044710

<400> SEQUENCE: 21

Met Ala Arg Cys Phe Ser Leu Val Leu Leu Leu Thr Ser Ile Trp

1 5 10 15

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

20 25 30

Ser Ile Gln Val Ser Cys Arg Ile Met Gly Ile Thr Leu Val Ser

35 40 45

Lys Lys Ala Asn Gln Gln Leu Asn Phe Thr Glu Ala Lys Glu Ala

50 55 60

Cys Arg Leu Leu Gly Leu Ser Leu Ala Gly Lys Asp Gln Val Glu

65 70 75

Thr Ala Leu Lys Ala Ser Phe Glu Thr Cys Ser Tyr Gly Trp Val

80 85 90

Gly Asp Gly Phe Val Val Ile Ser Arg Ile Ser Pro Asn Pro Lys

95 100 105

Cys Gly Lys Asn Gly Val Gly Val Leu Ile Trp Lys Val Pro Val

110 115 120

Ser Arg Gln Phe Ala Ala Tyr Cys Tyr Asn Ser Ser Asp Thr Trp

125 130 135

Thr Asn Ser Cys Ile Pro Glu Ile Ile Thr Thr Lys Asp Pro Ile

140 145 150

Phe Asn Thr Gln Thr Ala Thr Gln Thr Thr Glu Phe Ile Val Ser

155 160 165

Asp Ser Thr Tyr Ser Val Ala Ser Pro Tyr Ser Thr Ile Pro Ala

170 175 180

Pro Thr Thr Thr Pro Pro Ala Pro Ala Ser Thr Ser Ile Pro Arg

185 190 195

Arg Lys Lys Leu Ile Cys Val Thr Glu Val Phe Met Glu Thr Ser

200 205 210

Thr Met Ser Thr Glu Thr Glu Pro Phe Val Glu Asn Lys Ala Ala

215 220 225

Phe Lys Asn Glu Ala Ala Gly Phe Gly Gly Val Pro Thr Ala Leu

230 235 240

Leu Val Leu Ala Leu Leu Phe Phe Gly Ala Ala Ala Gly Leu Gly

245 250 255

Phe Cys Tyr Val Lys Arg Tyr Val Lys Ala Phe Pro Phe Thr Asn

260 265 270

Lys Asn Gln Gln Lys Glu Met Ile Glu Thr Lys Val Val Lys Glu

275 280 285

Glu Lys Ala Asn Asp Ser Asn Pro Asn Glu Glu Ser Lys Lys Thr

290 295 300

Asp Lys Asn Pro Glu Glu Ser Lys Ser Pro Ser Lys Thr Thr Val

305 310 315

Arg Cys Leu Glu Ala Glu Val

320

<210> SEQ ID NO 22

<211> LENGTH: 402

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<221> NAME/KEY: unsure

<222> LOCATION: 328, 374, 379

<223> OTHER INFORMATION: unknown, or other

<220> FEATURE: -

<223> OTHER INFORMATION: 3507515

<400> SEQUENCE: 22

Met Ala Glu Leu Gln Glu Val Gln Ile Thr Glu Glu Lys Pro Leu

1 5 10 15

Leu Pro Gly Gln Thr Pro Glu Ala Ala Lys Thr His Ser Val Glu

20 25 30

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

35 40 45

Pro Lys Arg Pro Ala Ile Leu Thr Tyr His Asp Val Gly Leu Asn

50 55 60

Tyr Lys Ser Cys Phe Gln Pro Leu Phe Gln Phe Glu Asp Met Gln

65 70 75

Glu Ile Ile Gln Asn Phe Val Arg Val His Val Asp Ala Pro Gly

80 85 90

Met Glu Glu Gly Ala Pro Val Phe Pro Leu Gly Tyr Gln Tyr Pro

95 100 105

Ser Leu Asp Gln Leu Ala Asp Met Ile Pro Cys Val Leu Gln Tyr

110 115 120

Leu Asn Phe Ser Thr Ile Ile Gly Val Gly Val Gly Ala Gly Ala

125 130 135

Tyr Ile Leu Ala Arg Tyr Ala Leu Asn His Pro Asp Thr Val Glu

140 145 150

Gly Leu Val Leu Ile Asn Ile Asp Pro Asn Ala Lys Gly Trp Met

155 160 165

Asp Trp Ala Ala His Lys Leu Thr Gly Leu Thr Ser Ser Ile Pro

170 175 180

Glu Met Ile Leu Gly His Leu Phe Ser Gln Glu Glu Leu Ser Gly

185 190 195

Asn Ser Glu Leu Ile Gln Lys Tyr Arg Asn Ile Ile Thr His Ala

200 205 210

Pro Asn Leu Asp Asn Ile Glu Leu Tyr Trp Asn Ser Tyr Asn Asn

215 220 225

Arg Arg Asp Leu Asn Phe Glu Arg Gly Gly Asp Ile Thr Leu Arg

230 235 240

Cys Pro Val Met Leu Val Val Gly Asp Gln Ala Pro His Glu Asp

245 250 255

Ala Val Val Glu Cys Asn Ser Lys Leu Asp Pro Thr Gln Thr Ser

260 265 270

Phe Leu Lys Met Ala Asp Ser Gly Gly Gln Pro Gln Leu Thr Gln

275 280 285

Pro Gly Lys Leu Thr Glu Ala Phe Lys Tyr Phe Leu Gln Gly Met

290 295 300

Gly Tyr Met Ala Ser Ser Cys Met Thr Arg Leu Ser Arg Ser Arg

305 310 315

Thr Ala Ser Leu Thr Ser Ala Ala Ser Val Asp Gly Xaa Arg Ser

320 325 330

Arg Ser Arg Thr Leu Ser Gln Ser Ser Glu Ser Gly Thr Leu Phe

335 340 345

Phe Gly Gly Pro Arg Gly His Thr Met Gly Gly Leu Leu Leu Asn

350 355 360

Gly Pro Cys Cys Pro Arg Val Gly Pro Ser Pro Gln Leu Xaa Gln

365 370 375

Ser Asn Leu Xaa Gly Ala Glu Arg Gly His Trp Gly His Arg Lys

380 385 390

Gln Arg Gly Lys Arg Ala Asp Ser Trp Arg Gly Arg

395 400

<210> SEQ ID NO 23

<211> LENGTH: 522

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE: -

<223> OTHER INFORMATION: 3688209

<400> SEQUENCE: 23

Met Ala Val Asn Lys Gly Leu Thr Leu Leu Asp Gly Asp Leu Pro

1 5 10 15

Glu Gln Glu Asn Val Leu Gln Arg Val Leu Gln Leu Pro Val Val

20 25 30

Ser Gly Thr Cys Glu Cys Phe Gln Lys Thr Tyr Thr Ser Thr Lys

35 40 45

Glu Ala His Pro Leu Val Ala Ser Val Cys Asn Ala Tyr Glu Lys

50 55 60

Gly Val Gln Ser Ala Ser Ser Leu Ala Ala Trp Ser Met Glu Pro

65 70 75

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

80 85 90

Ala Cys Arg Gly Leu Asp His Leu Glu Glu Lys Ile Pro Ala Leu

95 100 105

Gln Tyr Pro Pro Glu Lys Ile Ala Ser Glu Leu Lys Asp Thr Ile

110 115 120

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

125 130 135

Ala Ser Thr Ser Asp Lys Val Leu Gly Ala Ala Leu Ala Gly Cys

140 145 150

Glu Leu Ala Trp Gly Val Ala Arg Asp Thr Ala Glu Phe Ala Ala

155 160 165

Asn Thr Arg Ala Gly Arg Leu Ala Ser Gly Gly Ala Asp Leu Ala

170 175 180

Leu Gly Ser Ile Glu Lys Val Val Glu Tyr Leu Leu Pro Ala Asp

185 190 195

Lys Glu Glu Ser Ala Pro Ala Pro Gly His Gln Gln Ala Gln Lys

200 205 210

Ser Pro Lys Ala Lys Pro Ser Leu Leu Ser Arg Val Gly Ala Leu

215 220 225

Thr Asn Thr Leu Ser Arg Tyr Thr Val Gln Thr Met Ala Arg Ala

230 235 240

Leu Glu Gln Gly His Thr Val Ala Met Trp Ile Pro Gly Val Val

245 250 255

Pro Leu Ser Ser Leu Ala Gln Trp Gly Ala Ser Val Ala Met Gln

260 265 270

Ala Val Ser Arg Arg Arg Ser Glu Val Arg Val Pro Trp Leu His

275 280 285

Ser Leu Ala Ala Ala Gln Glu Glu Asp His Glu Asp Gln Thr Asp

290 295 300

Thr Glu Gly Glu Asp Thr Glu Glu Glu Glu Glu Leu Glu Thr Glu

305 310 315

Glu Asn Lys Phe Ser Glu Val Ala Ala Leu Pro Gly Pro Arg Gly

320 325 330

Leu Leu Gly Gly Val Ala His Thr Leu Gln Lys Thr Leu Gln Thr

335 340 345

Thr Ile Ser Ala Val Thr Trp Ala Pro Ala Ala Val Leu Gly Met

350 355 360

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

365 370 375

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

380 385 390

Thr Asp Asn Val Val Asp Thr Val Val His Tyr Val Pro Leu Pro

395 400 405

Arg Leu Ser Leu Met Glu Pro Glu Ser Glu Phe Arg Asp Ile Asp

410 415 420

Asn Pro Pro Ala Glu Val Glu Arg Arg Glu Ala Glu Arg Arg Ala

425 430 435

Ser Gly Ala Pro Ser Ala Gly Pro Glu Pro Ala Pro Arg Leu Ala

440 445 450

Gln Pro Arg Arg Ser Leu Arg Ser Ala Gln Ser Pro Gly Ala Pro

455 460 465

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

470 475 480

Arg Pro Gly Phe Pro Ala Val Pro Arg Glu Lys Pro Lys Arg Arg

485 490 495

Val Ser Asp Ser Phe Phe Arg Pro Ser Val Met Glu Pro Ile Leu

500 505 510

Gly Arg Thr His Tyr Ser Gln Leu Arg Lys Lys Ser

515 520