US20020169139A1

US20020169139A1 - Single gene encoding aortic-specific and striated-specific muscle cell isoforms and uses thereof

Info

Publication number: US20020169139A1
Application number: US10/160,865
Authority: US
Inventors: Mu-En Lee; Chung-Ming Hsieh
Original assignee: Individual
Current assignee: Harvard College
Priority date: 1995-06-22
Filing date: 2002-06-03
Publication date: 2002-11-14
Also published as: US6399753B1

Abstract

Aortic-preferentially-expressed gene-1 (APEG-1) and striated muscle preferentially expressed (SPEG) polypeptide, DNA sequences encoding and controlling the transcription of the APEG-1/SPEG encoding gene, methods of diagnosing vascular injury, methods of conferring smooth muscle-cell specific expression, and methods of inhibiting vascular smooth muscle cell proliferation by increasing the level of APEG-1 at the site of vascular injury.

Description

This application is a continuation-in-part of U.S. Ser. No. 08/795,868, filed on Feb. 6, 1997, which is a continuation-in-part of U.S. Ser. No. 08/494,577, filed on Jun. 22, 1995, which issued as U.S. Pat. No. 5,786,171 on Jul. 28, 1998.[0001]

BACKGROUND OF THE INVENTION

The invention relates to diagnosis and treatment of vascular injury.

Atherosclerosis and its subsequent complications, such as myocardial infarction, stroke, and peripheral vascular diseases, are the major causes of death in developed countries. Vascular endothelial and smooth muscle cells have important roles in the regulation of normal vascular tone. Damage or dysfunction of these cells can lead to vascular diseases, such as atherosclerosis and restenosis.

Atherosclerosis is believed to be a consequence of a response of the vascular wall to injury (Ross, R., 1993, Nature 362:801-9). Upon vascular injury and various other stimuli, cytokines and growth factors from activated vascular cells promote growth and migration of vascular smooth muscle cells in a dedifferentiated status, resulting in the formation of atherosclerotic plaques.

The pathogenesis of atherosclerosis is not fully understood, and an effective therapeutic regime has not been developed to prevent or cure atherosclerosis (Ross, R., The Pathogenesis of Atherosclerosis, in Heart Disease, a textbook of cardiovascular medicine, E. Braunwald, Editor, 1992, W. B. Saunders Company: Philadelphia. pp. 1106-24; and Ross, R.: The Pathogenesis of Atherosclerosis: a Perspective for the 1990s, 1993, Nature 362:801-9). Despite extensive research, the molecular mechanisms responsible for the regulation of gene expression in vascular endothelial and smooth muscle cells are largely unknown. In particular, trans-acting factors and cis-acting elements mediating vascular cell-specific gene expression have not been identified, mainly due to the fact that only a few vascular specific genes have been identified. Furthermore, of the genes that have been characterized as endothelial cell-specific (e.g. von Willebrand factors, VEGF receptor flk-1, VCAM-1, and E-selection (Hunter, J. J., et al., 1993, Hypertension 22:608-17) or smooth muscle cell-specific (e.g., CHIP28, SM22, and gax (Gorski, D. H., et al., 1993, Mol. Cell. Biol. 13(6):3722-33), many have been found in other cell types at various levels.

SUMMARY OF THE INVENTION

The invention is based on the discovery of a novel gene the expression of which gives rise to variant isoforms, one which is specific to aortic cells, and others which are found in striated muscle cells. Accordingly, the invention features an aortic cell-specific gene, and therefore provides a substantially pure DNA (e.g., genomic DNA, cDNA or synthetic DNA) encoding an aortic-preferentially-expressed gene-1 (APEG-1) polypeptide. By “substantially pure DNA” is meant DNA that is free of the genes which, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the APEG-1 gene. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote at a site other than its natural site; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.

Hybridization is carried out using standard techniques such as those described in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, (1989). “High stringency” refers to DNA hybridization and wash conditions characterized by high temperature and low salt concentration, e.g., wash conditions of 65° C. at a salt concentration of approximately 0.1×SSC. “Low” to “moderate” stringency refers to DNA hybridization and wash conditions characterized by low temperature and high salt concentration, e.g. wash conditions of less than 60° C. at a salt concentration of at least 1.0×SSC. For example, high stringency conditions may include hybridization at about 42° C., and about 50% formamide; a first wash at about 65° C., about 2×SSC, and 1% SDS; followed by a second wash at about 65° C. and about 0.1%×SSC. Lower stringency conditions suitable for detecting DNA sequences having about 50% sequence identity to an APEG-1 gene are detected by, for example, hybridization at about 42° C. in the absence of formamide; a first wash at about 42° C., about 6×SSC, and about 1% SDS; and a second wash at about 50° C., about 6×SSC, and about 1% SDS.

A substantially pure DNA having at least 50% sequence identity (preferably at least 70%, more preferably at least 80%, and most preferably at least 90%) to SEQ ID NO:1, 2, or 11, and encoding a polypeptide having a biological activity of an APEG-1 polypeptide is also within the invention. The percent sequence identity of one DNA to another is determined by standard means, e.g., by the Sequence Analysis Software Package developed by the Genetics Computer Group (University of Wisconsin Biotechnology Center, Madison, Wis.) (or an equivalent program), employing the default parameters thereof. “Biological activity of an APEG-1 polypeptide” is defined as the ability to inhibit the proliferation or migration of smooth muscle cells at the site of vascular injury.

The invention also includes a substantially pure DNA containing a constitutive or inducible, vascular cell-specific promoter, e.g., an APEG-1 promoter which is preferably in a vector into which an heterologous gene may be or has been cloned, and under the control of which the gene may be expressed. The promoter is preferably specific for arterial cells (e.g., cells of the aorta), and most preferably specific for vascular smooth muscle cells. DNA encoding APEG-1 may be operably linked to such regulatory sequences for expression of the APEG-1 polypeptide in vascular cells.

By “promoter” is meant a minimal DNA sequence sufficient to direct transcription. Promoters may be constitutive or inducible, and may be coupled to other regulatory sequences or “elements” which render promoter-dependent gene expression cell-type specific, tissue-specific or inducible by external signals or agents; such elements may be located in the 5′ or 3′ region of the native gene, or within an intron. By “heterologous promoter” is meant a promoter other than a naturally occurring APEG-1 promoter.

By “operably linked” is meant that a coding sequence and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s).

The invention also provides a method of directing vascular cell-specific expression of a protein by introducing into a vascular cell an isolated DNA containing a sequence encoding the protein operably linked to the vascular cell-specific promoter. A cell containing the DNA or vector of the invention is also within the invention.

The invention also features a substantially pure APEG-1 polypeptide (e.g., rat APEG-1 (SEQ ID NO:3) or human APEG-1 (e.g., human APEG-1 (SEQ ID NO:12)) and an antibody which specifically binds to an APEG-1 polypeptide. By a “substantially pure polypeptide” is meant a polypeptide which is separated from those components (proteins and other naturally-occurring organic molecules) which naturally accompany it. Typically, the polypeptide is substantially pure when it constitutes at least 60%, by weight, of the protein in the preparation. Preferably, the protein in the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, APEG-1 polypeptide. A substantially pure APEG-1 polypeptide may be obtained, for example, by extraction from a natural source (e.g., an aortic cell); by expression of a recombinant nucleic acid encoding an APEG-1 polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

A protein is substantially free of naturally associated components when it is separated from those contaminants which accompany it in its natural state. Thus, a protein which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components. Accordingly, substantially pure polypeptides include recombinant polypeptides derived from a eukaryote but produced in E. coli or another prokaryote, or in a eukaryote other than that from which the polypeptide was originally derived.

In another aspect, the invention provides a method of detecting injury in a sample of vascular tissue by determining the level of APEG-1 gene expression in the tissue; a decrease in the level of expression detected in the tissue sample compared to that detected in uninjured control vascular tissue indicates the presence of a vascular injury.

The invention also includes a method of inhibiting smooth muscle cell proliferation in an animal by contacting an artery of the animal with an APEG-1 polypeptide or a biologically active fragment thereof or with a compound that stimulates the APEG-1 promoter, e.g., stimulates APEG-1 expression.

In yet another aspect, the invention includes a method of making an APEG-1 polypeptide, e.g., a rat or human APEG-1 polypeptide, involving providing a cell containing DNA encoding an APEG-1 polypeptide and culturing the cell under conditions permitting expression of the APEG-1-encoding DNA, i.e., production of the recombinant APEG-1 by the cell.

The invention further features a substantially pure DNA having an APEG-1 derived promoter/enhancer sequence which regulates vascular smooth muscle cell-specific transcription of a polypeptide-encoding sequence to which it is operably linked. By “promoter/enhancer sequence” is meant a DNA sequence which contains one or more cis-acting elements which regulate transcription, e.g., cell specific transcription. The elements may be contiguous or separated by DNA not involved in the regulation of transcription, e.g., an enhancer element may be in a position immediately adjacent to the promoter element or up to several kilobases upstream or downstream of the transcriptional start site. The promoter/enhancer DNA is preferably derived from the 5′ region of a mammalian APEG-1 gene, such as that of the mouse (SEQ ID NO:17), and regulates preferential expression in vascular smooth muscle cells, e.g., aortic smooth muscle cells, of a polypeptide-encoding DNA to which it is operably linked. More preferably, the promoter/enhancer includes the 73 nucleotides of SEQ ID NO:20, which is located within the sequence of SEQ ID NO:17. In some embodiments, the promoter/enhancer contains SEQ ID NO:20 and is less than 2.7 kb, 1.0 kb, 500 bp, 250 bp, or 100 bp in length. The promoter/enhancer may also include a plurality of copies of SEQ ID NO:20.

The promoter/enhancer including SEQ ID NO:20 may be immediately contiguous to a polypeptide-encoding DNA. Alternatively, the promoter/enhancer may be separated by 5, 10, 20, 30, 40, 50, 75, or 100 nucleotides from the polypeptide-encoding DNA. In addition to or alternatively, the promoter/enhancer may be contiguous to, or be separated by 5, 10, 20, 30, 40, 50, 75, or 100 nucleotides from a heterologous promoter.

Preferably, expression of a polypeptide under the control of the APEG promoter/enhancer (e.g., SEQ ID NO:17) is at least 50% greater (e.g., as measured in the amount of polypeptide-encoding mRNA transcript), preferably at least 100% greater, more preferably at least 200% greater, and still more preferably at least 400% greater in vascular smooth muscle cells than in non-vascular smooth muscle cells. Most preferably, the APEG-1 promoter/enhancer directs vascular smooth muscle cell-specific polypeptide expression and directs negligible polypeptide expression in non-smooth muscle cell types. The promoter/enhancer sequence may in addition regulate developmental stage-specific expression, e.g., preferential expression in embryonic cells, of a polypeptide-encoding sequence.

The DNA of the invention (promoter/enhancer sequence) may be operably linked to a DNA sequence encoding a polypeptide that is not APEG-1 (i.e., a heterologous polypeptide), and function to regulate vascular smooth muscle cell-specific transcription of the polypeptide-encoding sequence. Examples of such polypeptides include tissue plasminogen activator (tPA), p21 cell cycle inhibitor, nitric oxide synthase, interferon-γ, and atrial natriuretic polypeptide.

The invention also includes a vector containing the promoter/enhancer DNA of the invention (operably linked to a polypeptide-encoding DNA sequence) and a vascular smooth muscle cell containing the vector. Also within the invention is a method of directing vascular smooth cell-specific expression of the polypeptide by introducing the vector into a vascular smooth muscle cell and maintaining the cell under conditions which permit expression of the polypeptide, e.g., introducing the vector into a human patient for gene therapy.

The vector of the invention can be used for gene therapy. For example, the vector can be introduced into a vascular smooth muscle cell to direct vascular smooth muscle cell-specific expression of a polypeptide. The vector of the invention can also be used for directing developmental stage-specific expression, e.g., preferential expression by embryonic cells, of a polypeptide, involving introducing into a vascular smooth muscle cell the vector of the invention.

The invention also features a method of inhibiting proliferation of vascular smooth muscle cells by administering to the cells an APEG-1 polypeptide.

The invention also features a striated muscle cell-specific variant gene product arising from the same genomic DNA encoding APEG-1, and therefore provides a substantially pure DNA (e.g., genomic DNA, cDNA or synthetic DNA) encoding a striated muscle preferentially-expressed gene (SPEG) polypeptide.

The DNA may encode a naturally occurring mammalian SPEG polypeptide such as a human SPEG polypeptide (SEQ ID NO:14) or mouse SPEG polypeptide (SEQ ID NO:16). For example, the invention includes degenerate variants of the human cDNA (SEQ ID NO:13) or the mouse cDNA (SEQ ID NO:15). The invention also includes a substantially pure DNA comprising a strand which hybridizes at high stringency to a DNA having the sequence of SEQ ID NO:13 or 15, or the complements thereof.

A substantially pure DNA having at least 50% sequence identity (preferably at least 70%, more preferably at least 80%, and most preferably at least 90%) to SEQ ID NO:13, or 15, and encoding a polypeptide having a biological activity of a SPEG polypeptide is also within the invention.

The invention also includes a substantially pure DNA containing a constitutive or inducible striated muscle cell-specific promoter, e.g., a SPEG promoter which is preferably in a vector into which an heterologous gene may be or has been cloned, and under the control of which promoter the gene may be expressed. The promoter is preferably specific for striated muscle cells (e.g., cells of skeletal or cardiac muscle). DNA encoding SPEG may be operably linked to such regulatory sequences for expression of the SPEG polypeptide in striated muscle cells.

The invention also provides a method of directing striated muscle cell-specific expression of a protein by introducing into a cell an isolated DNA containing a sequence encoding the protein operably linked to the striated cell-specific promoter. A cell containing the DNA or vector of the invention is also within the invention.

The invention also features a substantially pure SPEG polypeptide (e.g., human (SEQ ID NO:14) or mouse SPEG (SEQ ID NO:16) and an antibody which specifically binds to a SPEG polypeptide.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

DETAILED DESCRIPTION

The drawings will first be described. [0032]
FIG. 1. is a flow chart of the differential mRNA display procedure for identifying APEG sequences. [0033]
FIG. 2A is a photograph of a differential mRNA display showing APEG-1 preferentially expressed in the rat aorta. The differential expression was tested among 6 rat tissues. Unique bands in the aorta that were eluted and reamplified for subsequent analysis are indicated (). [0034]
FIG. 2B is a photograph of a differential mRNA display showing APEG-2 preferentially expressed in the rat aorta. The differential expression was tested among 6 rat tissues. Unique bands in the aorta that were eluted and reamplified for subsequent analysis are indicated (). [0035]
FIG. 2C is a photograph of a differential mRNA display showing APEG-3 preferentially expressed in the rat aorta. The differential expression was tested among 6 rat tissues. Unique bands in the aorta that were eluted and reamplified for subsequent analysis are indicated (). [0036]
FIG. 2D is photograph of a differential mRNA display showing APEG-4 preferentially expressed in the rat aorta. The differential expression was tested among 6 rat tissues. Unique bands in the aorta that were eluted and reamplified for subsequent analysis are indicated (). [0037]
FIG. 2E is a photograph of a Northern blot analysis showing tissue expression of APEG-1. Ten micrograms of total RNA from each tissue were used in Northern analysis. The loading of each tissue RNA was normalized by comparing 18s rRNA hybridization signals (shown in FIG. 2F). [0038]
FIG. 2F is a photograph of a Northern blot analysis showing 18s rRNA. [0039]
FIG. 2G is a photograph of a Northern blot analysis showing tissue expression of APEG-2. Ten micrograms of total RNAs from each tissue were used in Northern analysis, and the loading of each tissue RNA was normalized by comparing 18s rRNA hybridization signals. [0040]
FIG. 2H is a photograph of a Northern blot analysis showing tissue expression of APEG-3. Ten micrograms of total RNAs from each tissue were used in Northern analysis, and the loading of each tissue RNA was normalized by comparing 18s rRNA hybridization signals. [0041]
FIG. 2I is a photograph of a Northern blot analysis showing tissue expression of APEG-4. Ten micrograms of total RNAs from each tissue were used in Northern analysis, and the loading of each tissue RNA was normalized by comparing 18s rRNA hybridization signals. [0042]
FIG. 3A is a photograph of a Northern blot analysis using full length cDNA of APEG-1 (APEG-1 full cDNA) as a probe. Samples of RNA from twelve rat organs were analyzed. The respective lanes are labelled in FIG. 3D. [0043]
FIG. 3B is a photograph of a Northern blot analysis using a 3′ cDNA fragment originally cloned by differential mRNA display (APEG-1 3′ D.D. frag.) as a probe. Samples of RNA from twelve rat organs were analyzed. [0044]
FIG. 3C is a photograph of a Northern blot showing 18s rRNA bands (18s rRNA) to which RNA loading was normalized. [0045]
FIG. 3D is a bar graph showing tissue distribution of APEG-1 gene expression. [0046]
FIG. 4 is a flow chart showing the cloning strategy for APEG-1. A rat aortic cDNA library established in the yeast expression vector pcJATA was screened to isolate full length APEG-1 cDNA. Southern analysis was carried out to confirm the presence of APEG-1 in this cDNA library. Restriction enzyme-digested (EcoRI and XhoI) cDNA fragments were separated on an agarose gel and the portions that contained APEG-1 cDNA, as determined by size markers and Southern analysis, were excised to elute the cDNA contents. Eluted cDNAs were ligated with linearized pSP72 vectors, and the ligated DNAs were used to transform competent [0047] E. coli DHα5 cells to establish a size-selected aortic cDNA sublibrary. This cDNA sublibrary was screened by the APEG-1 cDNA 3′ fragment to obtain its full length cDNA.
FIG. 5 is a diagram of the nucleotide sequence of rat APEG-1 cDNA (SEQ ID NO:1). The longest open reading frame is located from nucleotide 169 to 511 (BOLD UPPERCASE) and the ATG flanking nucleotides that match the Kozak consensus sequence are indicated (UPPERCASE). A very short upstream open reading frame is present from nucleotide 102 to 116 (italic). There is a polyadenylation signal (underline) 21 nucleotides upstream of the poly-A tail. The primer annealing site of the 5′ arbitrary primer used in the initial differential display PCR is also indicated (ITALIC UPPERCASE). [0048]
FIG. 6 is a diagram of the amino acid sequence (SEQ ID NO:3) deduced from the longest APEG-1 cDNA open reading frame (SEQ ID NO:2). Possible phosphorylation sites of protein kinase C and casein kinase-2 are indicated (bold). An integrin binding site, RGD, is also shown (bold italic). “***” represents a stop codon. [0049]
FIG. 7A is a photograph of in vitro transcription products of the APEG-1 gene. The 1.3 kb APEG-1 cDNA and a positive control DNA template were transcribed by T7 RNA polymerase. 1 μl of the 20 μl RNA products were resolved on a 1.2% denaturing agarose gel. [0050]
FIG. 7B is a photograph of in vitro translation products of the APEG-1 gene. In vitro transcribed APEG-1 mRNA was translated by wheat germ extract in the presence of [[0051] ³⁵S]-methionine, and separated on a 10% tricine-SDS-polyacrylamide gel. In the mock reaction, mRNA template was absent.
FIG. 8 is an alignment of amino acid sequences of APEG-1 (SEQ ID NO:8), the myosin light chain kinase of chicken (ChkMLCK; SEQ ID NO:5) and of rabbit (RabMLCK; SEQ ID NO:7), and telokin of chicken (ChkTelo; SEQ ID NO:4) and of rabbit (RabTelo; SEQ ID NO:6). A consensus sequence (SEQ ID NO:9) is also shown to indicate the amino acid residues that are identical among these proteins. The conserved serine residue that is phosphorylated by cAMP-dependent protein kinase is marked by an asterisk (*). [0052]
FIG. 9A is a diagram of APEG-1 cDNA. APEG-1 cDNA was divided into four fragments by EcoR I, BamHI, Hind III, and XhoI restriction enzyme digestion. The three large fragments (405, 299, and 432 bp) were used to probe six rat tissue RNAs to show their different hybridization patterns. [0053]
FIG. 9B is a photograph of a Northern analysis using the 405 bp fragment of APEG-1 cDNA as a probe. [0054]
FIG. 9C is a photograph of a Northern analysis using the 299 bp fragment of APEG-1 cDNA as a probe. [0055]
FIG. 9D is a photograph of a Northern analysis using the 432 bp fragment of APEG-1 cDNA as a probe. [0056]
FIG. 10 is a photograph of a genomic Southern analysis of the APEG-1 gene. Genomic DNA from cultured rat aortic smooth muscle cells was harvested and digested with EcoRI, HindIII, or BamHI. APEG-1 full length cDNA was used as probe in the Southern analysis. The size of each band (indicated on the right) was determined according to the size markers (indicated on the left). [0057]
Fig. 11A is a photograph of ethidium bromide staining of the 3 clones of human homologues of rat APEG-1. Clone 1 (1.1, 1.2), clone 2 (2.1, 2.2), and clone 3 (3.1) were 1.45, 2.0, and 2.7 kb in size, respectively. [0058]
Fig. 11B is a photograph of a Southern analysis showing hybridization of these human homologues with a rat APEG-1 cDNA probe. [0059]
FIG. 12 is a photograph of a Northern analysis of APEG-1 expression in vitro. RNAs from rat aortic smooth muscle cells (RASMC) and from microvascular endothelial cells (RMEC) were purified and separated on a 1.2% denaturing agarose gel. RNA from normal rat aorta was used as a positive control. APEG-1 cDNA was used as probe in Northern analysis to examine its expression in these two cell types. [0060]
FIG. 13A is a photograph of a Northern analysis showing expression of APEG-1 in rat carotid artery during balloon injury. RNAs were purified from rat [0061] carotid arteries 2, 5, 8 days after balloon injury. Three injured rats were used in each time point and two uninjured rats were used as control. The APEG-1 cDNA was used in Northern analysis and the band intensities were normalized by 18s rRNA signal.
FIG. 13B is a bar graph showing expression of APEG-1 in rat carotid artery during balloon injury. Each column represents the mean expression of APEG-1 in the Northern analysis bands shown in FIG. 13A, expressed as a percent of control ± one standard error. [0062]
FIG. 14A is a photograph of a Coomassie blue stained 10% tricine-SDS-PAGE gel showing the purified FLAG-APEG-1 fusion protein. M, protein size marker. Ext, induced bacterial cell extracts. FT, cell extract that flowed through the FLAG peptide affinity column. [0063]
FIG. 14B is a photograph of a Western analysis of the purified fusion protein. A monoclonal anti-FLAG peptide antibody, M2 (IBI), was used to identify the purity of the fusion protein. Un, uninduced bacterial cell extracts. In, induced bacterial cell extracts. FT, cell extract that flowed through the FLAG peptide affinity column. [0064]
FIG. 15 is a bar graph comparing APEG-1 expression in diabetic rats and control rats. APEG-1 expression was decreased in diabetic rats (unpaired T test: T[0065] ₁₀=3.284, p value=0.0033).
FIG. 16 is a diagram showing the cDNA sequence of human APEG-1 (SEQ ID NO:11) [0066]
FIG. 17 is a diagram showing the amino acid sequence of human APEG-1 (SEQ ID NO:12). “*” represents a stop codon. [0067]
FIG. 18A is a photograph showing the results of an in situ hybridization experiment. The lumen of a rat aorta was sectioned and hybridization carried out using a rat APEG-1 sense strand DNA probe as a control. [0068]
FIG. 18B is a photograph showing APEG-1 mRNA expression in the lumen of a rat aorta. In situ hybridization was carried out using a rat antisense strand DNA probe to measure rat APEG-1 expression in aortic tissue. [0069]
FIGS. [0070] 19A-C are diagrams showing the pattern of exon usage in the APEG-1 and SPEG transcripts. FIG. l9A is a diagram showing the intron/exon arrangement of the APEG/SPEG locus. FIG. 19B is a diagram showing APEG-1 exon usage. FIG. 19C is a diagram showing SPEG exon usage.
FIG. 20 is a diagram showing the cDNA sequence of human SPEG (SEQ ID NO:13). [0071]
FIG. 21 is a diagram showing the amino acid sequence of human SPEG (SEQ ID NO:14). [0072]
FIG. 22 is a diagram showing the cDNA sequence of mouse SPEG (SEQ ID NO:15). [0073]
FIG. 23 is a diagram showing the amino acid sequence of mouse SPEG (SEQ ID NO:16). [0074]
FIG. 24A is a diagram showing the PGL-3 construct. [0075]
FIG. 24B is a bar graph showing the results of reporter transfection assays using 3.3 kb of APEG-1 5′ sequence. Cell lines used: RASMC, rat aortic smooth muscle cells; BAEC, bovine aortic endothelial cells; HeLa, human epidermoid carcinoma cell line; U-2 OS, human osteosarcoma cells; HepG2, human hepatoma cells; NIH 3T3 mouse fibroblasts. [0076]
FIG. 25 is a diagram showing the sequence of a 2.7 kb fragment containing the APEG-1 5′ vascular smooth muscle cell-specific promoter activity (SEQ ID NO:17). [0077]
FIG. 26 is a diagram showing a comparison of the full-length APEG-1 amino acid sequences of the human, mouse and rat. [0078]
FIG. 27 is a diagram showing a comparison of partial SPEG amino acid sequences in human and mouse. represents a stop codon. [0079]
FIG. 28 is a diagram showing the sequence of a 73 nucleotide DNA (SEQ ID NO:20) containing APEG-1 vascular smooth muscle cell-specific promoter activity. [0080]
FIG. 29 is a bar graph showing the results of reporter transfection assays using a 73 bp fragment of the APEG-1 5′ sequence. Cell lines used: RASMC, rat aortic smooth muscle cells; U-2 OS, human osteosarcoma cells; HeLa, a human epidermoid carcinoma cell line; and BAEC, bovine aortic endothelial cells.[0081]

PURIFICATION OF TOTAL RNAS

Total RNA was harvested from male Sprague-Dawley rat organs. The dissected organs were washed in phosphate buffered saline and snap-frozen in liquid nitrogen. The adventitia of the aorta was stripped, and the contents of the small intestine were removed before freezing. The frozen organs were homogenized and RNAs were harvested by acid guanidinium thiocyanate-phenol-chloroform extraction (Chomczynski, P. et al., 1987, Anal. Biochem. 162(1):156-9). Mouse embryo RNA was harvested from whole embryos. The cell culture RNAs were purified by guanidinium/CsCl ultracentrifugation. [0082]

DIFFERENTIAL MRNA DISPLAY

Fifty micrograms of total RNA were treated with DNase I (Boehringer Mannheim) to remove contaminating genomic DNA in the presence of RNase inhibitor RNasin (Promega). After phenol/chloroform extraction and ethanol precipitation, the RNA concentration was adjusted to 0.1 μg/ml in DEPC-treated dH2O. First strand cDNA was synthesized using MMLV reverse transcriptase (GIBCO, BRL) with the 3′ poly-A-anchoring primer T[0083] ₁₂VG (5′-TTTTTTTTTTTTVG-3′) (SEQ ID NO:10). Subsequently the reaction was heated at 95° C. to inactivate reverse transcriptase, and the cDNA products were stored at −20° C. Two microliters of the cDNA were used in 20 μl PCR reactions (2 μl cDNA, 0.2 μM 5′ arbitrary primer, 1 μM 3′ T₁₂VG primer, 1.5 mM Mg²⁺, 2.5 μM dNTP, 12.5 μCi ³⁵S-dATP, 1 unit Taq DNA polymerase; 94° C. for 15 sec, the thermal cycling was 40° C. for 30 sec and 72° C. for 30 sec; the thermal cycling was repeated for 40 cycles) following the reverse transcription. Sample loading buffer (98% formamide, 0.05% bromophenol blue, and 0.05% xylene cyanol) was added, and the samples were heated at 95° C. before loading onto a 6% sequencing gel. Overnight exposure of the dried sequencing gels to X-OMAT films (Kodak) was usually sufficient to display the differential mRNA patterns.

REAMPLIFICATION OF ELUTED CDNAS

Bands of interest on the dried gel were excised, soaked in 200 μl dH[0084] ₂O for 10 minutes at room temperature, and eluted by heating at 95° C. for 15 minutes. After a brief centrifugation, the supernatants were transferred into fresh tubes, and the eluted DNAs were ethanol-precipitated at −20° C. in the presence of 20 μg glycogen and 300 mM sodium acetate. The precipitated DNAs were collected by centrifugation and washed with 70% ethanol. Dried DNA pellets were resuspended in 10 μl dH₂O and nonradioactively reamplified by PCR with the same initial PCR primers and condition, except that the reaction volume was scaled up to 100 μl with 25 μM dNTP. Reamplified cDNAs were resolved on 1% agarose gel to determine their sizes and amounts.

RNA GEL ELECTROPHORESIS AND NORTHERN BLOTTING

Ten micrograms of total RNA were heat-denatured and loaded on a denaturing agarose gel (1.2% agarose, 1.1% formaldehyde, 0.5 μg/ml ethidium bromide in MOPS buffer). Electrophoresis was carried out at 10 V/cm for three to four hours. A photograph of the ethidium bromide staining pattern of the RNAs was taken under UV light illumination. The RNAs were then transferred onto a Nitropure membrane (Micron Separation Inc.) by standard blotting procedure (Ausubel, F. M., et al., ed. Current Protocols in Molecular Biology. ed. K. Janssen., 1994, Vol. 1., Current Protocols:4.9.1-14). [0085]

DNA GEL ELECTROPHORESIS AND SOUTHERN BLOTTING

DNAs were loaded and separated on a 1% agarose gel, followed by standard Southern blotting (Ausubel, F. M., et al., ed. Current Protocols in Molecular Biology. ed. K. Janssen., 1994, Vol. 1, Current Protocols: 2.9.1-15). The DNAs in the gel were denatured in denaturation buffer (1.5 M NaCl, 0.5 N NaOH), then neutralized in neutralization buffer (1.5 M NaCl, 1 M TrisCl, pH 7.4) prior to being transferred onto a Nitropure membrane in 20×SSC solution overnight. [0086]

RANDOM PRIMING AND HYBRIDIZATION

Radioactive DNA probes were generated by random priming (Boehringer Mannheim) with 25 to 50 ng of the DNA fragment. Hybridization to the DNA or RNA blots was carried out in QuikHyb solution (Stratagene) with 1×10[0087] ⁶cpm/ml of radioactive probes and 0.2 mg/ml herring sperm DNA (Boehringer Mannheim) at 68° C. for one to two hours. The blots were washed and exposed to X-ray films for permanent records.

QUANTITATION OF HYBRIDIZATION SIGNALS

To quantitate the hybridization signals, DNA and RNA blots were exposed to phosphor screens (Molecular Dynamics) overnight. The phosphor screens were then scanned by a PhosphoImager scanner (Molecular Dynamics) operated by the ImageQuant program (Molecular Dynamics) running on a PC-DOS/MS Windows computer system (Compaq). Intensities of the signals were quantified by the same ImageQuant program following the manufacturer's instructions. [0088]

DNA SEQUENCING AND SEQUENCE ANALYSIS

Dideoxynucleotide chain termination DNA sequencing method was used to sequence DNAs. One microliter of DMSO was always included to reduce the DNA template secondary structures that may interfere with the Sequenase (USB) enzymatic activity. The sequences were resolved on 8% sequencing gel (National Diagnostics). The DNA sequences were stored into a local computer mainframe (mbcrr.harvard.edu), and analyzed by a sequence analysis software package (Genetics Computer Group). [0089]

FUSION PROTEIN EXPRESSION AND PURIFICATION

Rat APEG-1 cDNA was cloned into pFLAG-2 vector, then transformed into [0090] E. coli BL21 cells. Transformed BL21 cells were grown in large scale to an optical density (OD₅₉₅) of 1.75. The cell pellet was resuspended in extraction buffer (20 mM TrisCl, pH 7.4, 0.2 mM EDTA, 1 M NaCl, 1 mM PMSF, 1 mM DTT) and sonicated on ice, after which the extract was frozen and thawed three times in liquid nitrogen and a 42° C. water bath. The soluble cell extract was collected by centrifugation (12,000×g, 4° C., 20 minutes) and used in purification of the fusion protein by affinity chromatography with a M2 anti-FLAG peptide mAb affinity column. The column, loaded twice with the soluble cell extract, was washed sequentially with 50 ml of each of the following solutions, TE/NaCl/NP-40 buffer (20 mM TrisCl pH 7.4, 0.2 mM EDTA, 150 mM NaCl, 0.5% NP-40), TE/NaCl buffer (20 mM TrisCl pH 7.4, 0.2 mM EDTA, 150 mM NaCl), and TE buffer (20 mM TrisCl pH 7.4, 0.2 mM EDTA). The FLAG-APEG-1 fusion protein was eluted with 10 ml glycine buffer (0.1 M glycine, pH 3.0) and the eluates were slowly collected in 0.8 ml fractions into microfuge tubes containing 50 μl 1 M TrisCl, pH 8.0, and 150 μl 5 M NaCl solutions. The purity of the purified fusion proteins was assayed by protein electrophoresis and Coomassie blue staining as well as western blotting with anti-FLAG mAb.

PROTEIN GEL ELECTROPHORESIS AND WESTERN BLOTTING

A 10% tricine-SDS-polyacrylamide gel system was used to separate bacterial-expressed pFLAG-APEG-1 fusion protein (Schägger, H. et al., 1987, Anal. Biochem. 166:368-79). This system was used because a 10% tricine-SDS-polyacrylamide gel has superior resolution for proteins less than approximately 14 kDa compared to a standard glycine-SDS-polyacrylamide gel. After electrophoresis, the protein gel was assembled in a semi-dry transfer apparatus (Hoefer) and the protein samples were transferred onto a PVDF membrane (Millipore) in transferring buffer (25 mM Tris base, 200 mM glycine, 20% methanol) at 125 mA for one hour. [0091]

IN VITRO TRANSCRIPTION AND TRANSLATION

Rat APEG-1 cDNA was cloned into the pSP72 vector and linearized so that RNA could be transcribed from its upstream T7 promoter with the T7 RNA polymerase. Transcription was carried out in a large-scale T7 transcription system (Novagen) in the presence of 7-[0092] ^meGpppGTP to produce capped mRNA. The in vitro transcribed mRNA was translated in an in vitro translation system of wheat germ extract (Promega) with the [³⁵S]-methionine to produce radiolabeled proteins.

CELLULAR LOCALIZATION

The expression plasmid c-myc-rAPEG-1/pCR3 was constructed by adding in frame a DNA sequence encoding a c-Myc peptide tag (EQKLISEED) to the rat APEG-1 open reading frame at the 5′ end by PCR techniques known in the art (any other detectable peptide can be used as a tag to localize APEG-1). This hybrid DNA fragment was then cloned in to the eukaryotic expression vector pCR3 (Invitrogen, San Diego, Calif.). COS-7 cells were transiently transfected with the c-myc-rAPEG-1 expressing plasmid by a standard DEAE-dextran method (e.g., the method described in Tan et al., 1994, Kidney Intern. 46:690). The U-2 OS cells were transiently transfected by the calcium phosphate method known in the art. Twenty-four hours after transfection, cells were transferred to two-well chamber slides. The cells were fixed with 4% paraformaldehyde in PBS after growing for an additional twenty-four hours. Immunostaining was performed with an anti-c-Myc monoclonal antibody (9E10, Oncogene, Cambridge, Mass.) followed by a rhodamine-conjugated goat anti-mouse IgG secondary antibody (Sigma, St. Louis, Mo.). Nuclear DNA counterstaining was performed with Hoechst 33258 at a concentration of 1 μ/ml. [0093]

CELL CULTURE

Primary rat aortic smooth muscle cells were maintained in DMEM medium supplied with 10% fetal calf serum, 4 mM L-glutamine, 10 mM HEPES, 100 U/ml penicillin and 100 ng/ml streptomycin. Primary rat microvascular endothelial cells were maintained in DMEM medium supplied with 20% fetal calf serum, 4 mM L-glutamine, 100 U/ml penicillin and 100 ng/ml streptomycin. [0094]
BAEC were isolated and cultured in Dulbecco's modified Eagle's medium (JRH Biosciences, Lenexa, Kans.) supplemented with 10% fetal calf serum (HyClone, Logan, Utah), 600 μg of glutamine/ml, 100 units of penicillin/ml, and 100 μg of streptomycin/ml. [0095]
HepG2 human hepatoma cells (ATCC HB-8065), U-2 OS human osteosarcoma cells (ATCC HTB-96), HeLa human epidermoid carcinoma cells (ATCC CRL-7923), HepG2 human hepatoma cells (ATCC HB-8065), and NIH 3T3 mouse fibroblasts (ATCC CRL-1658) are available from the American Type Culture Collection. [0096]

PLASMID DNA PURIFICATION

The mini- (<20 μg) and midiscale (<200 μg) preparations of plasmid DNA were purified by DNA-affinity chromatography (Qiagen). Large scale purification of plasmid DNA was carried out according to the alkaline lysis/CsCl ultracentrifugation methods (Ausubel, F. M., et al., ed. Current Protocols in Molecular Biology. ed. K. Janssen., 1994, Vol. 1, Current Protocols: 1.7.1-11). [0097]

PURIFICATION OF RECOMBINANT λGT11 DNA

Single positive plaques were picked and soaked in the suspension medium (0.1 M NaCl, 10 mM MgSO[0098] ₄, 50 mM TrisCl, pH 7.5, and 0.01% gelatin) with one drop of CHCl₃. Freshly prepared E. coli strain Y1090 competent cells were mixed and incubated briefly with the resuspended phage. The infected cells were grown overnight in LB medium with 10 mM MgSO₄and 0.2% maltose. The next morning one drop of chloroform was added into the medium to lyse the bacterial cells for 15 minutes. Bacterial debris was collected by centrifugation, and to the clear supernatant 100 U DNase I and 100 ng RNase A were added to digest E. coli genomic DNA and RNA. The solutions of EDTA, TrisCl (pH 8.0), NaCl, and proteinase K were added subsequently to final concentrations of 50 mM, 100 mM, 200 mM, and 100 ng/ml, respectively. The mixture was incubated at 42° C. for 30 minutes. Phage DNA was then phenol/chloroform extracted once and precipitated by adding 0.6×volume of isopropanol in the presence of 300 mM NaOAc. Precipitated phage DNA was recovered by centrifugation and washed with 70% ethanol, air dried, then dissolved in 250 μl TE buffer (10 mM TrisCl, pH 8.0, 1 mM EDTA).

CLONING APEG-1 GENES

To clone genes that are preferentially expressed in the aorta, total organ RNA was prepared from rat aorta with the adventitia removed, and from brain, skeletal muscle, esophagus, heart, and intestine. Using the differential mRNA display technique, a technique that systematically amplifies mRNAs by means of RT-PCR with different sets of 5′ arbitrary primers and 3′ oligo-dT anchoring primers, the mRNA patterns of different organs were compared. The PCR products were resolved on a denaturing polyacrylamide sequencing gel to display mRNA patterns that distinguish one organ from another. The bands that were separated by gel electrophoresis represent the 3′-termini of the cDNAs. Therefore, a band that is present in one organ but not in the others suggests that the gene is only expressed in that particular organ (FIG. 1). Specific mRNAs that were present solely in the aorta were identified and cloned. [0099]
The organ RNAs were screened with thirty-three 5′ arbitrary primers in combination with a T[0100] ₁₂VG 3′ oligo-dT anchoring primer. This initial screening covered 21 percent of the 160 primer combinations needed to screen all possible mRNAs to be displayed by this technique. This estimate is based on the assumption that one primer combination displays about 100 different mRNAs from approximately 15,000 different mRNA species present in each cell.
From the initial screening, seventeen bands that were present solely in the aorta were identified. These bands were cut from the gel and the cDNA fragments eluted and reamplified by PCR with the same primers that were used in their original RT-PCRs. These reamplified cDNAs were [0101] ³²P-labeled, then used in Northern blot analyses to confirm their aortic specificity. Four cDNA fragments were found to be aorta-specific (FIGS. 2A-2I). After cloning these four cDNA fragments by TA-cloning methods, the clones were designated APEG-1, APEG-2, APEG-3, and APEG-4. Their DNA sequences were determined by the dideoxynucleotide chain termination method and compared to known DNA sequences listed in the GENBANK® database. APEG-2 showed identical sequences to the rat SM22 gene (Shanahan, C. M., et al., 1993, Circ. Res. 73(1):193-204), a smooth muscle cell specific gene. APEG-4 was found to have a near-identical sequence to chicken and mouse TIMP-3 genes (tissue inhibitor of metalloproteinase-3) (Sun, Y., et al., 1994, Cancer Res. 54:1139-44; Leco, K. J., et al., 1994, J. Biol. Chem. 269(12):9352-60). APEG-1 and APEG-3 did not match any known genes. Further examination of the tissue distribution of expression showed that APEG-3 was also expressed in the lung, a result not seen in the initial Northern blot analysis. In contrast, APEG-1 showed the highest expression in the aorta among twelve rat organs (FIGS. 3A-3D), thus confirming the specificity of tissue expression.

CLONING AND SEQUENCE ANALYSIS OF RAT APEG-1 CDNA

The APEG-1 3′ cDNA fragment, derived from differential mRNA display, was used to screen a rat aortic cDNA library (FIG. 4). The cloned APEG-1 cDNA was determined to be 1,308 base pairs, consistent with the size of the message seen in Northern blot analysis. Sequences of both cDNA strands were determined by dideoxynucleotide chain termination sequencing with fragment-subcloning and oligonucleotide-walking strategies. The complete cDNA sequence had no homologous counterpart in the GENBANK® database. [0102]
The rat APEG-1 cDNA can then be used to screen a genomic library to obtain the vascular cell-specific promoter sequences which regulate expression cell-specific expression of APEG-1. [0103]
To analyze the protein encoded in APEG-1 cDNA, the sequence was searched for possible ATG initiation codons for translation from the 5′ end of the sequence. The longest open reading frame in the rat APEG-1 cDNA (SEQ ID NO:1) spans from 169 to 511 nucleotides (SEQ ID NO:2) downstream of the 5′ end of the cDNA. Another ATG sequence was found at nucleotide 102 to 104 (FIG. 5), but the possible translation from this preceding ATG codon is terminated after four amino acid residues, thus making it unlikely to be the initiation codon used in viva. The longest open reading frame has a Kozak consensus sequence (Kozak, M., 1987, J. Mol. Biol. 196:947-50) and encodrs a protein of 113 amino acids (SEQ ID NO:3) with a predicted molecular weight of 12,667 daltons and an estimated pI of 9.125 (FIG. 6). This predicted translation product was confirmed by in vitro transcription and in vitro translation of the APEG-1 cDNA, which yielded a major translation product of 12.7 kDa as predicted by the deduced amino acid sequence from the longest open reading frame (FIGS. [0104] 7A-7B). Comparison of the APEG-1 deduced amino acid sequence to the SwissProt protein database again showed no identical protein sequence. However, a region was identified that is homologous to proteins of the myosin light chain kinase family, which includes myosin light chain kinases and telokin (FIG. 8).
The myosin light chain kinases (MLCKs), present in all eukaryotic cells, are members of the Ca[0105] ²⁺-calmodulin-dependent protein kinases. They phosphorylate the 20 kDa light chain subunit of myosin, a protein that is important in regulating contraction of smooth muscle cells, secretory vesicle movement, cellular locomotion, and changes in cellular morphology (Gallagher, P. J., et al., 1991, J. Biol. Chem. 266(35):23945-52). The structure of MLCKs is highly conserved and composed of several modular domains. The MLCK carboxyl terminus is the calmodulin-binding domain and has a regulatory function mediated by two specific serines residues which become phosphorylated by cAMP-dependent protein kinase. Phosphorylation at these two sites downregulates MLCK kinase activity by decreasing the affinity of MLCK for Ca²⁺-calmodulin. One of the two phosphorylated serine residues in the MLCK C-terminus is conserved in APEG-1 (Ser12), suggesting a regulatory site of APEG-1.
Telokin, originally purified as an acidic protein from turkey gizzard, is a protein that has the same peptide sequence as the carboxyl terminal domain of MLCKs. Its mRNA transcription initiates from a promoter that is located in one of the MLCK introns. Telokin transcription regulation is independent from that of MLCK despite having a sequence identical to the MLCK carboxyl terminal domain. Telokin has been proposed to be a calmodulin-binding protein (Holden, H. M., et al., 1992, J. Mol. Biol. 227:840-51), and it is expressed in almost every smooth muscle cell, except in the aortic smooth muscle cell. It is not expressed in any non-muscle cells (Gallagher, P. J., et al., supra). [0106]
When the APEG-1 polypeptide sequence was compared with those of MLCKs, there was a 33% identity at the amino acid level. However, several lines of evidence indicate that APEG-1 is not a rat homologue of a MLCK. First, peptide sequence comparison of APEG-1 to rat smooth muscle MLCK has only 24% identity, significantly less than the identity between APEG-1 and rabbit or chicken MLCKs. Second, the APEG-1 protein is predicted to be a basic protein, whereas the telokin protein is acidic. Third, rabbit telokin is not expressed in the aorta, in contrast to the specific expression pattern of APEG-1. [0107]
When the APEG-1 protein was analyzed to identify sequence motifs, several residues were identified as capable of being phosphorylated by protein kinase C and casein kinase-2. An arg-gly-asp (RGD) peptide sequence was found at position 90-92. This motif is present in many proteins involved in cell adhesion as well as signaling, and it interacts with its cell surface receptor, an integrin (Hynes, R. O., 1992, Cell 69:11-25, Ruoslahti, E., et al., 1987, Science 238:491-6). This observation suggests that APEG-1 protein plays role in cell signaling. The motif of two cysteine residues, four residues upstream and six residues downstream of the integrin-binding RGD sequence, are also conserved in the disintegrins, a family of platelet aggregation inhibitors found in snake venom (Blobel, C. P., et al., 1992, Curr. Opin. Cell. Biol. 4:760-5). The [0108] cysteine residue 6 residues downstream of the RGD sequence was also found to be present in the APEG-1 protein.

CLONING OF MOUSE APEG-1

The mouse cDNA encoding an APEG-1 open reading frame was first amplified from mouse aortic RNA by reverse transcription polymerase chain reaction (RT-PCR) with primers conserved between the rat and human sequences. Using nested primers designed according to the open reading frame of mAPEG-1, the 3′ end of the mouse cDNA was obtained by 3′ RACE. Both strands of the entire mouse APEG-1 cDNA were sequenced at least once by the dideoxy chain termination sequencing method. [0109]

NORTHERN AND GENOMIC SOUTHERN ANALYSES OF APEG-1

The APEG-1 full length cDNA was used as the probe to hybridize a 12-organ RNA Northern blot. In addition to the 1.3 kb message that appeared in the aorta, two other much larger messages (10-20 kb) were observed in skeletal muscle, esophagus, and heart. These two large messages were not initially identified by the APEG-1 3′-probe; therefore, it is likely the 5′ sequence of APEG-1 cDNA hybridized to these new signals. To test this possibility further, three different probes from the 5′, the middle, and the 3′ portions of the APEG-1 cDNA sequence were used in Northern analysis (FIG. 9A). The result indicated that these 10-20 kb messages were recognized by the 5′ but not by the 3′ portion of the APEG-1 cDNA (FIGS. [0110] 9B-9D).
To determine the relationship of the 1.3 kb aortic transcript and the larger transcripts, a series of probes spanning the APEG-1 gene was used in Northern blot hybridization analyses of RNA isolated from rat aorta, heart, and skeletal muscle. This analysis revealed that the APEG-1 gene defines a muscle cell-specific protein family that encodes both smooth muscle cell-specific proteins and striated muscle cell-specific proteins. The APEG-1 transcripts were detected only in aortic RNA. The large transcripts correspond to variant isoforms, which have been named SPEGs. SPEGs are detected in striated muscle RNA (skeletal and cardiac tissue) but were not seen in aortic RNA. [0111]
The patterns of exon usage in APEG-1 and SPEGs are shown in FIGS. [0112] 19A-C. The APEG-1 gene spans 4.5 kb and is composed of five exons and four introns. SPEG-specific probes detect transcripts 10 and 12 kb in size that are composed of at least seven exons. Three of these exons are shared with the APEG-1 gene, while at least four are unique. The first exon of APEG-1 is separated from the closest upstream SPEG exon by 7 kb. The differential tissue expression patterns of APEG-1 and SPEG arise from utilization of different promoters, alternative splicing, or a combination of the two mechanisms.
The partial nucleotide and amino acid sequences of human SPEG are shown in FIG. 20 and FIG. 21, respectively. The partial nucleotide and amino acid sequences of mouse SPEG are shown in FIG. 22 and [0113] 23, respectively. A comparison of the human and mouse partial SPEG amino acid sequences is shown in FIG. 27.

CHROMOSOMAL LOCATION OF THE APEG-1 GENE

The APEG-1 gene was mapped to human chromosome 2q33-34, which is a region containing several genes involved in cardiovascular disease. [0114]

IDENTIFICATION OF APEG-1 ASSOCIATED SEQUENCES CONFERRING VASCULAR SMOOTH MUSCLE CELL GENE EXPRESSION

To determine whether a smooth muscle cell specific promoter exists 5′ to the first APEG-1 exon, a 3.3 kb APEG-1 5′ flanking sequence was used in a reporter gene transfection analysis using the luciferase reporter plasmid pGL3-C. As shown in FIGS. [0115] 24A-B, a high level of promoter activity directed by the APEG-1 5′ flanking sequence was detected in both rat aortic smooth muscle cells human aortic smooth muscle cells. In contrast, as shown in FIG. 24A-B, minimal activity was detected in five non-smooth muscle cell types, including human cell lines HeLa, HepG2, and U-2 OS.
The sequences responsible for directing a high level of promoter activity have been further localized within the 3.3 kb fragment to the 2.7 kb sequence shown in FIG. 25. [0116]
APEG-1 sequences able to confer vascular smooth muscle cell specific gene expression have been still further localized within the 2.7 kb sequence to a 73 nucleotide sequence (SEQ ID NO:20) shown in FIG. 28. The sequence corresponds to nucleotides 2666 to 2738 of the sequence shown in FIG. 25 (SEQ ID NO:17), and to nucleotides +4 to +76 of the APEG-1 transcript, wherein +1 is the first transcribed nucleotide. The 73 nucleotide sequence includes two AP-2 binding motifs and one E-box binding motif. [0117]
To demonstrate its ability to confer VSMC specific transcription, the 73 bp sequence was cloned in both orientations into the SmaI site of the pGL3-Promoter to generate pGL3-E1box and pGL3-E1Ebox.Rev, respectively. The pGL3-E1box and pGL3-E1Ebox.Rev constructs are shown schematically in the left-hand portion of FIG. 29. The APEG-1 derived sequence is shown as a filled oval, to denote the E-box containing region, and with an arrowhead to indicate the relative orientation of the 73 bp sequence in the two plasmids. Both plasmids in addition contain a promoter (P) derived from SV40. [0118]
Also shown in the schematic diagram are control constructs pGL3-Promoter, which contains the SV40-derived promoter (P) but lacks an enhancer element, and pGL3Control, which contains the SV40 promoter (P) and an SV40 enhancer region (En). The SV40 enhancer region is able to direct transcription in a variety of cell types. [0119]
The constructs were each transfected into rat aortic smooth cells (RASMC), U-2 OS, HeLa, and BAEC cells. Their ability to activate transcription of the SV40 promoter was determined by measuring luciferase activity. The luciferase activity for each construct in the respective cell types is shown in the right-hand portion of FIG. 29. For the data shown, luciferase activity was measured in each cell type as a percent of the luciferase activity of the pGL3-Control. Each bar represents the mean ± SEM. [0120]
In all of the cell types examined, the PGL3-Promoter construct demonstrated negligible luciferase activity. In contrast, the pGL3-Control plasmid, which contains the SV40 enhancer, was active in all cell lines. Both pGL3-E1 Ebox and pGL3-E1 Ebox.Rev expressed levels of luciferase activity comparable to control pGL3-Control only in RASMC. The promoters directed little or no luciferase activity in the U2-OS, HeLa, or BAEC cell lines. These results demonstrate that the 73 bp sequence from APEG-1 activates RASMC-specific transcription in an manner that does not depend on the orientation of the 73 bp sequence with respect to the SV40 promoter. [0121]
The 73 bp sequence was further characterized in gel mobility shift assays for binding activity upon incubation with nuclear extracts. Studies were done using two [0122] 18 bp double-stranded oligonucleotides derived from the mouse APEG-1 exon 1 sequence. One 18-mer oligonucleotide, named the E oligonucleotide, had the sequence 5′- GGGCCTCAGCTGGGTCAG-3′ (SEQ ID NO:21). This sequence corresponds to the E box motif in the 73 bp fragment, as well as 6 nucleotides upstream and downstream of the E box. The second oligonucleotide, named the Emut oligonucleotide, had the sequence 5′-GGGCCTCAGCACGGTCAG-3′ (SEQ ID NO:22). The Emut oligonucleotide was identical in sequence to the E oligonucleotide except that the nucleotide TG in the E box sequence changed to AC in the corresponding positions in the Emut sequence.
Each oligonucleotide was end-labeled with [γ-[0123] ³²P]ATP and incubated with or without RASMC nuclear extract. Omission of RASMC nuclear extract resulted in each labeled oligonucleotide's migrating at the position expected for the free oligonucleotide.
Incubation of the E oligonucleotide with the RASMC retarded the mobility of the oligonucleotide relative to its migration as a free nucleotide. No altered mobility was observed if the labeled E oligonucleotide was incubated with RASMC nuclear extract in the presence of a 100-fold molar excess of unlabeled E oligonucleotide. In contrast, an altered mobility was still observed following incubation of the labeled E oligonucleotide with RASMC nuclear extract in the presence of a 100-fold molar excess of unlabeled Emut oligonucleotide, or with an unlabeled oligonucleotide having a sequence unrelated to the E oligonucleotide. [0124]
No altered mobility was observed upon incubation of labeled Emut oligonucleotide and the RASMC nuclear extract. These results show that the Ebox-containing motif binds to one or more components of RASMC nuclear extracts in a sequence-specific manner. [0125]
Binding to the 73 nucleotide region by a component of RASMC nuclear extracts was also detected in a DNase I footprint assay. An APEG-1 genomic DNA sequence corresponding to the nucleotides from −132 to +76 bp was radiolabeled at either the 5′ or 3′ end with Klenow fragment and [α-[0126] ³²P]dNTP. The end-labeled probes were incubated with either bovine serum albumin (BSA) or RASMC nuclear extract and subjected to varying amounts of DNaseI digestion. Incubation with RASMC nuclear extract resulted in protected regions corresponding to the AP2 and Sp1 binding motifs in the APEG-1 genomic sequence. No protection of these regions was observed upon incubation with BSA.
The AP2 and SP1 regions were similarly protected when the DNaseI studies were performed on a fragment corresponding to nucleotides −490 to +76 of the genomic APEG-1 sequence. Together, the DNase I footprint studies reveal that VSMC nuclear extracts have one or more components that bind to the APEG-1 promoter region.—[0127]
Sequences capable of directing striated muscle specific expression of the SPEGs exons are determined by performing the above-described cell transfection [0128] assays using sequences 5′ to the first SPEG exon.

EXPRESSION OF APEG-1 AND SPEG IN MOUSE DEVELOPMENT

The full-length APEG-1 cDNA was used to probe RNA isolated from mouse embryos at different times in embryonic development. RNA was isolated from the entire embryo for these experiments. The APEG-1 probe hybridized to a 1.3 kb RNA from embryos beginning 9.5 days post-coitus (p.c.) and continuing to 20 days p.c. Strong hybridization was observed to RNA from embryos 11.5 to 20 days p.c. [0129]
APEG-1 transcript levels were also examined postnatally in RNA isolated from rat heart tissue. Hybridization to the APEG-1 probe was detectable in RNA from two-day old rats, but only faint hybridization was detected in RNA from rats aged 14 and 28 days. In situ hybridization experiments of post-natal heart tissue using the APEG-1 probe also revealed a decreased level of APEG-1 RNA. Interestingly, as APEG-1 RNA levels decreased, the levels of SPEG RNAs in striated muscle increased. [0130]
When considered with the tissue specific expression data, these results suggest that APEG-1 transcript levels are high during embryonic development, particularly at day 11.5 p.c. and thereafter. Post-natally, APEG-1 transcript levels, e.g., in cardiac muscle. was generally found to decrease. As global APEG-1 levels decreased, SPEG transcript levels in striated muscle cells increased. [0131]
Southern blot analysis suggested that APEG-1 has a single copy in the rat genome, because there was only one 17.1 kb band in the EcoR I-digested rat genomic DNA (FIG. 10). This result further indicated that the large messages are unlikely to be products of other genes, unless these other genes are closely linked with APEG-1 without any intervening EcoR I sites. From the APEG-1 cDNA sequence two BamH I and one Hind III site were located (FIG. 9A). This correlated with the Southern analysis data in that three bands (18.7, 2.4, and 1.4 kb) in BamH I- and two bands (12.0 and 6.4 kb) in HindIII-digested genomic DNA were identified. [0132]

CLONING OF THE HUMAN APEG-1 CDNA

The APEG-1 cDNA probe was used to screen a human λgt11 aortic5′-stretch cDNA library (Clontech). Four positive clones were purified, and the insert cDNA was sized by EcoRI digestion of the phage DNA and sequenced. The sequence of the human APEG-1 cDNA and the predicted amino acid sequence of the open reading frame encoding human APEG-1 are shown in FIG. 16 and FIG. 17, respectively. [0133]
The human APEG-1 cDNA can then be used to screen a genomic library to obtain the vascular cell-specific promoter sequences which regulate expression cell-specific expression of APEG-1. [0134]

COMPARISON OF THE HUMAN, MOUSE, AND RAT APEG-1 PEPTIDE SEQUENCES

FIG. 26 shows the aligned human, mouse, and rat APEG-1 peptide sequences, along with a derived consensus sequence (SEQ ID NO: 12, 18, 13, and 19). A comparison of the human and rat open reading frames revealed 90% identity at the cDNA level and 97% identity at the amino acid level. Comparison of the open reading frames of mouse and rat APEG-1 revealed 95% identity at the cDNA level and 98% identity at the amino acid level. Thus, APEG-1 is highly conserved across species. [0135]

DEPOSIT

A plasmid containing DNA encoding rat APEG-1 (rat APEG-1 cDNA in pSP72 vector) has been deposited with the American Type Culture Collection (ATCC) under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure on Mar. 3, 1995, and bears the accession number ATCC 97071. A plasmid containing DNA encoding human APEG-1 (human APEG-1 cDNA in pUC18 vector) was deposited with the American Type Culture Collection under the terms of the Budapest Treaty on Jun. 1, 1995, and bears the accession number ATCC 97180. A deposit of a plasmid clone containing 2.7 kb of 5′ flanking sequence of the mouse APEG-1 gene was deposited with the ATCC. on Feb. 5, 1997. Applicants' assignee, President and Fellows of Harvard College, acknowledges its duty to replace the deposit should the depository be unable to furnish a sample when requested due to the condition of the deposit before the end of the term of a patent issued hereon, and its responsibility to notify the ATCC of the issuance of such a patent, at which time the deposit will be made available to the public. Prior to that time, the deposit will be made available to the Commissioner of Patents under the terms of CFR §1.14 and 35 U.S.C. §112. [0136]

THE ABSENCE OF APEG-1 EXPRESSION IN PRIMARY CULTURE CELLS

As discussed above, APEG-1 was initially identified in adventitia-removed aortic tissue, a tissue composed of smooth muscle cells and endothelial cells. To identify which of these two cell types express APEG-1 gene, total RNAs were harvested from primary cultured rat aortic smooth muscle cells and microvascular endothelial cells, both at the second passage, and these RNAs were used in Northern analysis. APEG-1 message was not detected in these cell types (FIG. 12). It is likely that the in vivo expression of APEG-1 was lost during in vitro cell culture. These data suggest that APEG-1 expression is strictly growth-regulated, such that its expression is downregulated when cells are growing in vitro, as has been observed with respect to gas1 gene expression (Sal, G. D., et al., 1992, Cell 70:595-607). Alternatively, since cultured smooth muscle cells are believed to exhibit a dedifferentiated phenotype (Pauly, R. R., et al., 1992, Circulation 86 (suppl III):III-68-73), APEG-1 may be expressed solely in fully differentiated endothelial or smooth muscle cells. Consistent with a role in maintaining a differentiated phenotype, which is characterized by the absence of cell division, microinjected APEG-1 inhibited BrdU uptake in rat arterial smooth muscle cells. APEG-1 expression in vivo was found to be vascular smooth muscle cell-specific, as shown in FIG. 18A and 18B. [0137]

APEG-1 EXPRESSION IN THE BALLOON INJURY ANIMAL MODEL

Since APEG-1 gene expression in vitro is different from that in vivo, APEG-1 expression in vivo was studied. A balloon injury model of the rat carotid artery, which has been used extensively to study vascular smooth muscle cells in atherogenesis and vascular remodeling (Clowes, A. W., et al., 1983, Lab. Invest. 49(2):208-15, Clowes, A. W. et al., 1985, Circ. Res. 56:139-45), was used to study the expression modulation of APEG-1. In this animal model, the rat left carotid artery was injured by a 2F balloon catheter, intimal arterial endothelial cells completely removed, and the medial smooth muscle cell layer distended. After the carotid injury, formation of the neointima was initiated. This involves smooth muscle cells proliferating and migrating from the media. With this model, medial and neointimal smooth muscle cells reach their respective highest rates of proliferation two days and four days after the balloon injury, declining rapidly thereafter. The total number of smooth muscle cells approaches a maximum and remains constant after two weeks (Clowes, A. W. et al., 1985, supra). [0138]
Total RNAs from rat [0139] carotid arteries 2, 5, and 8 days after balloon injury were collected and used in Northern analysis with an APEG-1 cDNA probe. The results showed that APEG-1 is downregulated to 15%-20% of non-injured carotid arteries after 2 and 5 days; the expression recovered to 40% of control after 8 days (FIGS. 13A and 13B). These data suggest that APEG-1 expression is involved in the regulation of smooth muscle cell proliferation and/or migration, and expression has to be suppressed for either or both events to occur.

PRODUCTION AND PURIFICATION OF RECOMBINANT APEG-1

Recombinant APEG-1 was expressed as a fusion protein and purified by the pFLAG expression system (IBI) and subsequently injected into rabbit to produce antiserum. The rat APEG-1 cDNA was cloned into pFLAG-2 expression vector and used to transform the [0140] E. coli BL21 cells. The transformed cells were grown and induced by IPTG (isopropyl-β-D-thio-galactopyroside) to express the vector-encoded fusion protein. The FLAG-APEG-1 fusion protein was then purified by anti-FLAG monoclonal antibody affinity chromatography from soluble cell extract, and the purity was monitored by both Coomassie blue staining (FIG. 14A) and Western analysis (FIG. 14B).

APEG-1 CELLULAR LOCALIZATION

To determine the cellular localization of APEG-1, a plasmid was generated, c-myc-rAPEG-1/pCR3, that would express a fusion protein of APEG-1 with an N-terminal c-Myc tag. COS-7 cells were then transiently transfected with the c-myc-rAPEG-1/pCR3 plasmid and immunostained with a monoclonal anti-c-Myc antibody, 9E10. The c-Myc-tagged protein was expressed predominantly in the nuclei of transfected COS-7 cells. The same result was obtained when U-2 OS cells were used as the host cells. [0141]

METHODS OF DIAGNOSIS

The invention includes a method of detecting injury in a sample of vascular tissue. A depressed level of APEG-1 would predict a high degree of smooth muscle cell proliferation indicative of vascular tissue injury, e.g., restenosis. The diagnostic method of the invention is carried out by determining the level of APEG-1 gene expression in a tissue, e.g, a vascular biopsy obtained at atherectomy. The level of gene expression may be measured using methods known in the art, e.g., in situ hybridization, Northern blot analysis, or Western blot analysis using APEG-1-specific monoclonal or polyclonal antibodies. A decrease in the level of APEG-1 expression per cell in the test sample of tissue compared to the level per cell in uninjured control vascular tissue indicates the presence of a vascular injury in the test sample. For example, tissue obtained at atherectomy could be tested for APEG-1 expression, e.g., the level of APEG-1 transcript or protein. A depressed level of APEG-1 (compared to normal tissue) correlates with a high degree of smooth muscle cell proliferation indicating a high probability of restenosis. Such diagnostic procedures are useful to identify patients in need of therapeutic intervention to reduce or prevent restenosis. [0142]

METHODS OF DETECTING SPECIFIC TYPES OF MUSCLE CELLS

Because APEG-1 and SPEG mRNAs are enriched in vascular smooth muscle cells and in striated muscle cells, respectively, the APEG-1 and SPEG nucleic acid sequences can be used as probes to identify these cell types. For example, an APEG-1 specific nucleic acid sequence, e.g., a probe corresponding to an APEG-1 specific exon, is hybridized, using methods well known in the art, to RNA sequences in Northern blot hybridization studies or using in situ hybridization assays. Reactivity to an APEG-1 specific probe is indicative of a vascular smooth muscle cell tissue. Similarly, a SPEG-specific nucleic acid sequence, e.g., a probe corresponding to a SPEG-specific exon, is used to identify striated muscle cells. [0143]
APEG-1 and SPEG DNA sequences can also be used to make recombinant APEG-1 and SPEG polypeptides, or fragments thereof. Monoclonal or polyclonal antibodies are then raised to the recombinant polypeptides using methods well-known in the art. The anti-SPEG antibodies are then used, e.g., in western or immunofluorescence experiments, to identify vascular smooth muscle cells, in the case of APEG-1, or striated muscle cells in the case of SPEG. [0144]

METHODS OF THERAPY

Upon vascular injury and other stimuli, cytokines and growth factors from activated vascular cells promote growth and migration of dedifferentiated vascular smooth muscle cells, resulting in atherosclerotic plaques and restenosis. Administration of APEG-1 polypeptide to vascular smooth muscle cells in vitro (by microinjection) resulted in a decrease in DNA synthesis, indicative of a decrease in cellular proliferation. Vascular injury such as that caused during surgery or balloon angioplasty can be treated by administering APEG-1 polypeptides or DNA encoding APEG-1 polypeptides operably linked to appropriate expression control sequences. Other vascular conditions, e.g., atherosclerosis, transplant arteriosclerosis, and diabetes, which are characterized by a decrease in APEG-1 expression (FIG. 15) may be treated in a similar manner. APEG-1 polypeptide, DNA encoding an APEG-1 polypeptide, or compositions which stimulate the APEG-1 promoter may administered to increase the level of APEG-1 polypeptide in the injured vascular tissue and thus inhibit the growth of smooth muscle cells. [0145]
APEG-1 polypeptides may be administered to the patient intravenously in a pharmaceutically acceptable carrier such as physiological saline. Standard methods for intracellular delivery of peptides can be used, e.g. packaged in liposomes. Such methods are well known to those of ordinary skill in the art. It is expected that an intravenous dosage of approximately 1 to 100 μmoles of the polypeptide of the invention would be administered per kg of body weight per day. The compositions of the invention are useful for parenteral administration, such as intravenous, subcutaneous, intramuscular, and intraperitoneal. [0146]
DNA (e.g., APEG-1-encoding DNA, vascular cell-specific promoters (e.g., SEQ ID NO:17 or SEQ ID NO:20), SPEG-encoding DNA, and striated muscle cell-specific promoters) and vectors of the invention may be introduced into target cells of the patient by standard vectors and/or gene delivery systems. Suitable gene delivery systems may include liposomes, receptor-mediated delivery systems, naked DNA, and viral vectors such as herpes viruses, retroviruses, and adenoviruses, among others. For example, the DNA under encoding an APEG-1 or SPEG polypeptide under the control of a strong constitutive promoter may be administered locally to a blood vessel during balloon angioplasty using an adenovirus delivery system. [0147]
A vascular cell-specific promoter or promoter/enhancer sequence derived from the APEG-1 gene (e.g., SEQ ID NO:17 or SEQ ID NO:20) may be used to direct the expression of APEG-1 or genes other than APEG-1. Thus, vascular diseases may be treated by administering a vascular cell-specific promoter/enhancer sequence of the invention operably linked to a sequence encoding a heterologous polypeptide, e.g., an APEG-1 promoter linked to DNA encoding a growth inhibitor gene such as Rb, p21 or p18. [0148]
The DNA may encode a naturally occurring mammalian APEG-1 polypeptide such as a rat APEG-1 polypeptide (SEQ ID NO:3) or human APEG-1 polypeptide (SEQ ID NO:12). For example, the invention includes degenerate variants of SEQ ID NO:2 or SEQ ID NO:11. The invention also includes a substantially pure DNA comprising a strand which hybridizes at high stringency to a DNA having the sequence of SEQ ID NO:1, 2, or 11, or the complements thereof. [0149]
Similarly, a striated muscle cell specific-promoter may be used to direct expression of SPEG or genes other than SPEG. Thus, striated muscle diseases may be treated by administering a striated muscle cell-specific promoter of the invention operably linked to a sequence encoding a heterologous polypeptide, e.g., a SPEG promoter linked to DNA encoding a therapeutic gene, e.g., dystrophin to treat Duchenne's or Becker's muscular dystrophy, or a growth inhibitor gene such as Rb, p21, or p18, to reduce undesirable proliferation of striated muscle cells. [0150]
The DNA of the invention may be administered in a pharmaceutically acceptable carrier. The therapeutic composition may also include a gene delivery system as described above. Pharmaceutically acceptable carriers are biologically compatible vehicles which are suitable for administration to an animal e.g., physiological saline. A therapeutically effective amount is an amount of the nucleic acid of the invention which is capable of producing a medically desirable result in a treated animal. [0151]
As is well known in the medical arts, dosage for any given patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Dosages for the compounds of the invention will vary, but a preferred dosage for intravenous administration is from approximately 10[0152] ⁶to 10²²copies of the nucleic acid molecule. Determination of optimal dosage is well within the abilities of a pharmacologist of ordinary skill.
Drugs which stimulate the APEG-1 promoter may also be administered as described above to increase the level of expression APEG-1 in vascular tissue. Such drugs can be identified by contacting the APEG-1 promoter linked to a reporter gene with a candidate compound and measuring the level of expression of the reporter gene in the presence and absence of the compound. An increased level of expression in the presence of the compound compared to that in its absence indicates that the compound stimulates the APEG-1 promoter. [0153]
The invention also includes cells transfected with the DNA of the invention. Standard methods for transfecting cells with isolated nucleic acid are well known to those skilled in the art of molecular biology. Preferably, the cells are vascular smooth muscle cells, and they express an APEG-[0154] 1 polypeptide of the invention encoded by the nucleic acid of the invention. Cells of the invention may be administered to an animal locally or systemically using intravenous, subcutaneous, intramuscular, and intraperitoneal delivery methods. Alternatively, prokaryotic or eukaryotic cells in culture can be transfected with the DNA of the invention operably linked to expression control sequences appropriate for high-level expression in the cell. Such cells are useful for producing large amounts of the APEG-1 polypeptide, which can be purified and used, e.g., as a therapeutic or for raising anti-APEG-1 antibodies.
Other embodiments are within the following claims. [0155]
1 20 1 1308 DNA Rattus rattus CDS (170)...(508) 1 gaattcggca cgagcagaga cttaaggaag gtgcagacgg ggtccgtttg cacagcctca 60 gggcgcgtcc acatccccct tcagcagccc aatcacctct gatgaggagt acctgagccc 120 cccagaggag ttcccagaac ctggggagac ctggtcccga acccctacc atg aag ccc 178 Met Lys Pro 1 agt ccc agc cag gat cga gat tcc tct gac tct tcc tcc aag gca ccc 226 Ser Pro Ser Gln Asp Arg Asp Ser Ser Asp Ser Ser Ser Lys Ala Pro 5 10 15 cca acc ttc aag gtc tca ctc atg gac caa tca gtg aga gaa ggt caa 274 Pro Thr Phe Lys Val Ser Leu Met Asp Gln Ser Val Arg Glu Gly Gln 20 25 30 35 gat gtc att atg agc atc cgc gtg cag ggg gag ccc aag cct gtg gtc 322 Asp Val Ile Met Ser Ile Arg Val Gln Gly Glu Pro Lys Pro Val Val 40 45 50 tcc tgg ctg agg aat cgg cag cct gtg cgc cca gac cag cgg cgc ttt 370 Ser Trp Leu Arg Asn Arg Gln Pro Val Arg Pro Asp Gln Arg Arg Phe 55 60 65 gca gag gag gcc gag ggt ggg ctc tgc cgg ttg agg atc ctg gct gct 418 Ala Glu Glu Ala Glu Gly Gly Leu Cys Arg Leu Arg Ile Leu Ala Ala 70 75 80 gag agg gga gat gct ggt ttc tac act tgc aag gcg gtc aac gaa tat 466 Glu Arg Gly Asp Ala Gly Phe Tyr Thr Cys Lys Ala Val Asn Glu Tyr 85 90 95 ggc gct cgg cag tgt gag gcc cgc ctg gag gtc cga ggc gag 508 Gly Ala Arg Gln Cys Glu Ala Arg Leu Glu Val Arg Gly Glu 100 105 110 tgagctcagg gggccacctg cgctgccccc gctaccctcc gagctgcacc cctgtctcag 568 gcacctcctg gacctcgctg tgtttcactg cctcctgccc acagacccag ccggctcgcc 628 ggcccggaca tagcccatgc tccccttccc tccctagccc atacagcacc ctggggtaac 688 ccatcgggcc cctgtggatc ctccctcccc aagtggatat gtggctgtgc agaccaggag 748 gcccccagaa ggactgagtg ttgagaaggg atggccatga ggttgtgaca agctcccccc 808 gtccccagcc tccatgtagg gagcatccag cgaatgcatg tgctatgctg ctacaggcca 868 ctgtctgtct ctctgtctgt ctgcctgtgt gtctgtgaca gtcagggaag aaaaccttcg 928 agctgaggtg ggataagaca gaataagatg atagaacaca gcatctgtga gatgcagggg 988 cccagagggg caggcacagt ggataggaga ctctctggga agggtagggc actgaccatt 1048 gcagaaatgg gttttaaatg gcacaacatt ttttattcca catgagacca aaagctagag 1108 gtctgggatt aagccctgac tgctggcaag cttaggacca agtggggtac ccttcttcac 1168 agacacatcc gacacgctct gtctgggaat gagagagtag ccagactgag cacaggagca 1228 ggtcatagtg ggactggagg tttggaaaca ctatttcgta gctcaaataa agtccagttt 1288 gtacccaaaa aaaaaaaaaa 1308 2 342 DNA Rattus rattus 2 atgaagccca gtcccagcca ggatcgagat tcctctgact cttcctccaa ggcaccccca 60 accttcaagg tctcactcat ggaccaatca gtgagagaag gtcaagatgt cattatgagc 120 atccgcgtgc agggggagcc caagcctgtg gtctcctggc tgaggaatcg gcagcctgtg 180 cgcccagacc agcggcgctt tgcagaggag gccgagggtg ggctctgccg gttgaggatc 240 ctggctgctg agaggggaga tgctggtttc tacacttgca aggcggtcaa cgaatatggc 300 gctcggcagt gtgaggcccg cctggaggtc cgaggcgagt ga 342 3 113 PRT Rattus rattus 3 Met Lys Pro Ser Pro Ser Gln Asp Arg Asp Ser Ser Asp Ser Ser Ser 1 5 10 15 Lys Ala Pro Pro Thr Phe Lys Val Ser Leu Met Asp Gln Ser Val Arg 20 25 30 Glu Gly Gln Asp Val Ile Met Ser Ile Arg Val Gln Gly Glu Pro Lys 35 40 45 Pro Val Val Ser Trp Leu Arg Asn Arg Gln Pro Val Arg Pro Asp Gln 50 55 60 Arg Arg Phe Ala Glu Glu Ala Glu Gly Gly Leu Cys Arg Leu Arg Ile 65 70 75 80 Leu Ala Ala Glu Arg Gly Asp Ala Gly Phe Tyr Thr Cys Lys Ala Val 85 90 95 Asn Glu Tyr Gly Ala Arg Gln Cys Glu Ala Arg Leu Glu Val Arg Gly 100 105 110 Glu 4 113 PRT Gallus gallus 4 Met Lys Pro Ser Pro Ser Gln Asp Arg Asp Ser Ser Asp Ser Ser Ser 1 5 10 15 Lys Ala Pro Pro Thr Phe Lys Val Ser Leu Met Asp Gln Ser Val Arg 20 25 30 Glu Gly Gln Asp Val Ile Met Ser Ile Arg Val Gln Gly Glu Pro Lys 35 40 45 Pro Val Val Ser Trp Leu Arg Asn Arg Gln Pro Val Arg Pro Asp Gln 50 55 60 Arg Arg Phe Ala Glu Glu Ala Glu Gly Gly Leu Cys Arg Leu Arg Ile 65 70 75 80 Leu Ala Ala Glu Arg Gly Asp Ala Gly Phe Tyr Thr Cys Lys Ala Val 85 90 95 Asn Glu Tyr Gly Ala Arg Gln Cys Glu Ala Arg Leu Glu Val Arg Gly 100 105 110 Glu 5 157 PRT Gallus gallus 5 Met Ala Met Ile Ser Gly Met Ser Gly Arg Lys Ala Ser Gly Ser Ser 1 5 10 15 Pro Thr Ser Pro Ile Asn Ala Asp Lys Val Glu Asn Glu Asp Ala Phe 20 25 30 Leu Glu Glu Val Ala Glu Glu Lys Pro His Val Lys Pro Tyr Phe Thr 35 40 45 Lys Thr Ile Leu Asp Met Glu Val Val Glu Gly Ser Ala Ala Arg Phe 50 55 60 Asp Cys Lys Ile Glu Gly Tyr Pro Asp Pro Glu Val Met Trp Tyr Lys 65 70 75 80 Asp Asp Gln Pro Val Lys Glu Ser Arg His Phe Gln Ile Asp Tyr Asp 85 90 95 Glu Glu Gly Asn Cys Ser Leu Thr Ile Ser Glu Val Cys Gly Asp Asp 100 105 110 Asp Ala Lys Tyr Thr Cys Lys Ala Val Asn Ser Leu Gly Glu Ala Thr 115 120 125 Cys Thr Ala Glu Leu Leu Val Glu Thr Met Gly Lys Glu Gly Glu Gly 130 135 140 Glu Gly Glu Gly Glu Glu Asp Glu Glu Glu Glu Glu Glu 145 150 155 6 155 PRT Leporidae 6 Met Ala Met Ile Ser Gly Leu Ser Gly Arg Lys Ser Ser Thr Gly Ser 1 5 10 15 Pro Thr Ser Pro Leu Thr Ala Glu Arg Leu Glu Thr Glu Glu Asp Val 20 25 30 Ser Gln Ala Phe Leu Glu Ala Val Ala Glu Glu Lys Pro His Val Lys 35 40 45 Pro Tyr Phe Ser Lys Thr Ile Arg Asp Leu Glu Val Val Glu Gly Ser 50 55 60 Ala Ala Arg Phe Asp Cys Lys Ile Glu Gly Tyr Pro Asp Pro Glu Val 65 70 75 80 Val Trp Phe Lys Asp Asp Gln Ser Ile Arg Glu Ser Arg His Phe Gln 85 90 95 Ile Asp Tyr Asp Glu Asp Gly Asn Cys Ser Leu Ile Ile Ser Asp Val 100 105 110 Cys Gly Asp Asp Asp Ala Lys Tyr Thr Cys Lys Ala Val Asn Ser Leu 115 120 125 Gly Glu Ala Thr Cys Thr Ala Glu Leu Ile Val Glu Thr Met Glu Glu 130 135 140 Gly Glu Gly Glu Gly Glu Glu Glu Glu Glu Glu 145 150 155 7 155 PRT Leporidae 7 Met Ala Met Ile Ser Gly Leu Ser Gly Arg Lys Ser Ser Thr Gly Ser 1 5 10 15 Pro Thr Ser Pro Leu Thr Ala Glu Arg Leu Glu Thr Glu Glu Asp Val 20 25 30 Ser Gln Ala Phe Leu Glu Ala Val Ala Glu Glu Lys Pro His Val Lys 35 40 45 Pro Tyr Phe Ser Lys Thr Ile Arg Asp Leu Glu Val Val Glu Gly Ser 50 55 60 Ala Ala Arg Phe Asp Cys Lys Ile Glu Gly Tyr Pro Asp Pro Glu Val 65 70 75 80 Val Trp Phe Lys Asp Asp Gln Ser Ile Arg Glu Ser Arg His Phe Gln 85 90 95 Ile Asp Tyr Asp Glu Asp Gly Asn Cys Ser Leu Ile Ile Ser Asp Val 100 105 110 Cys Gly Asp Asp Asp Ala Lys Tyr Thr Cys Lys Ala Val Asn Ser Leu 115 120 125 Gly Glu Ala Thr Cys Thr Ala Glu Leu Ile Val Glu Thr Met Glu Glu 130 135 140 Gly Glu Gly Glu Gly Glu Glu Glu Glu Glu Glu 145 150 155 8 113 PRT Rattus rattus 8 Met Lys Pro Ser Pro Ser Gln Asp Arg Asp Ser Ser Asp Ser Ser Ser 1 5 10 15 Lys Ala Pro Pro Thr Phe Lys Val Ser Leu Met Asp Gln Ser Val Arg 20 25 30 Glu Gly Gln Asp Val Ile Met Ser Ile Arg Val Gln Gly Glu Pro Lys 35 40 45 Pro Val Val Ser Trp Leu Arg Asn Arg Gln Pro Val Arg Pro Asp Gln 50 55 60 Arg Arg Phe Ala Glu Glu Ala Glu Gly Gly Leu Cys Arg Leu Arg Ile 65 70 75 80 Leu Ala Ala Glu Arg Gly Asp Ala Gly Phe Tyr Thr Cys Lys Ala Val 85 90 95 Asn Glu Tyr Gly Ala Arg Gln Cys Glu Ala Arg Leu Glu Val Arg Gly 100 105 110 Glu 9 35 PRT Artificial Sequence synthetic consensus sequence 9 Ser Arg Ser Ser Pro Pro Phe Asp Val Glu Gly Gly Pro Pro Val Trp 1 5 10 15 Gln Glu Gly Cys Leu Ile Asp Tyr Thr Cys Lys Ala Val Asn Gly Cys 20 25 30 Ala Leu Val 35 10 14 DNA Artificial Sequence synthetic poly T anchoring primer 10 tttttttttt ttvg 14 11 1238 DNA Homo sapiens CDS (88)...(426) misc_feature (1)...(1238) n = A,T,C or G 11 tcacctctga tgaggaatac ctgagccccc cagaggagtt cccagagcct ggggagacct 60 ggggagacct ggccgcgaac ccccacc atg aag ccc agt ccc agc cag gac cgc 114 Met Lys Pro Ser Pro Ser Gln Asp Arg 1 5 cgt tct tct gac act ggc tcc aag gca ccc ccc acc ttc aag gtc tca 162 Arg Ser Ser Asp Thr Gly Ser Lys Ala Pro Pro Thr Phe Lys Val Ser 10 15 20 25 ctt atg gac cag tca gta aga gaa ggc caa gat gtc atc atg agc atc 210 Leu Met Asp Gln Ser Val Arg Glu Gly Gln Asp Val Ile Met Ser Ile 30 35 40 cgc gtg cag ggg gag ccc aag cct gtg gtc tcc tgg ctg aga aac cgc 258 Arg Val Gln Gly Glu Pro Lys Pro Val Val Ser Trp Leu Arg Asn Arg 45 50 55 cag ccc gtg cgc cca gac cag cgg cgc ttt gcg gag gag gct gag ggt 306 Gln Pro Val Arg Pro Asp Gln Arg Arg Phe Ala Glu Glu Ala Glu Gly 60 65 70 ggg ctg tgc cgg ctg cgg atc ctg gct gca gag cgt ggc gat gct ggt 354 Gly Leu Cys Arg Leu Arg Ile Leu Ala Ala Glu Arg Gly Asp Ala Gly 75 80 85 ttc tac act tgc aaa gcg gtc aat gag tat ggt gct cgg cag tgc gag 402 Phe Tyr Thr Cys Lys Ala Val Asn Glu Tyr Gly Ala Arg Gln Cys Glu 90 95 100 105 gcc cgc ttg gag gtc cga ggc gag tgagctcagg gggccacctg cgctcccccc 456 Ala Arg Leu Glu Val Arg Gly Glu 110 gctaccctcc gagccgcgcc cctgtctcag gcacctctcg gacctcgctg tgtttcactg 516 cctcctgccc acagacccag gcctgccggc ccggacccgt cccagcctcc cctccccacc 576 ccatgcagcc cccaggggga tagcccatgg gcccctgtgg acactccctc cccaagtgga 636 cacatggctg tgcaggccag gaggcccaca gatggactga gtgctgggaa ggggcggctt 696 cgaggggtat caaccccccg agtctctccc tgaaggggag caccgggcga gtgcatgtgc 756 tactgctgct acaggcctgt ctatctgttt gtctgtctgt gtgtctgtga cagtcaggga 816 aggatgcctc ggagctgagg tggggtgaga cagagtggga gagattacgg catggcatgg 876 aggggcccaa ggagcagggg ctgttgacaa aggccttacc aggaagggtt aggacactga 936 ccattctaga aatgggtttc gaatggcaca acactttcta tttcacaaaa gaccaaaagc 996 cagaggcccc aggctctgtg ctgatgaaca gcctggctga gccctggccc tggcaggttt 1056 agggcccatt tggggccccc tccttctctg tcagggctgg ggtgctctgt ctgggaatga 1116 gggagttaac caagtttggt gcaggagcag gggcaggggg ccactgtagt gagcgtggat 1176 gaaatttgga nacacctatn tcttaantca aataaagtcc agtttgtacc taaaaaaaaa 1236 aa 1238 12 113 PRT Homo sapiens 12 Met Lys Pro Ser Pro Ser Gln Asp Arg Arg Ser Ser Asp Thr Gly Ser 1 5 10 15 Lys Ala Pro Pro Thr Phe Lys Val Ser Leu Met Asp Gln Ser Val Arg 20 25 30 Glu Gly Gln Asp Val Ile Met Ser Ile Arg Val Gln Gly Glu Pro Lys 35 40 45 Pro Val Val Ser Trp Leu Arg Asn Arg Gln Pro Val Arg Pro Asp Gln 50 55 60 Arg Arg Phe Ala Glu Glu Ala Glu Gly Gly Leu Cys Arg Leu Arg Ile 65 70 75 80 Leu Ala Ala Glu Arg Gly Asp Ala Gly Phe Tyr Thr Cys Lys Ala Val 85 90 95 Asn Glu Tyr Gly Ala Arg Gln Cys Glu Ala Arg Leu Glu Val Arg Gly 100 105 110 Glu 13 2793 DNA Homo sapiens CDS (3)...(1985) 13 ga att ccg gtc caa atc cgc gct gct ccc ccc acc gtc ccc tcg ggt 47 Ile Pro Val Gln Ile Arg Ala Ala Pro Pro Thr Val Pro Ser Gly 1 5 10 15 cgg gaa gcg gtc ccc gcc ggg acc ccc ggc cca gcc cgc ggc cac ccc 95 Arg Glu Ala Val Pro Ala Gly Thr Pro Gly Pro Ala Arg Gly His Pro 20 25 30 cac gtc gcc cca ccg tcg cac tca gga gcc tgt gct gcc cga gga cac 143 His Val Ala Pro Pro Ser His Ser Gly Ala Cys Ala Ala Arg Gly His 35 40 45 cac cac cga aga gaa gcg agg gaa gaa gtc caa gtc gtc cgg gcc ctc 191 His His Arg Arg Glu Ala Arg Glu Glu Val Gln Val Val Arg Ala Leu 50 55 60 cct ggc ggg cac cgg gaa tcc cga ccc cag acg cca ctg agc gag gcc 239 Pro Gly Gly His Arg Glu Ser Arg Pro Gln Thr Pro Leu Ser Glu Ala 65 70 75 tca ggc cgc ctg tgg gcg ttg ggc cga tcg cct agg ctg gtg cgc gcc 287 Ser Gly Arg Leu Trp Ala Leu Gly Arg Ser Pro Arg Leu Val Arg Ala 80 85 90 95 ggc tcc cgc atc ctg gac aag ctg cag ttc ttc gag gag cga cgg cgc 335 Gly Ser Arg Ile Leu Asp Lys Leu Gln Phe Phe Glu Glu Arg Arg Arg 100 105 110 agc ctg gag cgc agc gac tcg ccg ccg gcg ccc ctg cgg ccc tgg gtg 383 Ser Leu Glu Arg Ser Asp Ser Pro Pro Ala Pro Leu Arg Pro Trp Val 115 120 125 ccc ctg cgc aag gcc cgc tct ctg gag cag ccc aag tcg gag cgc ggc 431 Pro Leu Arg Lys Ala Arg Ser Leu Glu Gln Pro Lys Ser Glu Arg Gly 130 135 140 gca ccg tgg ggc acc ccc ggg gcc tcg cag gaa gaa ctg cgg gcg cca 479 Ala Pro Trp Gly Thr Pro Gly Ala Ser Gln Glu Glu Leu Arg Ala Pro 145 150 155 ggc agc gtg gcc gag cgg cgc cgc ctg ttc cag cag aaa gcg gcc tcg 527 Gly Ser Val Ala Glu Arg Arg Arg Leu Phe Gln Gln Lys Ala Ala Ser 160 165 170 175 ctg gac gag cgc acg cgt cag cgc agc ccg gcc tca gac ctc gag ctg 575 Leu Asp Glu Arg Thr Arg Gln Arg Ser Pro Ala Ser Asp Leu Glu Leu 180 185 190 cgc ttc gcc cag gag ctg ggc cgc atc cgc cgc tcc acg tcg cgg gag 623 Arg Phe Ala Gln Glu Leu Gly Arg Ile Arg Arg Ser Thr Ser Arg Glu 195 200 205 gag ctg gtg cgc tcg cac gag tcc ctg cgc gcc acg ctg cag cgt gcc 671 Glu Leu Val Arg Ser His Glu Ser Leu Arg Ala Thr Leu Gln Arg Ala 210 215 220 cca tcc cct cga gag ccc ggc gag ccc ccg ctc ttc tct cgg ccc tcc 719 Pro Ser Pro Arg Glu Pro Gly Glu Pro Pro Leu Phe Ser Arg Pro Ser 225 230 235 acc ccc aag aca tcg cgg gcc gtg agc ccc gcc gcc gcc cag ccg ccc 767 Thr Pro Lys Thr Ser Arg Ala Val Ser Pro Ala Ala Ala Gln Pro Pro 240 245 250 255 tct ccg agc agc gcg gag aag ccg ggg gac gag cct ggg agg ccc agg 815 Ser Pro Ser Ser Ala Glu Lys Pro Gly Asp Glu Pro Gly Arg Pro Arg 260 265 270 agc cgc ggg ccg gcg ggc agg aca gag ccg ggg gaa ggc ccg cag cag 863 Ser Arg Gly Pro Ala Gly Arg Thr Glu Pro Gly Glu Gly Pro Gln Gln 275 280 285 gag gtt agg cgt cgg gac caa ttc ccg ctg acc cgg agc aga gcc atc 911 Glu Val Arg Arg Arg Asp Gln Phe Pro Leu Thr Arg Ser Arg Ala Ile 290 295 300 cag gag tgc agg agc cct gtg ccg ccc ccc gcc gcc gat ccc cca gag 959 Gln Glu Cys Arg Ser Pro Val Pro Pro Pro Ala Ala Asp Pro Pro Glu 305 310 315 gcc agg acg aaa gca ccc ccc ggt cgg aag cgg gag ccc ccg gcg cag 1007 Ala Arg Thr Lys Ala Pro Pro Gly Arg Lys Arg Glu Pro Pro Ala Gln 320 325 330 335 gcc gtg cgc ttc ctg ccc tgg gcc acg ccg ggc ctg gag ggc gct gct 1055 Ala Val Arg Phe Leu Pro Trp Ala Thr Pro Gly Leu Glu Gly Ala Ala 340 345 350 gta ccc cag acc ttg gag aag aac agg gcg ggg cct gag gca gag aag 1103 Val Pro Gln Thr Leu Glu Lys Asn Arg Ala Gly Pro Glu Ala Glu Lys 355 360 365 agg ctt cgc aga ggg ccg gag gag gac ggt ccc tgg ggg ccc tgg gac 1151 Arg Leu Arg Arg Gly Pro Glu Glu Asp Gly Pro Trp Gly Pro Trp Asp 370 375 380 cgc cga ggg gcc cgc agc cag ggc aaa ggt cgc cgg gcc cgg ccc acc 1199 Arg Arg Gly Ala Arg Ser Gln Gly Lys Gly Arg Arg Ala Arg Pro Thr 385 390 395 tcc cct gag ctc gag tct tcg gat gac tcc tac gtg tcc gct gga gaa 1247 Ser Pro Glu Leu Glu Ser Ser Asp Asp Ser Tyr Val Ser Ala Gly Glu 400 405 410 415 gag ccc cta gag gcc cct gtg ttt gag atc ccc ctg cag aat gtg gtg 1295 Glu Pro Leu Glu Ala Pro Val Phe Glu Ile Pro Leu Gln Asn Val Val 420 425 430 gtg gca cca ggg gca gat gtg ctg ctc aaa tgt atc atc act gcc aac 1343 Val Ala Pro Gly Ala Asp Val Leu Leu Lys Cys Ile Ile Thr Ala Asn 435 440 445 ccc ccg ccc caa gtg tcc tgg cac aag gat ggg tca gcg ctg cgc agc 1391 Pro Pro Pro Gln Val Ser Trp His Lys Asp Gly Ser Ala Leu Arg Ser 450 455 460 gag ggc cgc ctc ctc ctc cgg gct gag ggt gag cgg cac acc ctg ctg 1439 Glu Gly Arg Leu Leu Leu Arg Ala Glu Gly Glu Arg His Thr Leu Leu 465 470 475 ctc agg gag gcc agg gca gca gat gcc ggg agc tat atg gcc acc gcc 1487 Leu Arg Glu Ala Arg Ala Ala Asp Ala Gly Ser Tyr Met Ala Thr Ala 480 485 490 495 acc aac gag ctg ggc cag gcc acc tgt gcc gcc tca ctg acc gtg aga 1535 Thr Asn Glu Leu Gly Gln Ala Thr Cys Ala Ala Ser Leu Thr Val Arg 500 505 510 ccc ggt ggg tct aca tcc cct ttc agc agc ccc atc acc tcc gac gag 1583 Pro Gly Gly Ser Thr Ser Pro Phe Ser Ser Pro Ile Thr Ser Asp Glu 515 520 525 gaa tac ctg agc ccc cca gag gag ttc cca gag cct ggg gag acc tgg 1631 Glu Tyr Leu Ser Pro Pro Glu Glu Phe Pro Glu Pro Gly Glu Thr Trp 530 535 540 ccg cga acc ccc acc atg aag ccc agt ccc agc cag aac cgc cgt tct 1679 Pro Arg Thr Pro Thr Met Lys Pro Ser Pro Ser Gln Asn Arg Arg Ser 545 550 555 tct gac act ggc tcc aag gca ccc ccc acc ttc aag gtc tca ctt atg 1727 Ser Asp Thr Gly Ser Lys Ala Pro Pro Thr Phe Lys Val Ser Leu Met 560 565 570 575 gac cag tca gta aga gaa ggc caa gat gtc atc atg agc atc cgc gtg 1775 Asp Gln Ser Val Arg Glu Gly Gln Asp Val Ile Met Ser Ile Arg Val 580 585 590 cag ggg gag ccc aag cct gtg gtc tcc tgg ctg aga aac cgc cag ccc 1823 Gln Gly Glu Pro Lys Pro Val Val Ser Trp Leu Arg Asn Arg Gln Pro 595 600 605 gtg cgc cca gac cag cgg cgc ttt gcg gag gag gct gag ggt ggg ctg 1871 Val Arg Pro Asp Gln Arg Arg Phe Ala Glu Glu Ala Glu Gly Gly Leu 610 615 620 tgc cgg ctg cgg atc ctg gct gca gag cgt ggc gat gct ggt ttc tac 1919 Cys Arg Leu Arg Ile Leu Ala Ala Glu Arg Gly Asp Ala Gly Phe Tyr 625 630 635 act tgc aaa gcg gtc aat gag tat ggt gct cgg cag tgc gag gcc cgc 1967 Thr Cys Lys Ala Val Asn Glu Tyr Gly Ala Arg Gln Cys Glu Ala Arg 640 645 650 655 ttg gag gtc cga ggc gag tgagctcagg gggccacctg cgctcccccc 2015 Leu Glu Val Arg Gly Glu 660 gctaccctcc gagccgcgcc cctgtctcag gcacctctcg gacctcgctg tgtttcactg 2075 cctcctgccc acagacccag ctgccggccc ggacccgtcc cagcctcccc tccccacccc 2135 atgcagcccc cagggggata gcccatgggc ccctgtggac cctccctccc caagtggaca 2195 catggctgtg cagccaggag gcccacagat ggactgagtg ctgggaaggg gcggctgcga 2255 ggggtatcaa ccccccgagt ctctccctga aggggagcac cgggcgagtg catgtgctac 2315 tgctgctaca ggcctgtcta tctgtttgtc tgtctgtgtg tctgtgacag tcagggaagg 2375 atgcctcgga gctgaggtgg ggtgagacag agtgggagag attacggcat ggcatggagg 2435 ggcccaagga gcaggggctg ttgacaaagg ccttaccagg aagggttagg acactgacca 2495 ttctagaaat gggtttcgaa tggcacaaca ctttctattt cacaaaagac caaaagccag 2555 aggccccagg ctctgtgctg atgaacagcc tggctgagcc ctggccctgg caggtttagg 2615 gcccatttgg ggccccctcc ttctctgtca gggctggggt gctctgtctg ggaatgaggg 2675 agttaaccaa gtttggtgca ggagcagggg cagggggcca ctgtagtgag cgtggagaaa 2735 tttggaaaca cctatttctt aactcaaata aagtccagtt tgtacctaaa aaaaaaaa 2793 14 661 PRT Homo sapiens 14 Ile Pro Val Gln Ile Arg Ala Ala Pro Pro Thr Val Pro Ser Gly Arg 1 5 10 15 Glu Ala Val Pro Ala Gly Thr Pro Gly Pro Ala Arg Gly His Pro His 20 25 30 Val Ala Pro Pro Ser His Ser Gly Ala Cys Ala Ala Arg Gly His His 35 40 45 His Arg Arg Glu Ala Arg Glu Glu Val Gln Val Val Arg Ala Leu Pro 50 55 60 Gly Gly His Arg Glu Ser Arg Pro Gln Thr Pro Leu Ser Glu Ala Ser 65 70 75 80 Gly Arg Leu Trp Ala Leu Gly Arg Ser Pro Arg Leu Val Arg Ala Gly 85 90 95 Ser Arg Ile Leu Asp Lys Leu Gln Phe Phe Glu Glu Arg Arg Arg Ser 100 105 110 Leu Glu Arg Ser Asp Ser Pro Pro Ala Pro Leu Arg Pro Trp Val Pro 115 120 125 Leu Arg Lys Ala Arg Ser Leu Glu Gln Pro Lys Ser Glu Arg Gly Ala 130 135 140 Pro Trp Gly Thr Pro Gly Ala Ser Gln Glu Glu Leu Arg Ala Pro Gly 145 150 155 160 Ser Val Ala Glu Arg Arg Arg Leu Phe Gln Gln Lys Ala Ala Ser Leu 165 170 175 Asp Glu Arg Thr Arg Gln Arg Ser Pro Ala Ser Asp Leu Glu Leu Arg 180 185 190 Phe Ala Gln Glu Leu Gly Arg Ile Arg Arg Ser Thr Ser Arg Glu Glu 195 200 205 Leu Val Arg Ser His Glu Ser Leu Arg Ala Thr Leu Gln Arg Ala Pro 210 215 220 Ser Pro Arg Glu Pro Gly Glu Pro Pro Leu Phe Ser Arg Pro Ser Thr 225 230 235 240 Pro Lys Thr Ser Arg Ala Val Ser Pro Ala Ala Ala Gln Pro Pro Ser 245 250 255 Pro Ser Ser Ala Glu Lys Pro Gly Asp Glu Pro Gly Arg Pro Arg Ser 260 265 270 Arg Gly Pro Ala Gly Arg Thr Glu Pro Gly Glu Gly Pro Gln Gln Glu 275 280 285 Val Arg Arg Arg Asp Gln Phe Pro Leu Thr Arg Ser Arg Ala Ile Gln 290 295 300 Glu Cys Arg Ser Pro Val Pro Pro Pro Ala Ala Asp Pro Pro Glu Ala 305 310 315 320 Arg Thr Lys Ala Pro Pro Gly Arg Lys Arg Glu Pro Pro Ala Gln Ala 325 330 335 Val Arg Phe Leu Pro Trp Ala Thr Pro Gly Leu Glu Gly Ala Ala Val 340 345 350 Pro Gln Thr Leu Glu Lys Asn Arg Ala Gly Pro Glu Ala Glu Lys Arg 355 360 365 Leu Arg Arg Gly Pro Glu Glu Asp Gly Pro Trp Gly Pro Trp Asp Arg 370 375 380 Arg Gly Ala Arg Ser Gln Gly Lys Gly Arg Arg Ala Arg Pro Thr Ser 385 390 395 400 Pro Glu Leu Glu Ser Ser Asp Asp Ser Tyr Val Ser Ala Gly Glu Glu 405 410 415 Pro Leu Glu Ala Pro Val Phe Glu Ile Pro Leu Gln Asn Val Val Val 420 425 430 Ala Pro Gly Ala Asp Val Leu Leu Lys Cys Ile Ile Thr Ala Asn Pro 435 440 445 Pro Pro Gln Val Ser Trp His Lys Asp Gly Ser Ala Leu Arg Ser Glu 450 455 460 Gly Arg Leu Leu Leu Arg Ala Glu Gly Glu Arg His Thr Leu Leu Leu 465 470 475 480 Arg Glu Ala Arg Ala Ala Asp Ala Gly Ser Tyr Met Ala Thr Ala Thr 485 490 495 Asn Glu Leu Gly Gln Ala Thr Cys Ala Ala Ser Leu Thr Val Arg Pro 500 505 510 Gly Gly Ser Thr Ser Pro Phe Ser Ser Pro Ile Thr Ser Asp Glu Glu 515 520 525 Tyr Leu Ser Pro Pro Glu Glu Phe Pro Glu Pro Gly Glu Thr Trp Pro 530 535 540 Arg Thr Pro Thr Met Lys Pro Ser Pro Ser Gln Asn Arg Arg Ser Ser 545 550 555 560 Asp Thr Gly Ser Lys Ala Pro Pro Thr Phe Lys Val Ser Leu Met Asp 565 570 575 Gln Ser Val Arg Glu Gly Gln Asp Val Ile Met Ser Ile Arg Val Gln 580 585 590 Gly Glu Pro Lys Pro Val Val Ser Trp Leu Arg Asn Arg Gln Pro Val 595 600 605 Arg Pro Asp Gln Arg Arg Phe Ala Glu Glu Ala Glu Gly Gly Leu Cys 610 615 620 Arg Leu Arg Ile Leu Ala Ala Glu Arg Gly Asp Ala Gly Phe Tyr Thr 625 630 635 640 Cys Lys Ala Val Asn Glu Tyr Gly Ala Arg Gln Cys Glu Ala Arg Leu 645 650 655 Glu Val Arg Gly Glu 660 15 2614 DNA Mus musculus CDS (1)...(1806) 15 gaa ttc cgg ctg gcg ggc aca gtg gag tcc cgg ccc cag acg cca ctg 48 Glu Phe Arg Leu Ala Gly Thr Val Glu Ser Arg Pro Gln Thr Pro Leu 1 5 10 15 agc gag gct tcg ggt cgc ctg tca gca ctg ggc cgc tcg ccc cgg ctg 96 Ser Glu Ala Ser Gly Arg Leu Ser Ala Leu Gly Arg Ser Pro Arg Leu 20 25 30 gtg cgc gcg ggg tcc cgc atc ctg gac aag cta cag ttc ttc gaa gag 144 Val Arg Ala Gly Ser Arg Ile Leu Asp Lys Leu Gln Phe Phe Glu Glu 35 40 45 cgg cga cgc agc ctg gag cgc agc gac tcg ccg cca gcg ccc ctg cgg 192 Arg Arg Arg Ser Leu Glu Arg Ser Asp Ser Pro Pro Ala Pro Leu Arg 50 55 60 ccc tgg gtg ccc ctg cgc aag gct cgc tcg ctg gag cag ccg aag tcc 240 Pro Trp Val Pro Leu Arg Lys Ala Arg Ser Leu Glu Gln Pro Lys Ser 65 70 75 80 gag ggc ggt gcg gcg tgg ggc aca ccc gag gcc tcg cag gag gag ctg 288 Glu Gly Gly Ala Ala Trp Gly Thr Pro Glu Ala Ser Gln Glu Glu Leu 85 90 95 cgg tca cct cgg ggc agt gtg gca gag cgg cgt cgc ctg ttc cag caa 336 Arg Ser Pro Arg Gly Ser Val Ala Glu Arg Arg Arg Leu Phe Gln Gln 100 105 110 aag gcg gcc tcg ttg gat gaa cgc acg cga caa cgc agt gca acc tcg 384 Lys Ala Ala Ser Leu Asp Glu Arg Thr Arg Gln Arg Ser Ala Thr Ser 115 120 125 gac ctc gaa ctc cgc ttc gcc cag gag ctg ggt cgc atc cgc cga tct 432 Asp Leu Glu Leu Arg Phe Ala Gln Glu Leu Gly Arg Ile Arg Arg Ser 130 135 140 acg tcg cgg gag gag ctg gtg cgt tcg cac gag tcc ctg cgt gcc acg 480 Thr Ser Arg Glu Glu Leu Val Arg Ser His Glu Ser Leu Arg Ala Thr 145 150 155 160 ctg cag cgc gcc cca tcc cct cgg gag ccc ggc gag ccc cca ctc ttc 528 Leu Gln Arg Ala Pro Ser Pro Arg Glu Pro Gly Glu Pro Pro Leu Phe 165 170 175 tcc cgg cct tcc aca ccc aag acc tca cgg gct gtg agc ccg gct gcc 576 Ser Arg Pro Ser Thr Pro Lys Thr Ser Arg Ala Val Ser Pro Ala Ala 180 185 190 acc cag ccg ccg cct cct agt ggt gcg ggc aaa tct ggg gac gag cct 624 Thr Gln Pro Pro Pro Pro Ser Gly Ala Gly Lys Ser Gly Asp Glu Pro 195 200 205 ggg agg ccc cga agc aga ggg ccg gtg ggc agg act gaa ccg ggg gaa 672 Gly Arg Pro Arg Ser Arg Gly Pro Val Gly Arg Thr Glu Pro Gly Glu 210 215 220 ggc ccg cag cag gag atc aag cgt cgg gac caa ttc ccg cta acc agg 720 Gly Pro Gln Gln Glu Ile Lys Arg Arg Asp Gln Phe Pro Leu Thr Arg 225 230 235 240 agc aga gcc atc cag gag tgc agg agc cct gtg ccg ccc tac acc gcg 768 Ser Arg Ala Ile Gln Glu Cys Arg Ser Pro Val Pro Pro Tyr Thr Ala 245 250 255 gat ccc ccg gag agc agg aca aaa gcc ccc tcc ggt cgc aag cgg gaa 816 Asp Pro Pro Glu Ser Arg Thr Lys Ala Pro Ser Gly Arg Lys Arg Glu 260 265 270 ccc cct gct caa gcg gtg cgc ttt ctg ccc tgg gcc act ccg gga gtg 864 Pro Pro Ala Gln Ala Val Arg Phe Leu Pro Trp Ala Thr Pro Gly Val 275 280 285 gag gac tct gtt ctg ccc caa acc ttg gag aag aat aga gcg gga ccc 912 Glu Asp Ser Val Leu Pro Gln Thr Leu Glu Lys Asn Arg Ala Gly Pro 290 295 300 gag gct gag aag agg ctt cgc aga gga cct gag gag gat ggc ccc tgg 960 Glu Ala Glu Lys Arg Leu Arg Arg Gly Pro Glu Glu Asp Gly Pro Trp 305 310 315 320 ggg ccc tgg gac cgc aga ggg acc cgc agc caa ggc aaa ggt cgc cgt 1008 Gly Pro Trp Asp Arg Arg Gly Thr Arg Ser Gln Gly Lys Gly Arg Arg 325 330 335 gct cgg cct act tcc ccc gag ctc gag tcc tca gac gac tcc tat gtg 1056 Ala Arg Pro Thr Ser Pro Glu Leu Glu Ser Ser Asp Asp Ser Tyr Val 340 345 350 tcc gct ggg gaa gag ccc ctg gag gca ccc gtg ttt gag atc cct ctg 1104 Ser Ala Gly Glu Glu Pro Leu Glu Ala Pro Val Phe Glu Ile Pro Leu 355 360 365 cag aat atg gtg gtg gcg cca gga gct gac gtg cta ctt aag tgt atc 1152 Gln Asn Met Val Val Ala Pro Gly Ala Asp Val Leu Leu Lys Cys Ile 370 375 380 atc acc gcc aac ccc cca ccc caa gtg tcc tgg aaa aag gat ggg tcc 1200 Ile Thr Ala Asn Pro Pro Pro Gln Val Ser Trp Lys Lys Asp Gly Ser 385 390 395 400 atg ttg cac agc gag ggt cgt ctt ctc atc cgg gct gaa ggt gaa cgg 1248 Met Leu His Ser Glu Gly Arg Leu Leu Ile Arg Ala Glu Gly Glu Arg 405 410 415 cac aca ctg ctg ctc aga gag gcc cag gct gct gat gct ggg agc tac 1296 His Thr Leu Leu Leu Arg Glu Ala Gln Ala Ala Asp Ala Gly Ser Tyr 420 425 430 aca gcc act gcc acc aac gaa ctg ggc caa gct acc tgt gct tct tca 1344 Thr Ala Thr Ala Thr Asn Glu Leu Gly Gln Ala Thr Cys Ala Ser Ser 435 440 445 ctg gct gtg aga cct ggc ggc tcc aca tcc cct ttc agc agc ccc atc 1392 Leu Ala Val Arg Pro Gly Gly Ser Thr Ser Pro Phe Ser Ser Pro Ile 450 455 460 acc tct gat gag gag tac ctg agc ccc cca gag gag ttc cca gag cct 1440 Thr Ser Asp Glu Glu Tyr Leu Ser Pro Pro Glu Glu Phe Pro Glu Pro 465 470 475 480 ggg gag acc tgg ccc cga acc cct acc atg aag ctc agt ccc agc cag 1488 Gly Glu Thr Trp Pro Arg Thr Pro Thr Met Lys Leu Ser Pro Ser Gln 485 490 495 gat cat gat tcc tcc gac tct tct tcc aag gca ccc cca acg ttc aag 1536 Asp His Asp Ser Ser Asp Ser Ser Ser Lys Ala Pro Pro Thr Phe Lys 500 505 510 gtc tca ctc atg gac caa tcg gtg aga gaa ggt caa gat gtc att atg 1584 Val Ser Leu Met Asp Gln Ser Val Arg Glu Gly Gln Asp Val Ile Met 515 520 525 agc atc cgt gtg cag gga gag ccc aag cct gtg gtt tcc tgg ctg agg 1632 Ser Ile Arg Val Gln Gly Glu Pro Lys Pro Val Val Ser Trp Leu Arg 530 535 540 aat cga cag ccc gtg cgc cca gac cag cgg cgc ttt gca gag gag gcc 1680 Asn Arg Gln Pro Val Arg Pro Asp Gln Arg Arg Phe Ala Glu Glu Ala 545 550 555 560 gag ggt ggg ctc tgc cgc ttg agg atc ctg gct gct gaa cgg ggc gat 1728 Glu Gly Gly Leu Cys Arg Leu Arg Ile Leu Ala Ala Glu Arg Gly Asp 565 570 575 gct ggt ttc tac aca tgc aag gcg gtc aac gaa tat ggc gct cgg cag 1776 Ala Gly Phe Tyr Thr Cys Lys Ala Val Asn Glu Tyr Gly Ala Arg Gln 580 585 590 tgc gag gcc cgc ctg gag gtc cga ggc gag tgagctcagg gggccacctg 1826 Cys Glu Ala Arg Leu Glu Val Arg Gly Glu 595 600 cgctgccccc gctaccctcc gagctgcacc cctgtctcag gcacctctcg gacctcgctg 1886 tgtttcactg cctcctgccc acagacccag ccggctcgcc ggcccggact tagcccatgc 1946 tccccttccc tccctagccc atacagcacc ctggggtaac ccaccgggcc cctgtggatc 2006 ctccctcccc aagtggatat gtggctgtgc agaccaggag gcccccagaa ggactgagtg 2066 ttgggaaggg atggccatga ggggtgccaa gctccctcgg tctccccata gggagcatcc 2126 agcgagtgca tgtgctatgc tgctacaggc cactgtctgt ctatctgttt gtccgtctgt 2186 gtgtctgtga cagtcaggga agaaagcctt tgagctgagg tgggctaaga cagaataaga 2246 tgacagagca cagcatccat gagatgcagg ggttcagagg ggtcaggtac agtggatatg 2306 aggctctctg ggaaggggca gggcactgac catttcagaa atgggtttta aatggcacaa 2366 cattttttat tccacaagag accaaaagct agaggtctag ggttaagccc tagctgctgg 2426 caagattagg accaagtggg gtacccttct ttacagacac atccgacacg cgctgtctga 2486 gaatgagaga ggtagccagg ctgaacacag gagcagggtc atagtggagg tggagatttg 2546 gaaacactat ttcgtagctc aaataaagtc cagtttgtac ccaaaaaaaa aaaaaaaaaa 2606 aaaaaaaa 2614 16 602 PRT Mus musculus 16 Glu Phe Arg Leu Ala Gly Thr Val Glu Ser Arg Pro Gln Thr Pro Leu 1 5 10 15 Ser Glu Ala Ser Gly Arg Leu Ser Ala Leu Gly Arg Ser Pro Arg Leu 20 25 30 Val Arg Ala Gly Ser Arg Ile Leu Asp Lys Leu Gln Phe Phe Glu Glu 35 40 45 Arg Arg Arg Ser Leu Glu Arg Ser Asp Ser Pro Pro Ala Pro Leu Arg 50 55 60 Pro Trp Val Pro Leu Arg Lys Ala Arg Ser Leu Glu Gln Pro Lys Ser 65 70 75 80 Glu Gly Gly Ala Ala Trp Gly Thr Pro Glu Ala Ser Gln Glu Glu Leu 85 90 95 Arg Ser Pro Arg Gly Ser Val Ala Glu Arg Arg Arg Leu Phe Gln Gln 100 105 110 Lys Ala Ala Ser Leu Asp Glu Arg Thr Arg Gln Arg Ser Ala Thr Ser 115 120 125 Asp Leu Glu Leu Arg Phe Ala Gln Glu Leu Gly Arg Ile Arg Arg Ser 130 135 140 Thr Ser Arg Glu Glu Leu Val Arg Ser His Glu Ser Leu Arg Ala Thr 145 150 155 160 Leu Gln Arg Ala Pro Ser Pro Arg Glu Pro Gly Glu Pro Pro Leu Phe 165 170 175 Ser Arg Pro Ser Thr Pro Lys Thr Ser Arg Ala Val Ser Pro Ala Ala 180 185 190 Thr Gln Pro Pro Pro Pro Ser Gly Ala Gly Lys Ser Gly Asp Glu Pro 195 200 205 Gly Arg Pro Arg Ser Arg Gly Pro Val Gly Arg Thr Glu Pro Gly Glu 210 215 220 Gly Pro Gln Gln Glu Ile Lys Arg Arg Asp Gln Phe Pro Leu Thr Arg 225 230 235 240 Ser Arg Ala Ile Gln Glu Cys Arg Ser Pro Val Pro Pro Tyr Thr Ala 245 250 255 Asp Pro Pro Glu Ser Arg Thr Lys Ala Pro Ser Gly Arg Lys Arg Glu 260 265 270 Pro Pro Ala Gln Ala Val Arg Phe Leu Pro Trp Ala Thr Pro Gly Val 275 280 285 Glu Asp Ser Val Leu Pro Gln Thr Leu Glu Lys Asn Arg Ala Gly Pro 290 295 300 Glu Ala Glu Lys Arg Leu Arg Arg Gly Pro Glu Glu Asp Gly Pro Trp 305 310 315 320 Gly Pro Trp Asp Arg Arg Gly Thr Arg Ser Gln Gly Lys Gly Arg Arg 325 330 335 Ala Arg Pro Thr Ser Pro Glu Leu Glu Ser Ser Asp Asp Ser Tyr Val 340 345 350 Ser Ala Gly Glu Glu Pro Leu Glu Ala Pro Val Phe Glu Ile Pro Leu 355 360 365 Gln Asn Met Val Val Ala Pro Gly Ala Asp Val Leu Leu Lys Cys Ile 370 375 380 Ile Thr Ala Asn Pro Pro Pro Gln Val Ser Trp Lys Lys Asp Gly Ser 385 390 395 400 Met Leu His Ser Glu Gly Arg Leu Leu Ile Arg Ala Glu Gly Glu Arg 405 410 415 His Thr Leu Leu Leu Arg Glu Ala Gln Ala Ala Asp Ala Gly Ser Tyr 420 425 430 Thr Ala Thr Ala Thr Asn Glu Leu Gly Gln Ala Thr Cys Ala Ser Ser 435 440 445 Leu Ala Val Arg Pro Gly Gly Ser Thr Ser Pro Phe Ser Ser Pro Ile 450 455 460 Thr Ser Asp Glu Glu Tyr Leu Ser Pro Pro Glu Glu Phe Pro Glu Pro 465 470 475 480 Gly Glu Thr Trp Pro Arg Thr Pro Thr Met Lys Leu Ser Pro Ser Gln 485 490 495 Asp His Asp Ser Ser Asp Ser Ser Ser Lys Ala Pro Pro Thr Phe Lys 500 505 510 Val Ser Leu Met Asp Gln Ser Val Arg Glu Gly Gln Asp Val Ile Met 515 520 525 Ser Ile Arg Val Gln Gly Glu Pro Lys Pro Val Val Ser Trp Leu Arg 530 535 540 Asn Arg Gln Pro Val Arg Pro Asp Gln Arg Arg Phe Ala Glu Glu Ala 545 550 555 560 Glu Gly Gly Leu Cys Arg Leu Arg Ile Leu Ala Ala Glu Arg Gly Asp 565 570 575 Ala Gly Phe Tyr Thr Cys Lys Ala Val Asn Glu Tyr Gly Ala Arg Gln 580 585 590 Cys Glu Ala Arg Leu Glu Val Arg Gly Glu 595 600 17 2738 DNA Mus musculus 17 gcgatagata acctggtgat ccaaacctgt aatcctaact actgtggagg ctgagataat 60 aacttgccag agatacagag tcagttcaag accaccctag gcaactaaag agatcttgtt 120 tcagactaag aaaaagaggc ctagcaaggc cctacattca atcccccaga aacaaatgac 180 tcagacagcc caagtccaga ctgtaaatca gagactacag gggaccatac cccaaagaac 240 tctctagaat tcctgtgctc agaaaacttt gaaacccaat caaccaaact gggcagtggt 300 gtcacatgct tttaatccca gtactcagga ggcagaggca ggcagatctc tgagttcaag 360 tccagcctga tttactgatt gagtcaaggc tacacagaga taccctgtct caaaaaacta 420 acaagcaaaa tacaaaaaca aaaaccaaaa aaaaaaaaaa aaaaaaaaaa aaaataagaa 480 gcccaaccat ataagaagca ttttgaaaaa aaactaatgt ttgaaatcgc tggcatgggg 540 ttaaagatct agttcaaatt gggaagctgg ctgctgtcat tggaatcaca agggctgtcg 600 aaccagactt agggatttac agccctgctc tgaagttgaa tggccaagag ctgtgagatt 660 cagtgaaatc acctcttaga gttcccatcc tcccatgagg atttgcctag gtctcaaaac 720 ttccatgtgc ctagggatct ctagagtgct tttgaaaaaa aattacagtg ttcgactcct 780 cactttagaa aatcaattct gtaggctgga taaggtctaa gaatctgtat ttcaaaacaa 840 gccccaagtg gtacccgtgt gggtggttca agcatcacgc acacagtcct ggtgtagatg 900 gccttgggtg atgctatccg tgtgctagaa actgggtgtc tgtcgtgaag agactacaga 960 cagctgggat gtcaggcttg actggatata ctggcctggg ggaaattcct gcttgtgggc 1020 tgtctaatgc cagttcttat tgaatgatac tggcctgaaa gaactgtcca aagggcagct 1080 agatgaatag agtcagctca tggagagctg ggtcaaatgt aatgaagtgg tcctttaatg 1140 ggaaggtttg ggatcaaaag aacactgccc ttgctggtgt tatctcccac agtgaaatct 1200 gggtttgtag atggatcagg cttgggatgt tacaaaaaaa tggctacaaa gttgctttag 1260 cccatgcggt ctgcagggct tgggattcta cagcttggtg gtgtactttg gggttatggc 1320 tggaacagag gccacttctt tttctcagag aggcattcca ttggagcttg agcctgcagc 1380 ctgacaagca atctcgccaa gactcttgac ctaggcttgc tgctgattgg ctggctagca 1440 cctaggttct atttccctgc tggccaccag gggtctctga agcaaacata gacctttggc 1500 aattcgagtt aaatgtttgc cccgccctcc tttccttagc ctgggagctt gcctcagcac 1560 tgtccagcct ggaggtgacc ctggagccag gaatctaaac tctgtagagg gaaaggagtc 1620 ccctcttcca agggctgtgc ctatgacctc agtatcagct ggtggccacg cccccggcca 1680 caaatgccat tcggatttct ctctcctccc caaccttgag actgccagcc tgaaagtggg 1740 ctgtcctctt ggcccccaca cttcttcatc actggcagtg ctggggaaca caggtcatag 1800 cttgggaatg tggccctggg tggagagagg ggatcaagga gggagagaga tttgtggcct 1860 ctgctcaaca cctctgcttc tattattctt cctgagcccc ttccctaccc actgggtgca 1920 aacggaagct ggggaggagc gaccattggg gaggagcggc ccacacttcc ctagctttga 1980 gccctggtgg gctgaggggt gaggggcagt ttgccagcag aaattcagta gaacccatgg 2040 ttgagcaggg tgcaggcctg tgtcctgaag tacctgctct cctgaacttg tctagggcag 2100 gacctgggag tctgcagcca tgggctcagt ttccttaggt tggcagggga caaatctgga 2160 aaggagggtc aagccctgac agttctttgg ttctctgtgt ctgaaaaagc tggttgtggc 2220 ctatttgggg gtttaaggct ggctagttat gtattcctag gtcaggattc ttcttggttt 2280 gggcaaagca tggcgcttgc tgtgctgtat gggtcaacac ttctggccca ggcaaggata 2340 ttaaatgccg cagtgcagtg ccacccctta gacccctctg aggaccgggg tccccacacc 2400 tgtagtctag gccctactga tgggttcagc tcttgtcagt gtcccaagct gtaaggagag 2460 gaaaggcaga cagctagctg cttggaatga tcagagtcta aattcagctg gtctgggctc 2520 cgcccctccc ccgttcctat tccaccactc caggggctgc tccctgtggt ctcagcaggc 2580 accaccttcc cagccagcgc ctgcctgctg cccagcctct tgctggccac ccccacctcc 2640 tccttccccc gctcctaggc tcacttcccc tccccccagg gctggctcag tgcggggcct 2700 cagctgggtc agcgagtgag tggggctggc caggctga 2738 18 113 PRT Homo sapiens 18 Met Lys Leu Ser Pro Ser Gln Asp His Asp Ser Ser Asp Ser Ser Ser 1 5 10 15 Lys Ala Pro Pro Thr Phe Lys Val Ser Leu Met Asp Gln Ser Val Arg 20 25 30 Glu Gly Gln Asp Val Ile Met Ser Ile Arg Val Gln Gly Glu Pro Lys 35 40 45 Pro Val Val Ser Trp Leu Arg Asn Arg Gln Pro Val Arg Pro Asp Gln 50 55 60 Arg Arg Phe Ala Glu Glu Ala Glu Gly Gly Leu Cys Arg Leu Arg Ile 65 70 75 80 Leu Ala Ala Glu Arg Gly Asp Ala Gly Phe Tyr Thr Cys Lys Ala Val 85 90 95 Asn Glu Tyr Gly Ala Arg Gln Cys Glu Ala Arg Leu Glu Val Arg Gly 100 105 110 Glu 19 114 PRT Artificial Sequence synthetic consensus sequence 19 Met Lys Xaa Ser Pro Ser Gln Asp Xaa Xaa Ser Ser Asp Xaa Xaa Ser 1 5 10 15 Lys Ala Pro Pro Thr Phe Lys Val Ser Leu Met Asp Gln Ser Val Arg 20 25 30 Glu Gly Gln Asp Val Ile Met Ser Ile Arg Val Gln Gly Glu Pro Lys 35 40 45 Pro Val Val Ser Trp Leu Arg Asn Arg Gln Pro Val Arg Pro Asp Gln 50 55 60 Arg Arg Phe Ala Glu Glu Ala Glu Gly Gly Leu Cys Arg Leu Arg Ile 65 70 75 80 Leu Ala Ala Glu Arg Gly Asp Ala Gly Phe Tyr Thr Cys Lys Ala Val 85 90 95 Asn Glu Tyr Gly Ala Arg Gln Cys Glu Ala Arg Leu Glu Val Arg Gly 100 105 110 Glu Xaa 20 73 DNA Mus musculus 20 tcccctcccc ccagggctgg ctcagtgcgg ggcctcagct gggtcagcga gtgagtgggg 60 ctggccaggc tga 73

Claims

What is claimed is:

1. A substantially pure DNA comprising SEQ ID NO:20 operably linked to a heterologous promoter and a polypeptide encoding sequence, wherein said polypeptide coding sequence does not encode APEG-1.

2. The DNA of claim 1, wherein said DNA is less than 2.7 kb in length.

3. The DNA of claim 1, wherein said promoter/enhancer sequence regulates developmental stage-specific expression of said polypeptide-encoding sequence.

4. The DNA of claim 1, wherein said polypeptide is chosen from a group consisting of tissue plasminogen activator, p21 cell cycle inhibitor, nitric oxide synthase, interferon-γ, and atrial natriuretic polypeptide.

5. A vector comprising the DNA of claim 4.

6. A method of directing vascular smooth muscle cell-specific expression of a polypeptide, comprising introducing into a vascular smooth muscle cell the vector of claim 4.

7. A method of directing developmental stage-specific expression of a polypeptide, comprising introducing into a vascular smooth muscle cell the vector of claim 4.

8. The DNA of claim 1, comprising a plurality of copies of SEQ ID NO:20.

9. The DNA of claim 1, wherein said DNA consists of Seq ID NO:20.

10. A substantially pure DNA comprising a sequence encoding a striated muscle-preferentially expressed gene-1 (SPEG-1) polypeptide.

11. The DNA of claim 10, wherein said polypeptide is human SPEG-1.

12. The DNA of claim 11, wherein said polypeptide comprises the amino acid sequence of SEQ ID NO:14.

13. A substantially pure DNA comprising a nucleotide sequence having at least 50% sequence identity to SEQ ID NO:13.

14. The DNA of claim 10, wherein said polypeptide is mouse SPEG.

15. The DNA of claim 14, wherein said polypeptide comprises the amino acid sequence of SEQ ID NO:16.

16. A substantially pure DNA comprising a strand which hybridizes at high stringency to a strand of DNA having the sequence of SEQ ID NO:16, or the complement thereof.

17. A substantially pure DNA having at least 50% sequence identity to SEQ ID NO:15, and encoding a polypeptide having the biological activity of a SPEG-1 polypeptide.

18. A substantially pure human SPEG-1 polypeptide.

19. The polypeptide of claim 18, wherein said polypeptide comprises the amino acid sequence of SEQ ID NO:14.