PATENT DOCUMENT

Abstract:
Mammalian α3(V) pro-collagen and collagen polypeptides and variants thereof are encoded by exemplified polynucleotides. Investigative, diagnostic and therapeutic methods employ the polypeptides, polynucleotides and related materials, such as antibodies, sense- or antisense oligonucleotides and polynucleotides, and the like.

Full Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
       [0001]    This application claims the benefit of U.S. provisional application 60/186,510, filed on Mar. 2, 2000. 
     
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT  
       [0002] This work was supported by National Institutes of Health Grants GM46846 and AR43621. The US Government retains certain rights in the invention. 
     
    
     
       BACKGROUND OF THE INVENTION  
         [0003]    Fibrils of the abundant collagen types I and II incorporate monomers of the low abundance fibrillar collagen types V and XI, respectively, which play a role in regulating type I collagen fibrillogenesis in vivo (1,2). Type V collagen helps regulate the size and shape of type I/V heterotypic fibrils (3-5). In some cases of classical Ehlers-Danlos Syndrome (EDS), a heritable connective tissue disorder, mutations in type V collagen genes (6-10) give rise to type I collagen fibrils of abnormal shape and diameter and cause connective tissue fragility, particularly in skin and joints. In chondrodysplasia, defects in a type XI collagen gene give rise to abnormal type II collagen fibrils (11).  
           [0004]    Fibrillar collagens are synthesized as procollagen precursors with— and C-propeptides that are proteolytically processed to yield mature monomers. Type V collagen is widely distributed in vertebrate tissues as an α1(V) 2 α2(V) heterotrimer (12,13) that helps regulate the diameters of fibrils of the abundant collagen type I. Previously, mutations in the human COL5A1 and COL5A2 genes, which encode the pro-α1(V) and pro-α2(V) chains, respectively, have been identified as the underlying defects in cases of the heritable connective tissue disorder classical EDS (formerly EDS types I and II, see Ref. 76). However, both COL5A1 and COL5A2 have been excluded in some cases of classical EDS I, while a locus has yet to be identified for the hypermobility type of EDS (formerly EDS type III), a condition marked by gross joint laxity, recurrent joint dislocation, and chronic diffuse musculoskeletal pain not attributable to joint involvement.  
           [0005]    Another type V collagen is an α1(V)α2(V)α3(V) heterotrimer, isolated primarily from placenta (17,18), but also reported in uterus, skin, and synovial membranes (12,19-21). The α1(V)α2(V)α3(V) heterotrimer has remained poorly characterized but has a lower melting temperature than the α1(V) 2 α2(V) heterotrimer and may be incorporated into heterotypic fibrils. Type XI collagen, in the form of an α1(XI)α2(XI)α3 (XI) heterotrimer (22), was first characterized as a minor collagen of cartilage. However, findings of type XI chains in noncartilaginous tissues (23), of type V chains in cartilage (24), and of cross-type heterotrimers composed of α2(V) and α1(XI) chains (25,26) now suggest that type V and type XI chains constitute a single collagen type in which different combinations of chains associate in a tissue-specific manner.  
           [0006]    Complete primary structures of the type V/XI procollagen chains pro-α1(V), pro-α2(V), pro-α1(XI), and pro-α2(XI) are known (27-35). The pro-α3(XI) chain is thought to be an alternatively spliced product of the gene that encodes the pro-al chain of type II collagen (13, 24). Full-length cDNA sequences have provided not only the inferred primary structure of each chain, but have also provided probes that have allowed fine mapping of the expression domains of cognate mRNAs (27,36-41). Such studies are important, as the low levels of collagen type V/XI chains have limited biochemical and histochemical analyses of expression in developing and adult tissues. Nucleic acid probes have also enabled those studies which established the causal links between defects in type V/XI chains and genetic diseases (6-11).  
           [0007]    Of the fibrillar procollagen chains, only the pro-α3(V) remains largely uncharacterized at the nucleotide and amino acid level. The α3(V) chain exhibits only limited distribution in mammals and is believed to be the least abundant fibrillar (type V/XI) collagen chain. The limited distribution may reflect a more specialized role than those of the other type V/XI chains. It is the only fibrillar (type V/XI) collagen or procollagen chain for which neither complete primary structure nor nucleic acid probes are available. About a third of the amino acid sequence of the major collagenous domain of the α3(V) chain was determined by N-terminal sequencing of proteolytic fragments (42). Nevertheless, a true understanding of the nature of mammalian type V/XI collagen and its roles in development, physiology, disease and treatment requires characterization the pro-α3(V) and α3(V) chains.  
         BRIEF SUMMARY OF THE INVENTION  
         [0008]    The present invention is summarized in that mammalian α3(V) polypeptides and variants thereof are disclosed, as are recombinant materials, including genetic constructs, and methods for their production. The invention is further summarized in that polynucleotides that encode the polypeptides and the variants are also disclosed. The invention is still further summarized in that investigative, diagnostic and therapeutic compositions and methods employing the polypeptides, polynucleotides and related materials, such as antibodies, sense- or antisense oligonucleotides and polynucleotides, and the like, are also disclosed. The chromosomal map positions in humans and mice of the polynucleotides that encode the mammalian α3(V) polypeptides are also disclosed.  
           [0009]    It is an object of the present invention to enable production of large quantities of mammalian α3(V) polypeptides chains for research, diagnostic and therapeutic use.  
           [0010]    It is an advantage of the present invention that collagen comprising mammalian pro-α3(V) or α3(V) chains can be synthesized for any such use.  
           [0011]    Other objects, features and advantages of the present invention will become apparent upon consideration of the following detailed description taken in conjunction with the accompanying drawings.  
         BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS  
         [0012]    Not applicable.  
         DETAILED DESCRIPTION OF THE INVENTION  
         [0013]    As used herein, a “mammalian α3(V) polypeptide” refers to a modified or unmodified polypeptide having an amino acid sequence characteristic of those shown in SEQ ID NO: 2 and SEQ ID NO: 4, or a novel fragment thereof, especially a fragment that is antigenic or has a biological activity. Preferably, a mammalian α3(V) polypeptide exhibits at least one biological activity of mammalian α3(V) procollagen or collagen. A mammalian α3(V) polypeptide can be a mature protein or a larger protein that can include native or non-native amino acid sequences at the N- or C-terminus or both, a propeptide sequence, or other sequence attached to the mature polypeptide sequence. These sequences can include amino acid sequences that assist in purification, detection, or stabilization of the mammalian α3(V) polypeptide.  
           [0014]    Within the scope of the invention are polypeptides that have at least 80% amino acid identity to that of either SEQ ID NO: 2 or SEQ ID NO: 4 over its entire length, and more particularly polypeptides having at least 90% identity, or more preferably at least 95% identity, to that of SEQ ID NO: 2 or SEQ ID NO: 4, when the sequences are aligned to obtain the highest order match using published techniques. Most preferred are polypeptides having between 97 and 99% amino acid identity to that of SEQ ID NO: 2 or SEQ ID NO: 4. The term “identity” is given its art recognized meaning. Sequence identity can be determined, for example, using the methods disclosed by Devereux et al. (83), incorporated herein by reference in its entirety.  
           [0015]    An polypeptide is, e.g., 80% “identical” if it contains up to 20 amino acid sequence differences, changes or alterations (including substitutions, deletions, or insertions) per each 100 amino acids in reference sequences SEQ ID NO: 2 or SEQ ID NO: 4. The differences, changes or alterations can be at any position in the amino acid sequence of either polypeptide and can be interspersed as individual changes or contiguous differences.  
           [0016]    A “mammalian α3(V) polynucleotide” refers to a polynucleotide that encodes any mammalian α3(V) polypeptide, or a polynucleotide fragment thereof, or a complement of any of the foregoing. A polynucleotide can be modified or unmodified DNA or RNA, whether fully or partially single-stranded or double-stranded or even triple-stranded. A modified polynucleotide can be chemically or enzymatically induced and can include so-called non-standard bases such as inosine. A preferred polynucleotide comprises any sequence that can encode a polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4, where the number of such polynucleotides is substantial, in view of the well-known degeneracy in the genetic code. In a most preferred embodiment, the polynucleotide comprises a sequence of polypeptide-encoding nucleotides shown in SEQ ID NO: 1 (bases 82 to 5298) or SEQ ID NO: 3 (bases 87 to 5321), or is a polynucleotide fragment or complement of any of the foregoing.  
           [0017]    Within the scope of the invention are polynucleotides that comprise nucleotide sequences having at least 80% identity to that of any of the foregoing over its entire length, and more preferably polynucleotides comprising sequences having at least 90% identity, or more preferably at least 95% identity, to that of SEQ ID NO: 1 or SEQ ID NO: 3, when the sequences are aligned to obtain the highest order match using published techniques. A polynucleotide sequence is, e.g., 80% identical if it contains up to 20 nucleotide differences, changes or alterations (including substitutions, deletions, or insertions) per each 100 nucleotides in reference sequences SEQ ID NO: 1 or SEQ ID NO: 3. The differences, changes or alterations can be at any position in the nucleotide sequence of either polynucleotide and can be interspersed as individual changes or contiguous differences.  
           [0018]    Identified herein are certain fragments of the mouse and human polypeptides that were not previously known. These include SEQ ID NO: 2 between amino acids 1 and 477, SEQ ID NO: 2 between amino acids 564 and 663, SEQ ID NO: 2 between amino acids 709 and 721, SEQ ID NO: 2 between amino acids 758 and 785, SEQ ID NO: 2 between amino acids 819 and 923, SEQ ID NO: 2 between amino acids 1008 and 1052, SEQ ID NO: 2 between amino acids 1086 and 1245, SEQ ID NO: 2 between amino acids 1287 and 1310, SEQ ID NO: 2 between amino acids 1334 and 1739, SEQ ID NO: 4 between amino acids 1 and 478, SEQ ID NO: 4 between amino acids 565 and 664, SEQ ID NO: 4 between amino acids 710 and 722, SEQ ID NO: 4 between amino acids 75 9 and 786 , SEQ ID NO: 4 between amino acids 820 and 924, SEQ ID NO: 4 between amino acids 1009 and 1053, SEQ ID NO: 4 between amino acids 1087 and 1246, SEQ ID NO: 4 between amino acids 1288 and 1311, and SEQ ID NO: 4 between amino acids 1335 and 1745. Polypeptides having at least 80% identity to those polypeptide fragments, and preferably having at least 90%, 95%, 97% and 99% identity, are also within the scope of the invention, as are polynucleotides that encode any such polypeptide fragment.  
           [0019]    The invention also includes polynucleotides that hybridize to any of the aforementioned polynucleotides under stringent conditions, such as overnight incubation at 42° C. in a solution comprising 50% formamide, 5×SSC (150 mM NaCl, 15 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt&#39;s solution, 10% Dextran Sulfate, and 20 micrograms/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC at about 65° C. Polypeptides encoded by any of the foregoing polynucleotides are also within the scope of the invention.  
           [0020]    The polynucleotide can also be a variant of any of the foregoing. A “variant” as the term is used herein, is a polynucleotide that differs from a reference polynucleotide but retains essential properties. Generally, differences are limited so that the sequences of the reference polypeptide or polynucleotide and the variant are closely similar overall and may be identical in part. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide differs in nucleotide sequence from a reference polynucleotide. A variant polynucleotide may or may not encode an amino acid sequence that differs from the amino acid sequence encoded by the reference polynucleotide. Nucleotide changes can, but need not, result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. A variant of a polynucleotide or polypeptide can be a naturally occurring allelic variant, or can be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides may be made by mutagenesis techniques or by direct synthesis or other method.  
           [0021]    In keeping with the present invention, exemplary amino acid sequences of mammalian pro-α3(V) proteins, precursors of mammalian α3(V) proteins, are disclosed, as are cDNA sequences that encode the exemplified human and murine amino acid sequences. Patterns of expression in developing and adult tissues are examined, and the chromosomal locations of the cognate mouse Col5a3 and human COL5A3 genes are mapped. Full length mammalian pro-α3(V) cDNA sequences from mice and humans are disclosed in SEQ ID NO: 1 and SEQ ID NO: 3, respectively. Pro-α3(V) chain encoded by the exemplified murine and human nucleic acid sequences are disclosed in SEQ ID NO: 2 and SEQ ID NO: 4, respectively. The full-length mouse pro-α3(V) cDNA and amino acid sequences will be available at GenBank Accession No. AF176645. The full-length human pro-α3(V) cDNA and amino acid sequences will be available at GenBank Accession No. AF 177941.  
           [0022]    The disclosed amino acid sequences have all of the indicia of procollagen chains. Signal peptide cleavage sites, predicted by the method of Nielsen et al. (82), are after amino acid residue 30 (Ala) in the mouse protein and after amino acid residue 29 (Ala) in the human protein. Pro-α3(V) is closely related to the α1(V) precursor, pro-α1(V), but with marked differences in N-propeptide sequences, and collagenous domain features that provide insights into the low melting temperature of α1(V)α2(V)α3(V) heterotrimers, lack of heparin binding by α3 (V) chains and the possibility that α1(V)α2(V)α3(V) heterotrimers are incorporated into heterotypic fibrils.  
           [0023]    In a related aspect, any polynucleotide sequence of the present invention, or an antisense version thereof, can be provided in a vector or genetic construct in a manner known to those skilled in the art. A polypeptide-encoding polynucleotide so provided in a vector can, but need not, be under the transcriptional control of one or more regulatory elements which can include a promoter not natively found adjacent to the polynucleotide such that the encoded polypeptide can be produced when the vector is provided in a compatible host cell or in a cell-free transcription and translation system. Such cell-based and cell-free systems are well known to the skilled artisan. Cells comprising a vector containing a polynucleotide of the invention are themselves within the scope of the invention.  
           [0024]    Collagen and derivatives of collagen (gelatin) have been used in medical, pharmaceutical and consumer products for about 40 years. Examples of approved use of collagen include hemostats, vascular sealants, tissue sealants, implant coatings, injectable for plastic surgery, food additives, dental implants, artificial dura, wound dressings, antiadhesion barriers, antibiotic wound dressing, and platelet analyzer reagents. Human and animal collagen can be recombinantly reproduced. The disclosure of the fall-length mouse pro-α3(V) cDNA and the full-length human pro-α3(V) cDNA in the present invention makes it possible to recombinantly reproduce human and animal collagen α3(V), which can be used in the applications described above. In addition, human pro-α3(V) has been found to express in many tissues including mammary gland, placenta, uterus, brain, fetal lung, and fetal and adult heart. The present invention allows the reproduction of collagen α3(V) for the purpose of matching its natural role in the body. Thus, if any of the above tissue is damaged, collagen α3(V) can be produced and used in the tissue repairing process.  
           [0025]    The polynucleotides of the invention can also be employed as diagnostic reagents in assays for diagnosing a disease or susceptibility to a disease associated with α3(V) chains in human or non-human animals. Assays for detecting mutations in protein-encoding sequences are well known to the skilled artisan and can include assaying for changes in primary structure of a fragment by nucleotide sequence analysis, by digesting mismatched hybrids with RNase or by measuring changes in hybrid melting temperatures. Changes in sequence length resulting from insertion or deletion can be observed as a change in electrophoretic mobility of amplified fragments. The present invention also enables other methods for diagnosing changes in an α3(V)-encoding polynucleotide, such as nuclease protection assays, for one of ordinary skill in the art. A skilled artisan understands that such assays for diagnosing genetic changes at a fine scale in polynucleotides that encode α3(V) chains can be facilitated by providing an array of fragments of the polynucleotides of the invention for systematic screening in parallel for changes at any of a plurality of positions. This methodology enables an association between one or more mutations and a susceptibility to a disease such as classical or hypermobility type of EDS or diseases of other tissues in which α3(V) expression is noted such as diseases of female reproductive tissues or the heart as well as various other genetic diseases of the musculoskeletal system, connective tissue or skin.  
           [0026]    The present invention also enables one to diagnostically determine whether a human or non-human animal exhibits an altered (e.g., increased or decreased) amount of an α3(V) chain or an mRNA that encodes α3(V) in one or more tissues of interest. Methods for measuring polynucleotide levels are well known in the art and include quantitative PCR, Northern blotting, dot blotting and others. Methods for measuring protein levels are also known and include ELISA, radioimmunoassay, competitive-binding assays and Western blotting.  
           [0027]    Thus, the invention is also embodied in a diagnostic kit comprising one or more of any polynucleotide of the invention, a complementary sequence (antisense) to any polynucleotide of the invention, a polypeptide of the invention, or an antibody or single chain antibody against a polypeptide of the invention or against an immunogenic fragment thereof. An antibody can be obtained in any of several well-known methods such as hybridoma or trioma techniques and can also have utility in purifying α3(V) polypeptides or in treating diseases associated with the presence of α3(V).  
           [0028]    An immunological response effective to protect a human or non-human mammal against undesired activities of wild type or mutant α3(V) polypeptides can also be raised in vivo by administering to the mammal an immunogenic polypeptide (either directly or by administering to the mammal a genetic vector comprising sequences that direct expression of the polypeptide under the control of a transcriptional promoter). A vaccine of this type can also include a suitable carrier or adjuvant and can be administered at standard dosages according to standard protocols. The vaccine is preferably administered parenterally by injection, but can also be administered by any route known to be effective for inducing an immune response.  
           [0029]    The polypeptides of the invention also enable a skilled artisan to screen for agonists and antagonists of the polypeptides that can be selected using standard screening protocols that include the steps of expressing the polypeptide in or on suitable host cells, exposing the cells to various test compounds, and observing whether any test compound binds to the polypeptide or stimulates or inhibits any biological activity of the polypeptide relative to the binding or activity of the polypeptide in or on untreated control cells. The host cells can be any cells capable of expressing the polypeptide and can include mammalian cells, insect cells, yeast cells, or bacterial cells. Envisioned agonists and antagonists can include, but are not limited to, fragments of the full-length pro-α3(V) or α3(V) polypeptides that compete biologically with the full-length polypeptides as well as ligands, enzymes, receptors and the like that block active sites on the polypeptides and prevent their interaction with other molecules.  
           [0030]    In another aspect, then, the invention extends to a screening kit for identifying agonists or antagonists of the polypeptides of the invention, where the kit contains at least one polypeptide of the invention, an isolated cell or portion of a cell (such as a cell membrane) that contains a polypeptide of the invention, or an antibody to a polypeptide of the invention. In yet another aspect, agonists and antagonists so obtained are within the scope of the invention.  
           [0031]    In a therapeutic method, an agonist or antagonist can also be administered along with a pharmaceutically acceptable carrier to enhance or inhibit, respectively, a biological activity of the pro-α3(V) or α3(V) polypeptides. If the agonist or antagonist is itself a polypeptide or oligopeptide, it can be administered directly (with or without a suitable pharmaceutical carrier) or can be produced in vivo after administration of an expressible genetic vector that encodes the agonist or antagonist or a cell that contains the expressible genetic vector. Alternatively, expression of the pro-α3(V) or α3(V) polypeptides can be inhibited by administering an antisense sequence of the present invention to interfere with normal polypeptide expression. The antisense sequence can be administered directly (with or without a carrier) or can be produced in vivo after administration of a genetic vector capable of transcribing antisense genetic sequences. Appropriate dosages of an agonist or antagonist will vary depending upon the route of administration and the activity of the administered compound, but can readily be determined and optimized by a skilled artisan. Dosages in the range of between about 0.1 and 100 μg/kg are generally appropriate. 
       
    
    
     EXAMPLE 1  
     Murine Pro-α3(V) cDNA Sequence  
       [0032]    The following steps were performed to obtain a full-length cDNA sequence of murine pro-α3(V). A BLAST search of the dbEST database of expressed sequences tags, using query sequence LGPPGEDGAXGSVGPTGLPGDLGPPGDPGVSGIDG (SEQ ID NO: 4; amino acids 1246-1280) from a human α3(V) peptide TSK5/K1 (42), located 459-bp of α3(V) triple helix-encoding sequences from a mouse mammary gland EST (IMAGE clone 1366609; GenBank Accession No. AI021711). The EST clone was obtained from the IMAGE Consortium, sequenced in its entirety, and found to contain an insert of 2259-bp corresponding to roughly the 3′-most third of the final full-length mouse pro-α3(V) cDNA sequence (SEQ ID NO: 1; nt 3850-6108).  
         [0033]    Primer 5′-GGTCCCACAGGACTCCCTGGAGATCT-3′ (forward, SEQ ID NO: 1, nt 3853-3878) and primer 5′-TAGCCCAGGAGGTCCCAGGAGACCTG-3′ (reverse, reverse complement of SEQ ID NO: 1, nt 4209-4184), corresponding to EST sequences, amplified a 357 bp PCR product, using a mouse 17 days postcoitus (dpc) embryo cDNA 5′ stretch λgt10 library (Clontech) as template. This product was used to screen the same λgt10 library, yielding one positive clone(ME7) with a 1742-bp insert.  
         [0034]    Sequences of clone ME7 overlapped those of the EST clone and contained an additional 422-bp at the 5′-end.  
         [0035]    A 304-bp EcoRI fragment from the 5′-portion of the clone ME7 insert was used as a probe for further screening of the 17 dpc embryo library, yielding two additional clones, ME8-11 (1059-bp insert) and ME3-5 (876-bp insert), with 606-bp and 423-bp of additional 5′ sequences, respectively.  
         [0036]    Next, 5′ rapid amplification of cDNA ends (RACE) was performed with two nested pro-α3(V)-specific reverse primers, 5′-CCTTCAAACCAATGGGTCCTGGGTCT-3′ (reverse complement of SEQ ID NO: 1; nt 3061-3036) and 5′-CAATGCCACCAGAGGGGCCTACAGGA-3′ (reverse complement of SEQ ID NO: 1; nt 3142-3117), corresponding to sequences near the 5′-end of clone ME8-11, using the Marathon cDNA Amplification Kit and mouse brain Marathon-Ready cDNA template, according to the manufacturer&#39;s protocol (Clontech). This nested 5′RACE produced a 613 bp product.  
         [0037]    To obtain further mouse sequences, two pro-α3(V)-specific reverse primers corresponding to sequences near the 5′-end of the 613 bp 5′ RACE product, 5′-CTTTCTCCCCCAGTGGTCCCAAGGGT-3′ (primer MSP3, reverse complement of SEQ ID NO: 1; nt 2530-2505) and 5′-CCGGTGTGCCGCGTTCTCCTTCCTCT-3′ (primer MSP4, reverse complement of SEQ ID NO: 1; nt 2584-2559), were used both for a further nested 5′ RACE, performed as above, but in addition using Advantage-GC cDNA Polymerase Mix (Clontech); and for nested PCR using 17 dpc embryo λgt10 library cDNA as template and a λgt10 vector-specific primer, 5′-TCCCCACCTTTTGAGCAAGTTCAGCCT-3′ (SEQ ID NO: 5).  
         [0038]    Nested PCR with the λgt10 primer and library yielded a product with 898 bp of pro-α3(V) sequences. The 5′-RACE products were subcloned into the pGEM-T vector (Promega). A forward PCR primer, 5′-GTGACAGGGAGTGATGGCGCACCA-3′ (SEQ ID NO: 1; nt 1930-1953), corresponding to sequences within the 898 bp PCR product, and reverse primer MSP3 (see above) were used as a primer set for PCR screening of the 5′-RACE product -pGEM-T clones. One clone, which contained a 2530 bp PCR insert, was found to contain the remainder of mouse pro-α3(V) coding sequences plus 81-bp of the 5′-untranslated region (UTR).  
       EXAMPLE 2  
     Human Pro-α3(V) cDNA Sequence  
       [0039]    To obtain human pro-α3(V) sequences, a human placenta cDNA λgt11 library (Clontech) was screened with a 562-bp EcoRI cleavage fragment of the mouse IMAGE clone, roughly corresponding to the complete pro-α3(V) C-propeptide coding sequences. One positive clone (HP3-2) had a 3382-bp insert that corresponded to the 3′-half of human pro-α3(V) coding sequences plus 820-bp of 3′-UTR. A BLAST search of the dbEST database, using mouse pro-α3(V) C-propeptide sequences as the query sequence, located human retina EST pro-α3(V) sequences (EST19755, clone HARAL32, GenBank Accession No. AA3 17772, ATCC Item No. 118234). The EST clone was obtained from the American Type Culture Collection, sequenced in its entirety, and found to have an insert of 1316-bp that overlapped the 3′-end of clone HP3-2 and included an additional 34-bp of 3′-UTR extending to a poly(A) tail.  
         [0040]    Pro-α3(V)-specific reverse primers 5′-TCACCTAGAGGTCCCACTTCTCCTGTCT-3′ (reverse complement of SEQ ID NO: 3; nt 2884-2857) and 5′-AGTTCTCCTCTCTGTCCAGGGTGCCCT-3′ (reverse complement of SEQ ID NO: 3; nt 2797-2771), corresponding to sequences near the 5′-end of λgt11 clone HP3-2, were used for nested 5′ RACE with Marathon-ready human fetal brain cDNA as template, resulting in a product containing 366-bp of pro-α3(V) sequences. A subsequent nested PCR with pro-α3(V)-specific reverse primers 5′-GCTGCCCTGTCTTTCCCGACTTCCCT-3′ (reverse complement of SEQ ID NO: 3; nt 2562-2537) and 5′-ACCGGGAAATCCAATAGATCCCTTAGGT-3′ (reverse complement of SEQ ID NO: 3; nt 2513-2486), corresponding to sequences near the 5′-end of the 366 bp RACE product, and using a λgt10 vector-specific primer 5′-AGATTGGGGGTAAATAACAGAGGTGGCT-3′ (SEQ ID NO: 6) and λgt10 human Fetal Heart cDNA library template, produced a product containing 774-bp of pro-α3(V) sequences.  
         [0041]    Next, nested 5′ RACE with pro-α3(V)-specific reverse primers 5′-ACCCTTCTCCCCAGGAGTGCCAATGAGT-3′ (reverse complement of SEQ ID NO: 3; nt 2081-2054) and 5′-ACCCATGGTTTCCCTGCTGTCCCGGA-3′ 0  (reverse complement of SEQ ID NO: 3; nt 2028-2003), corresponding to sequences near the 5′-end of the 774-bp product, and using Marathon-Ready human heart cDNA template, yielded a 1532-bp product. This was followed by another nested 5′ RACE with pro-α3(V)-specific reverse primers 5′-TCACAAGCCTGGAAGGCGGCCTGAGGA-3′ (reverse complement of SEQ ID NO: 3; nt 739-713) and 5′-GGGTCCCCAGCACAGTGAGTCCAGCTA-3′ (reverse complement of SEQ ID NO: 3; nt 654-628), and using Marathon-Ready human heart cDNA template, which yielded a 551-bp product.  
         [0042]    A final nested 5′ RACE with pro-α3(V)-specific reverse primers 5′-AGTTCTCAGGAAAGTGGCCTTCTGGAA-3′ (reverse complement of SEQ ID NO: 3; nt 354-328) and 5′-GCACACCCAGGGCCTTCAGGACATCCA-3′ (reverse complement of SEQ ID NO: 3; nt 207-181), corresponding to sequences near the 5′-end of the 551-bp product, and using Marathon-Ready human placenta cDNA template and Advantage-GC cDNA Polymerase Mix (Clontech), produced a 207-bp product that contained remaining pro-α3(V) coding sequences plus 86-bp of 5′-UTR.  
         [0043]    First rounds of nested RACE PCRs were performed in 50 μl reactions with 20 pmol of each primer, 5 μl of Marathon cDNA, and 1 μl of Advantage cDNA Polymerase Mix (Clontech) at 95° C./3 min followed by 40 cycles of 95° C./20 sec, 68° C./30 sec, 72° C./2-4 min and final extension at 72° C./7 min. When Advantage-GC cDNA Polymerase Mix was used, GC-Melt was added to a final concentration of 1 M per reaction. First rounds of nested PCRs using λgt10 primers were performed the same way as first round RACE PCRs, except that the annealing temperature was 70° C., and template was 5 ul of a λgt10 library that had been diluted 12-fold with water and heat-denatured by boiling for 10 min. The second nested rounds of RACE PCRs and second nested rounds of PCRs using λgt10 primers, were performed the same way as first rounds, except that 25, rather than 40, cycles were used and template was 5 ul of first round PCR products diluted 50-fold with water.  
         [0044]    The full-length mouse and human prepro-α3(V) collagen chain sequences, inferred from cDNA clones and PCR products described in Experimental Procedures, are presented in SEQ ID NO: 1 and SEQ ID NO: 3, respectively. The human and mouse prepro-α3(V) chains comprise 1745 and 1739 amino acid residues, respectively. In each case, the mature form of the protein extends from amino acid 30 to the terminal residue 1745 or 1739, respectively.  
         [0045]    These sequences show pro-α3(V) to be related to the pro-α1(V), pro-α1(XI), and pro-α2(XI) chains, with sequence similarities and differences that provide insights into the nature and biology of the pro-α3(V) chain. As an example, a conserved Lys at position 84 of the COL1 domain suggests that α1(V)α2(V)α3(V) heterotrimers may be incorporated into heterotypic fibers, while differences in N-propeptide/telopeptide sequences suggest that such heterotypic fibrils would have different surface charge properties than heterotypic fibrils which incorporate α1(V) 2 α2(V) heterotrimers, likely to influence fibril shape/diameters and interactions with other macromolecules.  
         [0046]    An unexpected finding was the expression of pro-α3(V) RNA primarily in the connective tissue sheaths (epimysia) of forming muscles and in the rudiments of ligamentous attachments adjacent to forming bones and within nascent joints during development. This observation coupled with the fact that pro-α3(V) chains combine with pro-α1(V) and pro-α2(V) chains to form heterotrimers, suggests COL5A3 as a possible candidate locus for at least some cases of classical EDS in which COL5A1 and COL5A2 have been excluded, and for at least some cases of the hypermobility type of EDS. Expression of pro-α3(V) in epimycium also raises the possibility that defects in COL5A3 and Col5a3 might result in some muscle myopathies, as has recently been shown to be the case with the genes for type VI collagen (81).  
         [0047]    The pro-α3(V) chains presented in SEQ ID NO: 2 and SEQ ID NO: 4 are most similar to, but are distinct from, the pro-α1(V), pro-α1(XI) and pro-α2(XI) fibrillar procollagen chains. The human and mouse prepro-α3(V) chains comprise 1745 and 1739 amino acid residues, respectively. Each includes a 1011 amino acid major collagenous domain (COL1), which is shorter than the COL1 domains of the other vertebrate fibrillar collagen chains. In COL1, for example, the pro-α3(V) COL1 domain is most similar to that of pro-α1(V) (76% similarity, 71% identity), but only slightly less similar to that of pro-α1(XI) (74% similarity, 70% identity) and only somewhat less similar to that of pro-α2(XI) (72% similarity, 67% identity) when comparison was via the Genetics Computer Group GAP program (83). The shorter COL1 domain, coupled with its smaller number of imino acid residues than are found in the pro-α1(V) and pro-α2(V) COL1 (215 PRO codons versus 249 and 223 PRO codons, respectively), helps explain the lower melting temperature of pepsinized α1(V)α2(V)α3(V) heterotrimers compared to that of pepsinized α1(V) 2 α2(V) heterotrimers (18,67).  
         [0048]    The 412 amino acid residue region between the signal peptide and COL1 domain can be divided into four subdomains. Immediately upstream of the COL1 domain is a short non-collagenous linker region, and immediately N-terminal of this is a short collagenous domain which corresponds to the NC2 (noncollagenous 2) and COL2 domains, respectively. The pro-α3(V) COL2 domain is likely to form a shorter triple helix than those formed by the COL2 domains of the other procollagen chains of this subfamily.  
         [0049]    A large globular region between the pro-α3(V) signal peptide and the COL2 domain corresponds to an NC3 domain found in other members of this subfamily. NC3 can be roughly divided into two subdomains, namely (1) an amino terminal PARP (proline/arginine-rich protein) between the signal peptide and two clustered cysteines and (2) a variable region between PARP and COL2.  
         [0050]    The pro-α3(V) PARP domain retains four cysteines conserved among all members of the subfamily, but has a markedly acidic pI of 4.4, unlike the highly basic pI predicted from the sequence of the PARP domain of pro-α2(XI). This suggests a somewhat different function for pro-α3(V) and α3(V) chains than for the other family members.  
         [0051]    Little or no homology exists between the related proteins in the variable region and may reflect differences in biological activities. The residues of the α3(V) variable region appear to protrude beyond the surface of heterotypic fibrils and may directly control fibrillogenesis by sterically hindering the further addition of collagen monomers to the fibril surface. These protruding sequences may also help modulate interactions between heterotypic collagen fibrils and other components of the extracellular matrix. The pro-α3(V) variable domain has a highly basic predicted pI (e.g. 10.3 for the human sequence) and a total absence of tyrosines. This is predicted to affect the charge properties of the α3(V) chains which may in turn alter the charge properties and surface characteristics of α1(V)α2(V)α3(V) heterotrimers from those of α1(V) 2 α2(V) heterotrimers.  
         [0052]    Canonical RX(K/R)R furin cleavage sites (56,57) just C-terminal of the COL1 in the human and mouse pro-α3(V) chains align with that of the pro-α1(V) chain. In pro-α1(V), a furin-like proprotein convertase (53) appears to act immediately downstream of the canonical site to cleave the C-propeptide. Thus, the C-propeptides of the α1/α3(V)/α1/α2(XI) subfamily of procollagen chains may all be cleaved by the same, or by similar, furin-like proprotein convertases.  
         [0053]    Seven cysteine residues seen at similar positions in the C-propeptides of all previously characterized fibrillar procollagen chains are conserved within the pro-α3(V) C-propeptide or NC1 domain. Sequence alignment also reveals that the pro-α3(V) C-telopeptide is shorter than those of the pro-α1(V), pro-α1(XI), and pro-α2(XI) chains, as is the portion of the pro-α3(V) C-propeptide immediately adjacent to the C-telopeptide. Both regions have previously been noted as areas of relative sequence variability among procollagen chains (74). A potential glycosylation site (NQT) between C-propeptide cysteines 6 and 7, is conserved in both mouse and human pro-α3(V) sequences but is not found in any other fibrillar procollagen C-propeptide. The site may be of specific importance to the structure or function of pro-α3(V) chains.  
         [0054]    The a3(V) sequences support the suggestion that basicity of the binding region is a determinant of heparin/heparan sulfate binding in type V/XI collagen chains. While α2(V) and α3(V) chains do not bind heparin under physiological or denaturing conditions (69-71) isolated α1(V) chains do. As evidence that α1(V) chains mediate heparin binding, while α2(V) and α3(V) chains do not (70,71), it is known that triple helical type V collagen trimers bind to heparin with decreasing affinity in the order α1(V)3&gt;α1(V) 2α2 (V)&gt;α1(V)α2(V)α3(V). It has been suggested that the low basicity of the region of α2(V) that corresponds to the α1(V) binding site prevents heparin binding. In contrast, high basicity in the corresponding region in type XI chains (69) promotes heparin binding. It can be seen in the sequences that α3(V), like α2(V), has fewer basic residues in this region than do α1(V), α1(XI), or α2(XI). Moreover, α3(V), like α2(V), has more acidic residues in this region than do the other chains, further reducing localized basicity.  
       EXAMPLE 3  
       [0055]    Pro-α3(V) Expression in Adult and Developing Tissues  
         [0056]    Patterns of mRNA expression for pro-α3(V) chains were examined and were compared against mRNA expression patterns for pro-α1(V), pro-α2(V), pro-α1(XI) and pro-α2(XI) chains by hybridizing sequence-specific probes, and a ubiquitin control probe, to a multiple tissue expression (MTE) array (Clontech) of dot-blotted poly(A)+RNA from a variety of adult and fetal human tissues. The MTE array included RNA from whole brain, cerebral cortex, frontal lobe, parietal lobe, occipital lobe, temporal lobe, paracentral gyrus of cerebral cortex, pons, cerebellum left, cerebellum right, corpus callosum, amygdala, caudate nucleus, hippocampus, medulla oblongata, putamen, substantia nigra, accumbens nucleus, thalamus, pituitary gland, spinal cord, heart, aorta, left and right atrium, left and right ventricle, interventricular septum, apex of heart, esophagus, stomach, duodenum, jejunum, ileum, ileocecum, appendix, ascending, transverse and decending colon, rectum, kidney, skeletal muscle, spleen, thymus, peripheral blood leukocyte, lymph node, bone marrow, trachea, lung, placenta, bladder, uterus, prostate, testis, ovary, liver, pancreas, adrenal gland, thyroid gland, salivary gland, mammary gland, leukemia HL-60, HeLa S3, leukemia K-562, leukemia MOLT-4, Burkitt&#39;s lymphoma, Raji, Burkitt&#39;s lymphoma, Daudi, colorectal adenocarcinoma SW480, lung carcinoma A549, fetal brain, fetal heart, fetal kidney, fetal liver, fetal spleen, fetal thymus and fetal lung. Blots were hybridized to random primed probes in ExpressHyb (Clontech) at 65° C.  
         [0057]    The probes were prepared as follows:  
         [0058]    α3(V): A 1.6-kb probe corresponding to 3′-UTR and C-propeptide sequences was an EcoRI and FspI fragment of clone HP3-2 (see above).  
         [0059]    α1(V): A 1815-bp probe containing mainly triple helical and C-propeptide sequences was an EcoRI fragment of cDNA clone CW32 (27).  
         [0060]    α2(V): A a 564-bp probe corresponding to C-propeptide sequences was an EcoRI-HindIII fragment of cDNA clone pBSL18 (43).  
         [0061]    α1(XI): A 1,004-bp probe corresponding to C-propeptide and 3′-UTR sequences was amplified from human heart Marathon cDNA with forward primer 5′-TCATCCTAACCAAGGTTGCTCAGG-3′ (SEQ ID NO: 7) and reverse primer 5′-GAGTCAGCGGAAATTCAGGGACACG-3′ (SEQ ID NO: 8) using Advantage cDNA polymerase Mix and conditions of 95° C./3 min followed by 35 cycles of 95° C./20 s, 58° C./30 s, 72° C./3 min and final extension at 72° C./7 min. PCR generated probes were cloned into pGEM-T, sequenced to confirm identity, and excised by restriction with SpeI and ApaI.  
         [0062]    α2(XI): An 890-bp probe corresponding to C-propeptide and 3′-UTR sequences was amplified by nested PCR. The first round was with primers 5′-AGGCGAGGTGATCCAGCCACTGC-3′ (forward; SEQ ID NO: 9) and 5′-GCTCTCTAACGGGTAACAGGCTCC-3′ (reverse; SEQ ID NO: 10) using the same conditions used for PCR amplification of the human α1(XI), except that annealing was at 55° C. The second, nested round was with primers 5′-ATGCAGGAAGATGAGGCCATACC-3′ (forward; SEQ ID NO: 11) and 5′-GCTCTCTAACGGGTAACAGGCTCC-3′ (reverse; SEQ ID NO: 12), using 5 ul of a 1/50 dilution of the first round PCR product as template, and conditions of 95° C./3 min followed by 25 cycles of 95° C./20 s, 58° C./30 s, 72° C./3° C./3 min and final extension at 72° C./7 min.  
         [0063]    Particularly high pro-α3(V) expression was detected in mammary gland, which correlates with the initial isolation of pro-α3(V) sequences as a mouse mammary gland EST and suggests a role for pro-α3(V) chains in this tissue in humans and mice. Relatively high pro-α3(V) mRNA levels were also seen in placenta and uterus, consistent with the results of previous protein studies (12,17-19). In addition, high expression of pro-α3(V) mRNA was found in fetal heart and lung, and moderately high levels were detected in certain structures of adult human heart.  
         [0064]    Relatively high levels of pro-α1(V) and pro-α2(V) RNA were found in most of the same human tissues noted for pro-α3(V) expression, which suggests the presence of α1(V)α2(V)α3(V) heterotrimers in these tissues. An exception was adult brain, in which relatively high levels of pro-α3(V) mRNA expression were not matched by high levels of either pro-α1(V) or pro-α2(V) mRNA. These data are consistent with the possibility that pro-α3(V) chains may combine with other procollagen chains or form homotrimers in these regions of adult human brain.  
         [0065]    Highest pro-α1(XI) and pro-α2(XI) mRNA levels were seen in trachea, probably reflecting the hyaline cartilage content of this structure. Surprisingly high levels of pro-α1(XI) and especially high levels of pro-α2(XI) mRNA were also found in structures of adult human brain. While this may suggest heterotrimer formation between pro-α3(V) and one or both type XI procollagen chains in brain, distributions of both type XI procollagen mRNAs in the different brain structures are quite different from that of pro-α3(V) mRNA.  
         [0066]    Patterns of mRNA expression for human pro-α3(V) and other fibrillar procollagen chains were further characterized by sequentially hybridizing sequence-specific probes, and a β-actin control probe, to multiple tissue Northern (MTN) blots I, containing approximately 2 μg poly(A)+ RNA per lane from a subset of the tissues examined by dot-blot assay, namely human pancreas, kidney, skeletal muscle, liver, lung, placenta, brain, heart, peripheral leukocytes, colon, small intestine, uterus, testis, prostate, thymus and spleen. Northern blots were washed in 2×SSC, 0.1% SDS at 65° C., followed by 0.1×SSC, 0.1% SDS at 55° C.  
         [0067]    The pro-α3(V) expression patterns were generally consistent with those seen by dot-blot, with particularly high levels of expression of a ˜6.0-kb band detected in heart, placenta and uterus. As in the dot-blot, pro-α1(V), pro-α2(V) and pro-α3(V) mRNAs were coexpressed in heart, placenta and uterus. Interestingly, pro-α3(V) mRNA in liver had a somewhat faster mobility (˜5.5-kb) than that detected in the other tissues, while the pro-α3(V) mRNA in brain had a considerably faster mobility (˜4.2-kb). The nature of the ˜4.2-kb transcript in brain is unclear, as the full-length pro-α3(V) coding sequence is 5235-bp and there is no evidence for alternative splicing of the pro-α3(V) N-propeptide.  
         [0068]    Spatiotemporal expression patterns of pro-α3(V) mRNA in embryonic murine tissues were examined by hybridizing procollagen-specific probes to a mouse embryo blot containing poly(A)+ RNA from 7, 11, 15, and 17 dpc mouse embryos (Clontech).  
         [0069]    Probes for the murine sequences were prepared as follows:  
         [0070]    α3(V): A 784 bp probe corresponding to 3′-UTR sequences was amplified from EST IMAGE clone 1366609 with forward primer 5′-TGAAGTTGTGAGGTGGGAAGGAAGCT-3′ and reverse primer 5′-GAGCACAGTTCCTTGGTTTATTCT-3′ using Advantage cDNA polymerase Mix and conditions of 94° C./3-5 min followed by 30-35 cycles of 94° C./ 30 s, 55-70° C./30 s, 72° C./3 min and final extension at 72° C./10 min. PCR-generated probes were cloned into pGEM-T, sequenced to confirm identity, and excised by restriction with SpeI and SacII.  
         [0071]    α1(V): A 1,206-bp probe corresponding to C-propeptide and 3′-UTR sequences was amplified from 17-dpc mouse embryo Marathon cDNA with forward primer 5′-GGAGAGCTACGTGGATTATGC-3′ (SEQ ID NO: 13) and reverse primer 5′-CCATCGGAAAGGCACGTGTGG-3′ (SEQ ID NO: 14), under the conditions noted just above. PCR-generated probes were cloned into pGEM-T, sequenced to confirm identity, and excised by restriction with SpeI and ApaI.  
         [0072]    α2(V): A 524-bp probe corresponding to 3′-UTR sequences was amplified from 17-dpc mouse embryo Marathon cDNA with forward primer 5′-CTTCAAGACACCTGCTCTAAGCT-3′ (SEQ ID NO: 17) and reverse primer 5′-ACATACCCCATCATGTAAGCTACC-3′ (SEQ ID NO: 18), with the probe gel-purified, direct-sequenced to check identity, and random-primed for blotting.  
         [0073]    α1(XI): A 948-bp probe corresponding to C-propeptide and 3′-UTR sequences was amplified from 17-dpc mouse embryo Marathon cDNA with forward primer 5′-GTTTGGATTTGAAGTCGGTCCAGC-3′ (SEQ ID NO: 19) and reverse primer 5′-TGGCATTACTGAAGCACGCTGAGG-3′ (SEQ ID NO: 20), under the conditions noted just above. PCR-generated probes were cloned into pGEM-T, sequenced to confirm identity, and excised by restriction with SpeI and ApaI.  
         [0074]    α2(XI): A 61 1-bp α2(XI) Northern blot corresponding to N-propeptide/telopeptide sequences was amplified from 17-dpc mouse embryo Marathon cDNA with forward primer 5′-ATGTGGCTTACCGTGTGGCACG-3′ (SEQ ID NO: 21) and reverse primer 5′-GCTCTGTGGCTTATGAAGTCTTGC-3′ (SEQ ID NO: 22), under the conditions noted just above. PCR-generated probes were cloned into pGEM-T, sequenced to confirm identity, and excised by restriction with SpeI and ApaI.  
         [0075]    The murine pro-α3(V) probe hybridized to a readily detectable single ˜6.3-kb band in the RNA of 7 dpc mid-gastrulation mouse embryos. This mRNA disappears at 11 dpc and was not visible even upon prolonged exposure of the blot, nor was signal for pro-α3(V) RNA detectable at this stage by in situ hybridization of 11 dpc mouse embryos. Pro-α3(V) mRNA reappears at 15 dpc and is further increased in abundance at 17 dpc, during a period of post-organogenesis fetal growth and development.  
         [0076]    Among the other fibrillar procollagen mRNAs, strong expression of both pro-α1(V) and pro-α2(V) mRNAs accompany that of pro-α3(V) mRNA at 15 and 17 dpc. Although pro-α2(V) mRNA expression is also strong at 7 dpc, expression of pro-α1(V) is not readily detectable at this stage of development, with low levels of pro-α1(V) mRNA just visible upon prolonged exposure of the blot.  
         [0077]    Pro-α1(XI) and pro-α2(XI) mRNAs are also readily detectable at 15 and 17 dpc, but even prolonged exposure of the blot did not reveal detectable levels at 7 and 11 dpc. These results suggest a role for type V, but not type XI collagen chains in mid-gastrulation mouse embryos. The results are also consistent with the possibility that pro-α3(V) chains may exist either as homotrimers or in heterotrimeric combination with pro-α2(V) chains, in the absence of pro-α1(V) chains, at this time. However, the possibility that α3(V) chains are found only in the context of α1(V)α2(V)α3(V) heterotrimers at 7 dpc, despite wide differences in RNA levels for the various chains, has not been excluded.  
         [0078]    To determine the distribution of expression of pro-α3(V) during mouse development, and to compare this to the expression domains of other type V/XI procollagen chains, a series of in situ hybridizations were performed on serial sagittal and parasagittal sections of 13.5 dpc and 15.5 dpc mouse embryos using anti-sense, and sense control, riboprobes specific for pro-α3(V), pro-α1(V), pro-α1(XI) and pro-α2(X) sequences. For in situ hybridization, uniform labeling of riboprobes with [35S]UTP, tissue preparation, and hybridization were performed as described (44), except that sections were 5 μm thick and mounted two to six/slide. For histological analysis, sections were prepared and stained with hematoxylin, eosin and alcian blue as described previously (45). Slides were analyzed using light- and dark-field optics of a Zeiss Axiophot 2 microscope.  
         [0079]    Probes were prepared as follows using a 17 dpc mouse embryo Marathon cDNA template:  
         [0080]    α1(V): A 475-bp probe corresponding to 3′-UTR sequences was amplified using forward primer 5′-TGAGCCCACCGGTCTCCAGAGC-3′ (SEQ ID NO: 15) and reverse primer 5′-CCATCGGAAAGGCACGTGTGG-3′ (SEQ ID NO: 16). Antisense and sense riboprobes were generated by linearizing with NotI and transcribing with T7 two different subclones in which the insert was in opposite orientations.  
         [0081]    α3(V): A 1,480-bp probe corresponding to N-propeptide/telopeptide sequences was amplified with forward primer 5′-AGACCAGTCCACATCCCCCTTGGCCT-3′ (SEQ ID NO: 1; nt 34-59) and reverse primer 5′-CTTTCATGGACAGCTGAGCCTGTTGCA-3′ (reverse complement of SEQ ID NO: 1; nt 1513-1487). Riboprobes were generated from this template by linearizing with ApaLI and transcribing with polymerase SP6 (antisense) or by linearizing with NotI and transcribing with polymerase T7 (sense).  
         [0082]    α1(XI): Antisense and sense riboprobes were generated from the pro-α1(XI) Northern blot probe by linearizing with NotI two subclones of the vector containing the insert in opposite orientations and transcribing with T7.  
         [0083]    α2(XI): Antisense and sense riboprobes were generated from the pro-α2(XI) Northern blot probe by linearizing the vector with NotI and transcribing with T7 (antisense) or by linearizing the vector with NcoI and transcribing with SP6 (sense).  
         [0084]    At 13.5 dpc pro-α3(V) RNA expression was barely detectable, although pro-α1(V) RNA expression was widely distributed throughout developing mesenchyme and intense pro-α1(XI) and pro-α2(XI) signals were already visible in nascent chondrified cartilaginous elements.  
         [0085]    At 15.5 dpc, however, pro-α3(V) expression was readily discernible and the pro-α3(V) expression domain was a subset of that of pro-α1(V). Interestingly, although pro-α1(V) expression was widely distributed throughout developing connective tissues, with especially high levels of expression seen in the perichondrium associated with cartilaginous primordia of future bones, expression of pro-α3(V) was not detected in perichondrium or other regions of bone primordia, but was instead most readily detectable in the superficial fascia and in the epimysia, or connective tissue sheaths, tracing the outlines of the developing muscles of the anterior chest wall, the cutaneous panniculus carnosus muscle and the developing musculature of the neck. In addition to its expression in epimysium, pro-α3(V) expression was also seen in the connective tissue sheath, or epineureum, of some nerves. Although pro-α3(V) was not expressed in perichondrium, high pro-α3(V) expression was observed closely apposed to the cartilage primordia of future bones in the soft tissue associated with a number of joints, in what appeared to be incipient ligamentous attachments (formation of ligaments and tendons first begins in mouse development, as mesenchymal condensations at 14 dpc, Ref. 75). Pro-α3(V) expression in nascent ligamentous attachments can be seen i) between the cartilage primordia of the bone at the base of the skull and the first two cervical vertebrae C1 (atlas) and C2 (axis), ii) apposed to the cartilage primordium of the exoccipital bone and, iii) between the cartilage primordia of the femoral head and acetabulum of the hip joint. Pro-α3(V) signal was also detectable in forming tendons within the hindlimb.  
       EXAMPLE 4  
       [0086]    Mapping the Human COL5A3 and Mouse Col5a3 Genes  
         [0087]    Chromosomal positions were established for the human COL5A3 and mouse Col5a3 genes that encode the human and murine pro-α3(V) chains, respectively. The human COL5A3 gene was mapped by radiation hybrid mapping (46), using PCR analysis of the Genebridge 4 radiation hybrid panel (Research Genetics). Primers (50 pmol each) were 5′-CTGCTTCAGCAGCTGAGAGTGTCC-3′ (forward, SEQ ID NO: 3; nt 5309-5332) and 5′-ACCACCTGGCATGGCAAGGTGAGC-3′ (reverse, reverse complement of SEQ ID NO: 3; nt 5946-5923), in 50-μl reactions with 100 ng template DNA and 2.5 U Taq polymerase (Sigma) at 95° C./5 min followed by 30 cycles of 94° C./30 s, 60° C./45 s, 72° C./2 min and final extension at 72° C./10 min. These conditions amplified a 615-bp product from human genomic DNA template, corresponding to 3′-UTR sequences. Scoring, submitted to the WICGR Mapping Service at the Whitehead Institute/MIT Center for Genome Research, clearly mapped COL5A3 to chromosome 19 p, 6.19 cR from WI-8049 and 2.02 cR from WI-7557 (Lod 2.68 relative to most likely). According to the Genome Database (http://gdbwww.gdb.org), WI-7557 amplifies from gene DNMT1, which has been cytogenetically mapped to 19p13.2 (77). The nearby polymorphic marker should be useful in analyzing linkage with EDS and other disease phenotypes.  
         [0088]    The murine Col5a3 gene was mapped by PCR analysis of 94 progeny of the C57BL/6J X  Mus spretus  (BSS) backcross from the Jackson Laboratory (47). Primers (20 pmol each) were 5′-CCTGGCAAGAGGGTGAGTGGTCTTCCA-3′ (forward; SEQ ID NO: 23) and 5′-GCATCCAGGTTTATGTCAAGAGTGGGCT-3′ (reverse; SEQ ID NO: 24), in 20-μl reactions with 25 ng template DNA and 0.4 μl Advantage cDNA polymerase mix (Clontech) at 95° C./3 min followed by 30 cycles of 94° C./30 s, 65° C./45 s, 72° C./30 sec and final extension at 72° C./5 min. These conditions amplified 315-bp (C57BL/6J) and 285-bp ( M. spretus ) products, corresponding to Col5a3 intronic sequences with differences in length mostly due to different alleles of a CA polymorphic repeat (25 and 9 CA repeats, respectively). Segregation of these products in the 94 BSS backcross progeny showed linkage of Col5a3 to a region of proximal chromosome 9, which is homologous to human 19p13.2.  
         [0089]    Mapping of the human and mouse sequences reported herein to homologous positions in the human and murine genomes, supports the contention that they are human and mouse homologues of the same gene, rather than genes for related, but genetically distinct procollagen chains. No connective tissue or musculoskeletal disorder that might readily arise from defects in the pro-α3(V) chain has yet been mapped to the same chromosomal region as either COL5A3 or Col5a3. However, the highly polymorphic simple sequence (CA) repeat D19S413, with a maximum heterozygosity of 0.78 (78) has, like COL5A3, been mapped to the ˜3.6 cM interval between WI-8049 and WI-7557 and, thus, should be of use in the initial analysis of linkage between COL5A3 and disease phenotypes in EDS and other affected families. The observed distribution of pro-α3(V) RNA, and the association of α1(V), α2(V), and α3(V) chains in heterotrimers, suggests the human α3(V) gene COL5A3 as a candidate locus for at least some cases of classical EDS in which the α1(V) and α2(V) genes have been excluded, and for at least some cases of the hypermobility type of EDS.  
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         1 
         
           
             24  
           
           
             1  
             6109  
             DNA  
             Mus musculus  
             
               CDS  
               (82)..(5298)  
             
           
            1 

gcctccggct gtccagagtg actgctccca ggaagaccag tccacatccc ccttggcctt     60 

ggtgcaccag gccccgctgg g atg aga agc tgc cgg aga ctg gat cag ctt      111 
                        Met Arg Ser Cys Arg Arg Leu Asp Gln Leu 
                          1               5                  10 

cag gcc ggc ctc tgc ctg ctc ctg gcc tcc ctg cag ctc gtg tcc tgg      159 
Gln Ala Gly Leu Cys Leu Leu Leu Ala Ser Leu Gln Leu Val Ser Trp 
                 15                  20                  25 

acg ctg gct gca gaa cct gtg gac gta ctg gaa gcc tgg ggt gtg cat      207 
Thr Leu Ala Ala Glu Pro Val Asp Val Leu Glu Ala Trp Gly Val His 
             30                  35                  40 

aga gac cag gct ggg gtg gct gaa ggg cct ggc ttc tgc ccc ctg agg      255 
Arg Asp Gln Ala Gly Val Ala Glu Gly Pro Gly Phe Cys Pro Leu Arg 
         45                  50                  55 

att cca cag ggt gac cga gca ttc agg gtg ggc aag tcc agc ctt ctc      303 
Ile Pro Gln Gly Asp Arg Ala Phe Arg Val Gly Lys Ser Ser Leu Leu 
     60                  65                  70 

agt gtc ccc acg tgg cag ctc ttc cca gat ggg cat ttt cct gag aac      351 
Ser Val Pro Thr Trp Gln Leu Phe Pro Asp Gly His Phe Pro Glu Asn 
 75                  80                  85                  90 

ttt tct gtg ctg ctc aca ctg agg gcc cag cca gcc aat cag tct gtc      399 
Phe Ser Val Leu Leu Thr Leu Arg Ala Gln Pro Ala Asn Gln Ser Val 
                 95                 100                 105 

ctt ctg tct att tat gat gag aag ggt gtc cgg cag ctg ggg ctg gca      447 
Leu Leu Ser Ile Tyr Asp Glu Lys Gly Val Arg Gln Leu Gly Leu Ala 
            110                 115                 120 

ctg ggg cca gct ctg ggc ctc ctt ggt gac tcc ttc agg ccc ctc ccc      495 
Leu Gly Pro Ala Leu Gly Leu Leu Gly Asp Ser Phe Arg Pro Leu Pro 
        125                 130                 135 

aag caa gtc aac att atg gat ggc agg tgg cac cgt gtg gca gtc agc      543 
Lys Gln Val Asn Ile Met Asp Gly Arg Trp His Arg Val Ala Val Ser 
    140                 145                 150 

atc agt ggt aac aag gtg acc ctg gtg gtt gac tgt gaa ccg cag ccc      591 
Ile Ser Gly Asn Lys Val Thr Leu Val Val Asp Cys Glu Pro Gln Pro 
155                 160                 165                 170 

cca aca ttt ggt cag ggg cct cgg ttt ata agt aca gct gga ctc act      639 
Pro Thr Phe Gly Gln Gly Pro Arg Phe Ile Ser Thr Ala Gly Leu Thr 
                175                 180                 185 

gtg atg gga acc cag gac acc agg gaa gag tct ttt gag gga gac atc      687 
Val Met Gly Thr Gln Asp Thr Arg Glu Glu Ser Phe Glu Gly Asp Ile 
            190                 195                 200 

cag gag ctg ctg tta att cca gac cct cag gct gcc ttc cag gcc tgt      735 
Gln Glu Leu Leu Leu Ile Pro Asp Pro Gln Ala Ala Phe Gln Ala Cys 
        205                 210                 215 

gag agc tac ctc cct ggt tgt gaa acc ctc gat tcc aca acc aca ggg      783 
Glu Ser Tyr Leu Pro Gly Cys Glu Thr Leu Asp Ser Thr Thr Thr Gly 
    220                 225                 230 

gcc ccc aaa gac gat gaa cca gaa acc cct gcc cct cgt cgt cga aag      831 
Ala Pro Lys Asp Asp Glu Pro Glu Thr Pro Ala Pro Arg Arg Arg Lys 
235                 240                 245                 250 

ggc aaa ggg aag aaa aaa ggg cgg ggt cga aag ggc aag gga aga aag      879 
Gly Lys Gly Lys Lys Lys Gly Arg Gly Arg Lys Gly Lys Gly Arg Lys 
                255                 260                 265 

aaa aac aag gag acc tca gag ctg agt ccg acc cct ggt gcc cct gag      927 
Lys Asn Lys Glu Thr Ser Glu Leu Ser Pro Thr Pro Gly Ala Pro Glu 
            270                 275                 280 

aac cag acc tcc ctc cac atc cct gag aca gag aag aca gtt ccc cac      975 
Asn Gln Thr Ser Leu His Ile Pro Glu Thr Glu Lys Thr Val Pro His 
        285                 290                 295 

ctg cct ctg act ccc aca cct ctg gcc atc acc acc act gtc acg att     1023 
Leu Pro Leu Thr Pro Thr Pro Leu Ala Ile Thr Thr Thr Val Thr Ile 
    300                 305                 310 

gga caa aat gcc aca gtc tcg cag ggg ttg gac tcc ggt act gaa acc     1071 
Gly Gln Asn Ala Thr Val Ser Gln Gly Leu Asp Ser Gly Thr Glu Thr 
315                 320                 325                 330 

gag cag acg act cca gag gtg gac tct act gag gag ggt gaa gga ggt     1119 
Glu Gln Thr Thr Pro Glu Val Asp Ser Thr Glu Glu Gly Glu Gly Gly 
                335                 340                 345 

ggc ccc acc atg ggc ccc aag ttc cgg gca gca gag cag tcc tta cag     1167 
Gly Pro Thr Met Gly Pro Lys Phe Arg Ala Ala Glu Gln Ser Leu Gln 
            350                 355                 360 

act gag ttc cag atc ttt cct ggt gct gga gaa aag gga gcg aaa gga     1215 
Thr Glu Phe Gln Ile Phe Pro Gly Ala Gly Glu Lys Gly Ala Lys Gly 
        365                 370                 375 

gag cct gcg aca gta gag cag gga cag cag ttt gag ggg cct gca gga     1263 
Glu Pro Ala Thr Val Glu Gln Gly Gln Gln Phe Glu Gly Pro Ala Gly 
    380                 385                 390 

gct cca gga ccc cgg gga ata tct ggt cct tca ggc cct cct ggg cct     1311 
Ala Pro Gly Pro Arg Gly Ile Ser Gly Pro Ser Gly Pro Pro Gly Pro 
395                 400                 405                 410 

ccg ggc ttc cct ggg gac cgt ggt cta ccg ggt cct gcc ggc ctc cca     1359 
Pro Gly Phe Pro Gly Asp Arg Gly Leu Pro Gly Pro Ala Gly Leu Pro 
                415                 420                 425 

gga atc cca ggc atc gat gga gcc cgg ggc ctg ccg ggc aca gtg att     1407 
Gly Ile Pro Gly Ile Asp Gly Ala Arg Gly Leu Pro Gly Thr Val Ile 
            430                 435                 440 

atg atg ccg ttc cat ttt gca agc agc tcg atg aag gga ccc cca gtg     1455 
Met Met Pro Phe His Phe Ala Ser Ser Ser Met Lys Gly Pro Pro Val 
        445                 450                 455 

tcc ttc cag cag gcc cag gcc cag gca gta ttg caa cag gct cag ctg     1503 
Ser Phe Gln Gln Ala Gln Ala Gln Ala Val Leu Gln Gln Ala Gln Leu 
    460                 465                 470 

tcc atg aaa ggg ccc cct ggt cca gta ggg ctc act ggg cgc cca ggc     1551 
Ser Met Lys Gly Pro Pro Gly Pro Val Gly Leu Thr Gly Arg Pro Gly 
475                 480                 485                 490 

cct gtg ggc ctc cct gga tat cca ggt ctg aaa ggt gaa ctg gga gaa     1599 
Pro Val Gly Leu Pro Gly Tyr Pro Gly Leu Lys Gly Glu Leu Gly Glu 
                495                 500                 505 

gtg ggg cca cag ggc ccc cga gga tta cag ggc cct cct ggg cct cct     1647 
Val Gly Pro Gln Gly Pro Arg Gly Leu Gln Gly Pro Pro Gly Pro Pro 
            510                 515                 520 

gga cgg gaa ggc aag aca ggc cga gct gga gca gat ggg gct cgg ggg     1695 
Gly Arg Glu Gly Lys Thr Gly Arg Ala Gly Ala Asp Gly Ala Arg Gly 
        525                 530                 535 

ctc ccg gga gac aca gga cct aag ggt gac agg ggc ttt gat ggc ctg     1743 
Leu Pro Gly Asp Thr Gly Pro Lys Gly Asp Arg Gly Phe Asp Gly Leu 
    540                 545                 550 

ccc ggg ctg cct ggt gag aag ggc caa agg ggt gac ttt gga cga gta     1791 
Pro Gly Leu Pro Gly Glu Lys Gly Gln Arg Gly Asp Phe Gly Arg Val 
555                 560                 565                 570 

ggg caa cct ggt ccc cca gga gag gat ggt gta aag ggc ctg cag gga     1839 
Gly Gln Pro Gly Pro Pro Gly Glu Asp Gly Val Lys Gly Leu Gln Gly 
                575                 580                 585 

cct cca ggg ccc act ggc cag gct gga gag ccg ggt ccc cga ggt ctg     1887 
Pro Pro Gly Pro Thr Gly Gln Ala Gly Glu Pro Gly Pro Arg Gly Leu 
            590                 595                 600 

att ggc ccc aga ggt ctc cca ggt ccc cta gga cgc ccg ggt gtg aca     1935 
Ile Gly Pro Arg Gly Leu Pro Gly Pro Leu Gly Arg Pro Gly Val Thr 
        605                 610                 615 

ggg agt gat ggc gca cca ggg gcc aaa ggc aac gtg ggt cct cct gga     1983 
Gly Ser Asp Gly Ala Pro Gly Ala Lys Gly Asn Val Gly Pro Pro Gly 
    620                 625                 630 

gaa cca gga ccc cca gga cag caa gga aac cac ggc tcc cag gga att     2031 
Glu Pro Gly Pro Pro Gly Gln Gln Gly Asn His Gly Ser Gln Gly Ile 
635                 640                 645                 650 

cca ggc ccc cag ggg ccc att ggc act ccc ggg gaa aag ggt ccc cct     2079 
Pro Gly Pro Gln Gly Pro Ile Gly Thr Pro Gly Glu Lys Gly Pro Pro 
                655                 660                 665 

gga aac ccc gga att cca ggt gtc cca gga tct gag ggc ccc ccg ggc     2127 
Gly Asn Pro Gly Ile Pro Gly Val Pro Gly Ser Glu Gly Pro Pro Gly 
            670                 675                 680 

cac cca ggc cac gag ggt ccc aca gga gaa aaa ggg gct cag ggc cca     2175 
His Pro Gly His Glu Gly Pro Thr Gly Glu Lys Gly Ala Gln Gly Pro 
        685                 690                 695 

cca gga tca gca ggc cct cgg ggc tat cct gga ctt cgt ggt gtg aag     2223 
Pro Gly Ser Ala Gly Pro Arg Gly Tyr Pro Gly Leu Arg Gly Val Lys 
    700                 705                 710 

ggt acc tct ggt aac cgg ggt ctc caa ggc gag aaa gga gaa agg gga     2271 
Gly Thr Ser Gly Asn Arg Gly Leu Gln Gly Glu Lys Gly Glu Arg Gly 
715                 720                 725                 730 

gag gat ggc ttt cct ggc ttc aag ggt gat gag gga cca aaa ggc gac     2319 
Glu Asp Gly Phe Pro Gly Phe Lys Gly Asp Glu Gly Pro Lys Gly Asp 
                735                 740                 745 

cgg gga aac ccc gga ccc cca ggt ccc aga gga gag gat ggt cca gaa     2367 
Arg Gly Asn Pro Gly Pro Pro Gly Pro Arg Gly Glu Asp Gly Pro Glu 
            750                 755                 760 

gga caa aag ggg cct ggg gga ctg cct ggt gat gag ggt cct cca gga     2415 
Gly Gln Lys Gly Pro Gly Gly Leu Pro Gly Asp Glu Gly Pro Pro Gly 
        765                 770                 775 

gca gca ggg gag aag ggc aag ctt ggg gtg cca ggt ctc cca ggt tat     2463 
Ala Ala Gly Glu Lys Gly Lys Leu Gly Val Pro Gly Leu Pro Gly Tyr 
    780                 785                 790 

cca gga cgc cca gga cct aag gga tct att gga ttt cct gga ccc ttg     2511 
Pro Gly Arg Pro Gly Pro Lys Gly Ser Ile Gly Phe Pro Gly Pro Leu 
795                 800                 805                 810 

gga cca ctg ggg gag aaa ggc aag cgg ggc aaa gca gga cag cca gga     2559 
Gly Pro Leu Gly Glu Lys Gly Lys Arg Gly Lys Ala Gly Gln Pro Gly 
                815                 820                 825 

gag gaa gga gaa cgc ggc aca ccg ggc acc cga gga gac agg gga cag     2607 
Glu Glu Gly Glu Arg Gly Thr Pro Gly Thr Arg Gly Asp Arg Gly Gln 
            830                 835                 840 

ccg ggg gcc aca ggc cag cct ggc ccc aag ggt gac gtg ggc cag aat     2655 
Pro Gly Ala Thr Gly Gln Pro Gly Pro Lys Gly Asp Val Gly Gln Asn 
        845                 850                 855 

ggg tct cct ggg ccc cct ggg gaa aag ggt cta ccc ggt ctt caa ggc     2703 
Gly Ser Pro Gly Pro Pro Gly Glu Lys Gly Leu Pro Gly Leu Gln Gly 
    860                 865                 870 

cca cca gga ttc ccc gga cca aaa ggc ccc ccg ggt cct cag ggg aaa     2751 
Pro Pro Gly Phe Pro Gly Pro Lys Gly Pro Pro Gly Pro Gln Gly Lys 
875                 880                 885                 890 

gac ggg ata tct ggg cac cct gga caa aga gga gaa ttg ggc ttc caa     2799 
Asp Gly Ile Ser Gly His Pro Gly Gln Arg Gly Glu Leu Gly Phe Gln 
                895                 900                 905 

ggt ctg aca ggc ccc cct gga cca gct ggc gtc ctt ggt cct cag gga     2847 
Gly Leu Thr Gly Pro Pro Gly Pro Ala Gly Val Leu Gly Pro Gln Gly 
            910                 915                 920 

aag gta ggg gac gtg ggg cct cta ggc gag aga ggc ccc cca ggg cct     2895 
Lys Val Gly Asp Val Gly Pro Leu Gly Glu Arg Gly Pro Pro Gly Pro 
        925                 930                 935 

cct gga cct cct ggt gaa caa ggt ctg cca ggc ata gaa ggc aga gaa     2943 
Pro Gly Pro Pro Gly Glu Gln Gly Leu Pro Gly Ile Glu Gly Arg Glu 
    940                 945                 950 

ggg gcc aag ggt gag cta gga ccc ctg ggg tcc gtc ggg aag gag ggg     2991 
Gly Ala Lys Gly Glu Leu Gly Pro Leu Gly Ser Val Gly Lys Glu Gly 
955                 960                 965                 970 

cca cct ggg ccc agg ggc ttc cct ggc ccc caa gga gcc ccc gga gac     3039 
Pro Pro Gly Pro Arg Gly Phe Pro Gly Pro Gln Gly Ala Pro Gly Asp 
                975                 980                 985 

cca gga ccc att ggt ttg aag ggt gac aaa ggt ccc cca ggc cct gtt     3087 
Pro Gly Pro Ile Gly Leu Lys Gly Asp Lys Gly Pro Pro Gly Pro Val 
            990                 995                1000 

ggg gca aat ggc tcc ccg gga gag cgt ggt cct gta ggc ccc tct ggt     3135 
Gly Ala Asn Gly Ser Pro Gly Glu Arg Gly Pro Val Gly Pro Ser Gly 
       1005                1010                1015 

ggc att ggg ctt cct ggc cag agt gga ggg caa ggc cct att ggt cct     3183 
Gly Ile Gly Leu Pro Gly Gln Ser Gly Gly Gln Gly Pro Ile Gly Pro 
   1020                1025                1030 

gct ggc gag aag ggg tcc ccg gga gaa cgg ggt act cct ggt cct act     3231 
Ala Gly Glu Lys Gly Ser Pro Gly Glu Arg Gly Thr Pro Gly Pro Thr 
1035               1040                1045                1050 

ggc aaa gat ggt att cca gga ccc ccg ggg ctt cag ggc ccc tct gga     3279 
Gly Lys Asp Gly Ile Pro Gly Pro Pro Gly Leu Gln Gly Pro Ser Gly 
               1055                1060                1065 

gct gcg ggg cct tct ggg gaa gaa gga gac aag ggg gaa gta ggg atg     3327 
Ala Ala Gly Pro Ser Gly Glu Glu Gly Asp Lys Gly Glu Val Gly Met 
           1070                1075                1080 

cct ggt cac aaa gga agc aaa ggg gat aaa gga gat gca ggc cca cct     3375 
Pro Gly His Lys Gly Ser Lys Gly Asp Lys Gly Asp Ala Gly Pro Pro 
       1085                1090                1095 

gga cca aca gga ata aga ggt cca gca ggc cat tca ggc ctc ccg ggt     3423 
Gly Pro Thr Gly Ile Arg Gly Pro Ala Gly His Ser Gly Leu Pro Gly 
   1100                1105                1110 

gct gat ggc gct cag ggt cgc cgg gga ccc cct ggc ctc ttc ggg cag     3471 
Ala Asp Gly Ala Gln Gly Arg Arg Gly Pro Pro Gly Leu Phe Gly Gln 
1115               1120                1125                1130 

aag ggg gat gac gga gtt cga ggc ttt gta ggt gta att ggt cct cca     3519 
Lys Gly Asp Asp Gly Val Arg Gly Phe Val Gly Val Ile Gly Pro Pro 
               1135                1140                1145 

ggt ctg cag ggg ctg ccg ggt cct ccg ggg gag aag ggc gag gtt gga     3567 
Gly Leu Gln Gly Leu Pro Gly Pro Pro Gly Glu Lys Gly Glu Val Gly 
           1150                1155                1160 

gac gta gga tcc atg ggt cca cat gga gct cca ggc cct cgg ggt ccc     3615 
Asp Val Gly Ser Met Gly Pro His Gly Ala Pro Gly Pro Arg Gly Pro 
       1165                1170                1175 

cct ggg ccc agt gga tca gag ggc ccc cca ggt ctg cct gga gga gta     3663 
Pro Gly Pro Ser Gly Ser Glu Gly Pro Pro Gly Leu Pro Gly Gly Val 
   1180                1185                1190 

gga cag cct ggt gct gtg ggc gag aag ggt gag cca ggg gat gct gga     3711 
Gly Gln Pro Gly Ala Val Gly Glu Lys Gly Glu Pro Gly Asp Ala Gly 
1195               1200                1205                1210 

gac gcc gga ccc cca gga att ccc ggc atc cct ggg ccc aaa ggt gaa     3759 
Asp Ala Gly Pro Pro Gly Ile Pro Gly Ile Pro Gly Pro Lys Gly Glu 
               1215                1220                1225 

att ggt gaa aag ggg gat tcg ggt cca tca ggg gct gct ggt ccc cca     3807 
Ile Gly Glu Lys Gly Asp Ser Gly Pro Ser Gly Ala Ala Gly Pro Pro 
           1230                1235                1240 

ggc aag aaa gga ccc cca gga gag gac ggc tct aag ggg aac atg ggt     3855 
Gly Lys Lys Gly Pro Pro Gly Glu Asp Gly Ser Lys Gly Asn Met Gly 
       1245                1250                1255 

ccc aca gga ctc cct gga gat cta ggg ccc cca gga gac cct gga gtt     3903 
Pro Thr Gly Leu Pro Gly Asp Leu Gly Pro Pro Gly Asp Pro Gly Val 
   1260                1265                1270 

ccg ggt att gat ggc atc cca ggg gag aag gga aat gct ggt gat att     3951 
Pro Gly Ile Asp Gly Ile Pro Gly Glu Lys Gly Asn Ala Gly Asp Ile 
1275               1280                1285                1290 

ggg gga ccg ggg cca cct gga gct tcc ggg gaa cct ggt gcc cgt ggc     3999 
Gly Gly Pro Gly Pro Pro Gly Ala Ser Gly Glu Pro Gly Ala Arg Gly 
               1295                1300                1305 

ctc cct ggc aag agg ggt tcc cct ggc cgc atg ggt cca gaa gga aga     4047 
Leu Pro Gly Lys Arg Gly Ser Pro Gly Arg Met Gly Pro Glu Gly Arg 
           1310                1315                1320 

gag ggc gag aaa ggc gcc aag gga gat gct ggt cct gat gga ccc cca     4095 
Glu Gly Glu Lys Gly Ala Lys Gly Asp Ala Gly Pro Asp Gly Pro Pro 
       1325                1330                1335 

ggc agg aca ggc ccc att ggg gct cga ggg ccc cct gga cga att ggg     4143 
Gly Arg Thr Gly Pro Ile Gly Ala Arg Gly Pro Pro Gly Arg Ile Gly 
   1340                1345                1350 

cct gat ggt ctt cca ggg atc cct ggt cct gtg ggt gaa cca ggt ctc     4191 
Pro Asp Gly Leu Pro Gly Ile Pro Gly Pro Val Gly Glu Pro Gly Leu 
1355               1360                1365                1370 

ctg gga cct cct ggg cta atc ggc cct cca ggg ccc ctg ggc cca cct     4239 
Leu Gly Pro Pro Gly Leu Ile Gly Pro Pro Gly Pro Leu Gly Pro Pro 
               1375                1380                1385 

ggc ctc cct ggc ctg aag gga gat gct ggc ccc aag ggg gag aag ggc     4287 
Gly Leu Pro Gly Leu Lys Gly Asp Ala Gly Pro Lys Gly Glu Lys Gly 
           1390                1395                1400 

cac att ggg cta ata ggc ctc att ggt ccc cca ggg gag gcc ggt gag     4335 
His Ile Gly Leu Ile Gly Leu Ile Gly Pro Pro Gly Glu Ala Gly Glu 
       1405                1410                1415 

aaa ggc gat cag ggg ttg cca ggt gtg cag ggc ccc cca ggc ctt cag     4383 
Lys Gly Asp Gln Gly Leu Pro Gly Val Gln Gly Pro Pro Gly Leu Gln 
   1420                1425                1430 

gga gac cct ggt ctc cct ggt cct gtt ggc tcg tta ggt cac cct ggg     4431 
Gly Asp Pro Gly Leu Pro Gly Pro Val Gly Ser Leu Gly His Pro Gly 
1435               1440                1445                1450 

ccc cca ggt gtg gtg ggc cct ctg gga cag aag ggc tcc aaa ggg tcc     4479 
Pro Pro Gly Val Val Gly Pro Leu Gly Gln Lys Gly Ser Lys Gly Ser 
               1455                1460                1465 

ccg gga tct ctt ggt cct cgt gga gac cct gga cca gcg ggt cct cct     4527 
Pro Gly Ser Leu Gly Pro Arg Gly Asp Pro Gly Pro Ala Gly Pro Pro 
           1470                1475                1480 

ggt ccc ccg ggt tct ccg gct gag gtg cat ggc ctg cgc agg cgc cga     4575 
Gly Pro Pro Gly Ser Pro Ala Glu Val His Gly Leu Arg Arg Arg Arg 
       1485                1490                1495 

tct gtg acg gac acc ctg gaa ggt ggc ctg gag gag gtg atg gcc tca     4623 
Ser Val Thr Asp Thr Leu Glu Gly Gly Leu Glu Glu Val Met Ala Ser 
   1500                1505                1510 

ctg aat tca ctg agc ttg gag ctg cag cag ttg cag aga cct ctg ggc     4671 
Leu Asn Ser Leu Ser Leu Glu Leu Gln Gln Leu Gln Arg Pro Leu Gly 
1515               1520                1525                1530 

aca gcc gag agc cca ggc ctc atg tgc cga gag ctt cac cgc gac cac     4719 
Thr Ala Glu Ser Pro Gly Leu Met Cys Arg Glu Leu His Arg Asp His 
               1535                1540                1545 

cca cac ctg ccc gat gga gag tac tgg att gac ccc aat cag ggc tgt     4767 
Pro His Leu Pro Asp Gly Glu Tyr Trp Ile Asp Pro Asn Gln Gly Cys 
           1550                1555                1560 

gca cgt gac gcc ttc aag gtt ttc tgc aac ttc acg gca gga ggt gag     4815 
Ala Arg Asp Ala Phe Lys Val Phe Cys Asn Phe Thr Ala Gly Gly Glu 
       1565                1570                1575 

acc tgt ctc tat cca gac aag aag ttt gag acg gtg aaa ctg gcc tcg     4863 
Thr Cys Leu Tyr Pro Asp Lys Lys Phe Glu Thr Val Lys Leu Ala Ser 
   1580                1585                1590 

tgg tcc cga gag aag cct gga ggc tgg tac agc acc ttc cgc cga ggg     4911 
Trp Ser Arg Glu Lys Pro Gly Gly Trp Tyr Ser Thr Phe Arg Arg Gly 
1595               1600                1605                1610 

aag aag ttc tcc tat gtg gat gct gat ggc tcc ccg gtg aat gtg gtc     4959 
Lys Lys Phe Ser Tyr Val Asp Ala Asp Gly Ser Pro Val Asn Val Val 
               1615                1620                1625 

cag ttg acc ttc ctg aag ttg ttg agt gct gca gcc cat cag agg ttc     5007 
Gln Leu Thr Phe Leu Lys Leu Leu Ser Ala Ala Ala His Gln Arg Phe 
           1630                1635                1640 

act tac atc tgc cag aac tcg gtg gca tgg ctg gat gaa gct gcg ggt     5055 
Thr Tyr Ile Cys Gln Asn Ser Val Ala Trp Leu Asp Glu Ala Ala Gly 
       1645                1650                1655 

gac cac agg cac tcc atc cgc ttc caa ggg acc aac tgg gaa gag ttg     5103 
Asp His Arg His Ser Ile Arg Phe Gln Gly Thr Asn Trp Glu Glu Leu 
   1660                1665                1670 

tcc ttc aac cag aca aca gca gct acc atc aag gtc tcc cat gat ggc     5151 
Ser Phe Asn Gln Thr Thr Ala Ala Thr Ile Lys Val Ser His Asp Gly 
1675               1680                1685                1690 

tgt cgg gtc cgg aag gga cag gcg aag acc ctc ttt gaa ttc agc tct     5199 
Cys Arg Val Arg Lys Gly Gln Ala Lys Thr Leu Phe Glu Phe Ser Ser 
               1695                1700                1705 

tct gtg ggt ttc ctg cct ctg tgg gat gtg gct gcc tct gac ttt ggt     5247 
Ser Val Gly Phe Leu Pro Leu Trp Asp Val Ala Ala Ser Asp Phe Gly 
           1710                1715                1720 

cag acg aac caa aag ttt ggg ttt gaa ctc ggc tcc atc tgc ttt agc     5295 
Gln Thr Asn Gln Lys Phe Gly Phe Glu Leu Gly Ser Ile Cys Phe Ser 
       1725                1730                1735 

agc tgaagttgtg aggtgggaag gaagctgaag ggagccccac atgggctcct          5348 
Ser 

tggtgctgag gctctgaggc cattctgttt atccccaggg actccagatc cagggtcacg   5408 

tgactctgac tattctttct cccttgtagg gggagagtgt ggagagccca gctccctctg   5468 

tctgttcacc ccaggtggta tacccagttg tctgctagct cccccctcca tccaactgtc   5528 

cattgtccac ctcaccccca gacctccatg cagtagactt ttaactcaga gctggtgaag   5588 

ccccacccct gcctctccac ccctccacca ggccttttgg tgctattcct ttccatagtt   5648 

gagcactgga tacctcctga tccctgcctg ggacccttcc ctcgcatact tcttctttct   5708 

ttgagtaaaa gaagtaaagc aagatcaaag ggggcgccct ccctgagctg cgccttcctt   5768 

ctgcttcctt gacccagtgc tgcacaatct cctctcccta ctctgcccca ctcctgtgcc   5828 

cccaagcctt caggggacca agatgttggg cataaatcag gatcctacat ggtgctgccc   5888 

tgctcataac tgggaactgt atgaaagggg gaatgaatgg tctgtggtct atttaatttg   5948 

cttccttctg aaggaagtct ggggtacggt gagagattcc agaaggatct gtaccctccc   6008 

ttacctacgc ggctctcctc cccaggacac agggcaaaat cgccatctca agaataaacc   6068 

aaggaactgt gctcttctaa aaaaaaaaaa aaaaaaaaaa a                       6109 

 
           
             2  
             1739  
             PRT  
             Mus musculus  
           
            2 

Met Arg Ser Cys Arg Arg Leu Asp Gln Leu Gln Ala Gly Leu Cys Leu 
  1               5                  10                  15 

Leu Leu Ala Ser Leu Gln Leu Val Ser Trp Thr Leu Ala Ala Glu Pro 
             20                  25                  30 

Val Asp Val Leu Glu Ala Trp Gly Val His Arg Asp Gln Ala Gly Val 
         35                  40                  45 

Ala Glu Gly Pro Gly Phe Cys Pro Leu Arg Ile Pro Gln Gly Asp Arg 
     50                  55                  60 

Ala Phe Arg Val Gly Lys Ser Ser Leu Leu Ser Val Pro Thr Trp Gln 
 65                  70                  75                  80 

Leu Phe Pro Asp Gly His Phe Pro Glu Asn Phe Ser Val Leu Leu Thr 
                 85                  90                  95 

Leu Arg Ala Gln Pro Ala Asn Gln Ser Val Leu Leu Ser Ile Tyr Asp 
            100                 105                 110 

Glu Lys Gly Val Arg Gln Leu Gly Leu Ala Leu Gly Pro Ala Leu Gly 
        115                 120                 125 

Leu Leu Gly Asp Ser Phe Arg Pro Leu Pro Lys Gln Val Asn Ile Met 
    130                 135                 140 

Asp Gly Arg Trp His Arg Val Ala Val Ser Ile Ser Gly Asn Lys Val 
145                 150                 155                 160 

Thr Leu Val Val Asp Cys Glu Pro Gln Pro Pro Thr Phe Gly Gln Gly 
                165                 170                 175 

Pro Arg Phe Ile Ser Thr Ala Gly Leu Thr Val Met Gly Thr Gln Asp 
            180                 185                 190 

Thr Arg Glu Glu Ser Phe Glu Gly Asp Ile Gln Glu Leu Leu Leu Ile 
        195                 200                 205 

Pro Asp Pro Gln Ala Ala Phe Gln Ala Cys Glu Ser Tyr Leu Pro Gly 
    210                 215                 220 

Cys Glu Thr Leu Asp Ser Thr Thr Thr Gly Ala Pro Lys Asp Asp Glu 
225                 230                 235                 240 

Pro Glu Thr Pro Ala Pro Arg Arg Arg Lys Gly Lys Gly Lys Lys Lys 
                245                 250                 255 

Gly Arg Gly Arg Lys Gly Lys Gly Arg Lys Lys Asn Lys Glu Thr Ser 
            260                 265                 270 

Glu Leu Ser Pro Thr Pro Gly Ala Pro Glu Asn Gln Thr Ser Leu His 
        275                 280                 285 

Ile Pro Glu Thr Glu Lys Thr Val Pro His Leu Pro Leu Thr Pro Thr 
    290                 295                 300 

Pro Leu Ala Ile Thr Thr Thr Val Thr Ile Gly Gln Asn Ala Thr Val 
305                 310                 315                 320 

Ser Gln Gly Leu Asp Ser Gly Thr Glu Thr Glu Gln Thr Thr Pro Glu 
                325                 330                 335 

Val Asp Ser Thr Glu Glu Gly Glu Gly Gly Gly Pro Thr Met Gly Pro 
            340                 345                 350 

Lys Phe Arg Ala Ala Glu Gln Ser Leu Gln Thr Glu Phe Gln Ile Phe 
        355                 360                 365 

Pro Gly Ala Gly Glu Lys Gly Ala Lys Gly Glu Pro Ala Thr Val Glu 
    370                 375                 380 

Gln Gly Gln Gln Phe Glu Gly Pro Ala Gly Ala Pro Gly Pro Arg Gly 
385                 390                 395                 400 

Ile Ser Gly Pro Ser Gly Pro Pro Gly Pro Pro Gly Phe Pro Gly Asp 
                405                 410                 415 

Arg Gly Leu Pro Gly Pro Ala Gly Leu Pro Gly Ile Pro Gly Ile Asp 
            420                 425                 430 

Gly Ala Arg Gly Leu Pro Gly Thr Val Ile Met Met Pro Phe His Phe 
        435                 440                 445 

Ala Ser Ser Ser Met Lys Gly Pro Pro Val Ser Phe Gln Gln Ala Gln 
    450                 455                 460 

Ala Gln Ala Val Leu Gln Gln Ala Gln Leu Ser Met Lys Gly Pro Pro 
465                 470                 475                 480 

Gly Pro Val Gly Leu Thr Gly Arg Pro Gly Pro Val Gly Leu Pro Gly 
                485                 490                 495 

Tyr Pro Gly Leu Lys Gly Glu Leu Gly Glu Val Gly Pro Gln Gly Pro 
            500                 505                 510 

Arg Gly Leu Gln Gly Pro Pro Gly Pro Pro Gly Arg Glu Gly Lys Thr 
        515                 520                 525 

Gly Arg Ala Gly Ala Asp Gly Ala Arg Gly Leu Pro Gly Asp Thr Gly 
    530                 535                 540 

Pro Lys Gly Asp Arg Gly Phe Asp Gly Leu Pro Gly Leu Pro Gly Glu 
545                 550                 555                 560 

Lys Gly Gln Arg Gly Asp Phe Gly Arg Val Gly Gln Pro Gly Pro Pro 
                565                 570                 575 

Gly Glu Asp Gly Val Lys Gly Leu Gln Gly Pro Pro Gly Pro Thr Gly 
            580                 585                 590 

Gln Ala Gly Glu Pro Gly Pro Arg Gly Leu Ile Gly Pro Arg Gly Leu 
        595                 600                 605 

Pro Gly Pro Leu Gly Arg Pro Gly Val Thr Gly Ser Asp Gly Ala Pro 
    610                 615                 620 

Gly Ala Lys Gly Asn Val Gly Pro Pro Gly Glu Pro Gly Pro Pro Gly 
625                 630                 635                 640 

Gln Gln Gly Asn His Gly Ser Gln Gly Ile Pro Gly Pro Gln Gly Pro 
                645                 650                 655 

Ile Gly Thr Pro Gly Glu Lys Gly Pro Pro Gly Asn Pro Gly Ile Pro 
            660                 665                 670 

Gly Val Pro Gly Ser Glu Gly Pro Pro Gly His Pro Gly His Glu Gly 
        675                 680                 685 

Pro Thr Gly Glu Lys Gly Ala Gln Gly Pro Pro Gly Ser Ala Gly Pro 
    690                 695                 700 

Arg Gly Tyr Pro Gly Leu Arg Gly Val Lys Gly Thr Ser Gly Asn Arg 
705                 710                 715                 720 

Gly Leu Gln Gly Glu Lys Gly Glu Arg Gly Glu Asp Gly Phe Pro Gly 
                725                 730                 735 

Phe Lys Gly Asp Glu Gly Pro Lys Gly Asp Arg Gly Asn Pro Gly Pro 
            740                 745                 750 

Pro Gly Pro Arg Gly Glu Asp Gly Pro Glu Gly Gln Lys Gly Pro Gly 
        755                 760                 765 

Gly Leu Pro Gly Asp Glu Gly Pro Pro Gly Ala Ala Gly Glu Lys Gly 
    770                 775                 780 

Lys Leu Gly Val Pro Gly Leu Pro Gly Tyr Pro Gly Arg Pro Gly Pro 
785                 790                 795                 800 

Lys Gly Ser Ile Gly Phe Pro Gly Pro Leu Gly Pro Leu Gly Glu Lys 
                805                 810                 815 

Gly Lys Arg Gly Lys Ala Gly Gln Pro Gly Glu Glu Gly Glu Arg Gly 
            820                 825                 830 

Thr Pro Gly Thr Arg Gly Asp Arg Gly Gln Pro Gly Ala Thr Gly Gln 
        835                 840                 845 

Pro Gly Pro Lys Gly Asp Val Gly Gln Asn Gly Ser Pro Gly Pro Pro 
    850                 855                 860 

Gly Glu Lys Gly Leu Pro Gly Leu Gln Gly Pro Pro Gly Phe Pro Gly 
865                 870                 875                 880 

Pro Lys Gly Pro Pro Gly Pro Gln Gly Lys Asp Gly Ile Ser Gly His 
                885                 890                 895 

Pro Gly Gln Arg Gly Glu Leu Gly Phe Gln Gly Leu Thr Gly Pro Pro 
            900                 905                 910 

Gly Pro Ala Gly Val Leu Gly Pro Gln Gly Lys Val Gly Asp Val Gly 
        915                 920                 925 

Pro Leu Gly Glu Arg Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Glu 
    930                 935                 940 

Gln Gly Leu Pro Gly Ile Glu Gly Arg Glu Gly Ala Lys Gly Glu Leu 
945                 950                 955                 960 

Gly Pro Leu Gly Ser Val Gly Lys Glu Gly Pro Pro Gly Pro Arg Gly 
                965                 970                 975 

Phe Pro Gly Pro Gln Gly Ala Pro Gly Asp Pro Gly Pro Ile Gly Leu 
            980                 985                 990 

Lys Gly Asp Lys Gly Pro Pro Gly Pro Val Gly Ala Asn Gly Ser Pro 
        995                1000                1005 

Gly Glu Arg Gly Pro Val Gly Pro Ser Gly Gly Ile Gly Leu Pro Gly 
   1010                1015                1020 

Gln Ser Gly Gly Gln Gly Pro Ile Gly Pro Ala Gly Glu Lys Gly Ser 
1025               1030                1035                1040 

Pro Gly Glu Arg Gly Thr Pro Gly Pro Thr Gly Lys Asp Gly Ile Pro 
               1045                1050                1055 

Gly Pro Pro Gly Leu Gln Gly Pro Ser Gly Ala Ala Gly Pro Ser Gly 
           1060                1065                1070 

Glu Glu Gly Asp Lys Gly Glu Val Gly Met Pro Gly His Lys Gly Ser 
       1075                1080                1085 

Lys Gly Asp Lys Gly Asp Ala Gly Pro Pro Gly Pro Thr Gly Ile Arg 
   1090                1095                1100 

Gly Pro Ala Gly His Ser Gly Leu Pro Gly Ala Asp Gly Ala Gln Gly 
1105               1110                1115                1120 

Arg Arg Gly Pro Pro Gly Leu Phe Gly Gln Lys Gly Asp Asp Gly Val 
               1125                1130                1135 

Arg Gly Phe Val Gly Val Ile Gly Pro Pro Gly Leu Gln Gly Leu Pro 
           1140                1145                1150 

Gly Pro Pro Gly Glu Lys Gly Glu Val Gly Asp Val Gly Ser Met Gly 
       1155                1160                1165 

Pro His Gly Ala Pro Gly Pro Arg Gly Pro Pro Gly Pro Ser Gly Ser 
   1170                1175                1180 

Glu Gly Pro Pro Gly Leu Pro Gly Gly Val Gly Gln Pro Gly Ala Val 
1185               1190                1195                1200 

Gly Glu Lys Gly Glu Pro Gly Asp Ala Gly Asp Ala Gly Pro Pro Gly 
               1205                1210                1215 

Ile Pro Gly Ile Pro Gly Pro Lys Gly Glu Ile Gly Glu Lys Gly Asp 
           1220                1225                1230 

Ser Gly Pro Ser Gly Ala Ala Gly Pro Pro Gly Lys Lys Gly Pro Pro 
       1235                1240                1245 

Gly Glu Asp Gly Ser Lys Gly Asn Met Gly Pro Thr Gly Leu Pro Gly 
   1250                1255                1260 

Asp Leu Gly Pro Pro Gly Asp Pro Gly Val Pro Gly Ile Asp Gly Ile 
1265               1270                1275                1280 

Pro Gly Glu Lys Gly Asn Ala Gly Asp Ile Gly Gly Pro Gly Pro Pro 
               1285                1290                1295 

Gly Ala Ser Gly Glu Pro Gly Ala Arg Gly Leu Pro Gly Lys Arg Gly 
           1300                1305                1310 

Ser Pro Gly Arg Met Gly Pro Glu Gly Arg Glu Gly Glu Lys Gly Ala 
       1315                1320                1325 

Lys Gly Asp Ala Gly Pro Asp Gly Pro Pro Gly Arg Thr Gly Pro Ile 
   1330                1335                1340 

Gly Ala Arg Gly Pro Pro Gly Arg Ile Gly Pro Asp Gly Leu Pro Gly 
1345               1350                1355                1360 

Ile Pro Gly Pro Val Gly Glu Pro Gly Leu Leu Gly Pro Pro Gly Leu 
               1365                1370                1375 

Ile Gly Pro Pro Gly Pro Leu Gly Pro Pro Gly Leu Pro Gly Leu Lys 
           1380                1385                1390 

Gly Asp Ala Gly Pro Lys Gly Glu Lys Gly His Ile Gly Leu Ile Gly 
       1395                1400                1405 

Leu Ile Gly Pro Pro Gly Glu Ala Gly Glu Lys Gly Asp Gln Gly Leu 
   1410                1415                1420 

Pro Gly Val Gln Gly Pro Pro Gly Leu Gln Gly Asp Pro Gly Leu Pro 
1425               1430                1435                1440 

Gly Pro Val Gly Ser Leu Gly His Pro Gly Pro Pro Gly Val Val Gly 
               1445                1450                1455 

Pro Leu Gly Gln Lys Gly Ser Lys Gly Ser Pro Gly Ser Leu Gly Pro 
           1460                1465                1470 

Arg Gly Asp Pro Gly Pro Ala Gly Pro Pro Gly Pro Pro Gly Ser Pro 
       1475                1480                1485 

Ala Glu Val His Gly Leu Arg Arg Arg Arg Ser Val Thr Asp Thr Leu 
   1490                1495                1500 

Glu Gly Gly Leu Glu Glu Val Met Ala Ser Leu Asn Ser Leu Ser Leu 
1505               1510                1515                1520 

Glu Leu Gln Gln Leu Gln Arg Pro Leu Gly Thr Ala Glu Ser Pro Gly 
               1525                1530                1535 

Leu Met Cys Arg Glu Leu His Arg Asp His Pro His Leu Pro Asp Gly 
           1540                1545                1550 

Glu Tyr Trp Ile Asp Pro Asn Gln Gly Cys Ala Arg Asp Ala Phe Lys 
       1555                1560                1565 

Val Phe Cys Asn Phe Thr Ala Gly Gly Glu Thr Cys Leu Tyr Pro Asp 
   1570                1575                1580 

Lys Lys Phe Glu Thr Val Lys Leu Ala Ser Trp Ser Arg Glu Lys Pro 
1585               1590                1595                1600 

Gly Gly Trp Tyr Ser Thr Phe Arg Arg Gly Lys Lys Phe Ser Tyr Val 
               1605                1610                1615 

Asp Ala Asp Gly Ser Pro Val Asn Val Val Gln Leu Thr Phe Leu Lys 
           1620                1625                1630 

Leu Leu Ser Ala Ala Ala His Gln Arg Phe Thr Tyr Ile Cys Gln Asn 
       1635                1640                1645 

Ser Val Ala Trp Leu Asp Glu Ala Ala Gly Asp His Arg His Ser Ile 
   1650                1655                1660 

Arg Phe Gln Gly Thr Asn Trp Glu Glu Leu Ser Phe Asn Gln Thr Thr 
1665               1670                1675                1680 

Ala Ala Thr Ile Lys Val Ser His Asp Gly Cys Arg Val Arg Lys Gly 
               1685                1690                1695 

Gln Ala Lys Thr Leu Phe Glu Phe Ser Ser Ser Val Gly Phe Leu Pro 
           1700                1705                1710 

Leu Trp Asp Val Ala Ala Ser Asp Phe Gly Gln Thr Asn Gln Lys Phe 
       1715                1720                1725 

Gly Phe Glu Leu Gly Ser Ile Cys Phe Ser Ser 
   1730                1735 

 
           
             3  
             6200  
             DNA  
             Homo sapiens  
             
               CDS  
               (87)..(5321)  
             
           
            3 

gcgagtgact gcaccgagcc cgagaagtcg ccgcgccccg cagccgcccc gactggttcc     60 

ccgccttgcc cgtgggcccc gccggg atg ggg aac cgc cgg gac ctg ggc cag     113 
                             Met Gly Asn Arg Arg Asp Leu Gly Gln 
                               1               5 

ccg cgg gcc ggt ctc tgc ctg ctc ctg gcc gcg ctg cag ctt ctg ccg      161 
Pro Arg Ala Gly Leu Cys Leu Leu Leu Ala Ala Leu Gln Leu Leu Pro 
 10                  15                  20                  25 

ggg acg cag gcc gat cct gtg gat gtc ctg aag gcc ctg ggt gtg cag      209 
Gly Thr Gln Ala Asp Pro Val Asp Val Leu Lys Ala Leu Gly Val Gln 
                 30                  35                  40 

gga ggc cag gct ggg gtc ccc gag ggg cct ggc ttc tgt ccc cag agg      257 
Gly Gly Gln Ala Gly Val Pro Glu Gly Pro Gly Phe Cys Pro Gln Arg 
             45                  50                  55 

act cca gag ggt gac cgg gca ttc aga att ggc cag gcc agc acg ctc      305 
Thr Pro Glu Gly Asp Arg Ala Phe Arg Ile Gly Gln Ala Ser Thr Leu 
         60                  65                  70 

ggc atc ccc acg tgg gaa ctc ttt cca gaa ggc cac ttt cct gag aac      353 
Gly Ile Pro Thr Trp Glu Leu Phe Pro Glu Gly His Phe Pro Glu Asn 
     75                  80                  85 

ttc tcc ttg ctg atc acc ttg cgg gga cag cca gcc aat cag tct gtc      401 
Phe Ser Leu Leu Ile Thr Leu Arg Gly Gln Pro Ala Asn Gln Ser Val 
 90                  95                 100                 105 

ctg ctg tcc att tat gat gaa agg ggt gcc cgg cag ttg ggc ctg gca      449 
Leu Leu Ser Ile Tyr Asp Glu Arg Gly Ala Arg Gln Leu Gly Leu Ala 
                110                 115                 120 

ctg ggg cca gcg ctg ggt ctc cta ggt gac ccc ttc cgc ccc ctc ccc      497 
Leu Gly Pro Ala Leu Gly Leu Leu Gly Asp Pro Phe Arg Pro Leu Pro 
            125                 130                 135 

cag cag gtc aac ctc aca gat ggc agg tgg cac cgt gtg gcc gtc agc      545 
Gln Gln Val Asn Leu Thr Asp Gly Arg Trp His Arg Val Ala Val Ser 
        140                 145                 150 

ata gat ggt gag atg gtg acc ctg gta gct gac tgt gaa gct cag ccc      593 
Ile Asp Gly Glu Met Val Thr Leu Val Ala Asp Cys Glu Ala Gln Pro 
    155                 160                 165 

cct gtt ttg ggc cat ggc ccc cgc ttc atc agc ata gct gga ctc act      641 
Pro Val Leu Gly His Gly Pro Arg Phe Ile Ser Ile Ala Gly Leu Thr 
170                 175                 180                 185 

gtg ctg ggg acc cag gac ctt ggg gaa aag act ttc gag gga gac att      689 
Val Leu Gly Thr Gln Asp Leu Gly Glu Lys Thr Phe Glu Gly Asp Ile 
                190                 195                 200 

cag gag ctg ctg ata agc cca gat cct cag gct gcc ttc cag gct tgt      737 
Gln Glu Leu Leu Ile Ser Pro Asp Pro Gln Ala Ala Phe Gln Ala Cys 
            205                 210                 215 

gag cgg tac ctc ccc gac tgt gac aac ctg gca ccg gca gcc aca gtg      785 
Glu Arg Tyr Leu Pro Asp Cys Asp Asn Leu Ala Pro Ala Ala Thr Val 
        220                 225                 230 

gct ccc cag ggt gaa cca gaa acc cct cgt cct cgg cgg aag ggg aag      833 
Ala Pro Gln Gly Glu Pro Glu Thr Pro Arg Pro Arg Arg Lys Gly Lys 
    235                 240                 245 

gga aaa ggg agg aag aaa ggg cga ggt cgc aag ggg aag ggc agg aaa      881 
Gly Lys Gly Arg Lys Lys Gly Arg Gly Arg Lys Gly Lys Gly Arg Lys 
250                 255                 260                 265 

aag aac aag gaa att tgg acc tca agt cca cct cct gac tcc gca gag      929 
Lys Asn Lys Glu Ile Trp Thr Ser Ser Pro Pro Pro Asp Ser Ala Glu 
                270                 275                 280 

aac cag acc tcc act gac atc ccc aag aca gag act cca gct cca aat      977 
Asn Gln Thr Ser Thr Asp Ile Pro Lys Thr Glu Thr Pro Ala Pro Asn 
            285                 290                 295 

ctg cct ccg acc ccc acg cct ttg gtc gtc acc tcc act gtg act act     1025 
Leu Pro Pro Thr Pro Thr Pro Leu Val Val Thr Ser Thr Val Thr Thr 
        300                 305                 310 

gga ctc aat gcc acg atc cta gag ggg agc ttg gac cct gac agt gga     1073 
Gly Leu Asn Ala Thr Ile Leu Glu Gly Ser Leu Asp Pro Asp Ser Gly 
    315                 320                 325 

acc gag ctg ggg acc ctg gag acc aag gca gcc agg gag gat gaa gaa     1121 
Thr Glu Leu Gly Thr Leu Glu Thr Lys Ala Ala Arg Glu Asp Glu Glu 
330                 335                 340                 345 

gga gat gat tcc acc atg ggc cct gac ttc cgg gca gca gaa tat cca     1169 
Gly Asp Asp Ser Thr Met Gly Pro Asp Phe Arg Ala Ala Glu Tyr Pro 
                350                 355                 360 

tct cgg act cag ttc cag atc ttt cct ggt gct gga gag aaa gga gca     1217 
Ser Arg Thr Gln Phe Gln Ile Phe Pro Gly Ala Gly Glu Lys Gly Ala 
            365                 370                 375 

aaa gga gag ccc gca gtg att gaa aag ggg cag cag ttt gag gga cct     1265 
Lys Gly Glu Pro Ala Val Ile Glu Lys Gly Gln Gln Phe Glu Gly Pro 
        380                 385                 390 

cca gga gcc cca gga ccc caa ggg gtg gtt ggc ccc tca ggc cct ccc     1313 
Pro Gly Ala Pro Gly Pro Gln Gly Val Val Gly Pro Ser Gly Pro Pro 
    395                 400                 405 

ggc ccc cca gga ttc cct ggc gac cct ggt cca ccg ggc cct gct ggc     1361 
Gly Pro Pro Gly Phe Pro Gly Asp Pro Gly Pro Pro Gly Pro Ala Gly 
410                 415                 420                 425 

ctc cca gga atc ccc ggc att gat ggg atc cga ggc cca ccg ggc act     1409 
Leu Pro Gly Ile Pro Gly Ile Asp Gly Ile Arg Gly Pro Pro Gly Thr 
                430                 435                 440 

gtg atc atg atg ccg ttc cag ttt gca ggc ggc tcc ttt aaa ggc ccc     1457 
Val Ile Met Met Pro Phe Gln Phe Ala Gly Gly Ser Phe Lys Gly Pro 
            445                 450                 455 

cca gtc tca ttc cag cag gcc cag gct cag gca gtt ctg cag cag act     1505 
Pro Val Ser Phe Gln Gln Ala Gln Ala Gln Ala Val Leu Gln Gln Thr 
        460                 465                 470 

cag ctc tct atg aaa ggc ccc cct ggt cca gtg ggg ctc act ggg cgc     1553 
Gln Leu Ser Met Lys Gly Pro Pro Gly Pro Val Gly Leu Thr Gly Arg 
    475                 480                 485 

cca ggc cct gtg ggt ctc ccc ggg cat cca ggt ctg aaa gga gag gag     1601 
Pro Gly Pro Val Gly Leu Pro Gly His Pro Gly Leu Lys Gly Glu Glu 
490                 495                 500                 505 

gga gca gaa ggg cca cag ggt ccc cga ggc ctg cag gga cct cat gga     1649 
Gly Ala Glu Gly Pro Gln Gly Pro Arg Gly Leu Gln Gly Pro His Gly 
                510                 515                 520 

ccc cct ggc cga gtg ggc aag atg ggc cgc cct gga gca gat gga gct     1697 
Pro Pro Gly Arg Val Gly Lys Met Gly Arg Pro Gly Ala Asp Gly Ala 
            525                 530                 535 

cgg ggc ctc cca ggg gac act gga cct aag ggt gat cgt ggc ttc gat     1745 
Arg Gly Leu Pro Gly Asp Thr Gly Pro Lys Gly Asp Arg Gly Phe Asp 
        540                 545                 550 

ggc ctc cct ggg ctg cct ggt gag aag ggc caa agg ggt gac ttt ggc     1793 
Gly Leu Pro Gly Leu Pro Gly Glu Lys Gly Gln Arg Gly Asp Phe Gly 
    555                 560                 565 

cat gtg ggg caa ccc ggt ccc cca gga gag gat ggt gag agg gga gca     1841 
His Val Gly Gln Pro Gly Pro Pro Gly Glu Asp Gly Glu Arg Gly Ala 
570                 575                 580                 585 

gag gga cct cca ggg ccc act ggc cag gct ggg gag ccg ggt cca cga     1889 
Glu Gly Pro Pro Gly Pro Thr Gly Gln Ala Gly Glu Pro Gly Pro Arg 
                590                 595                 600 

gga ctg ctt ggc ccc aga ggc tct cct ggc ccc acg ggt cgc ccg ggt     1937 
Gly Leu Leu Gly Pro Arg Gly Ser Pro Gly Pro Thr Gly Arg Pro Gly 
            605                 610                 615 

gtg act gga att gat ggt gct cct ggt gcc aaa ggc aat gtg ggt cct     1985 
Val Thr Gly Ile Asp Gly Ala Pro Gly Ala Lys Gly Asn Val Gly Pro 
        620                 625                 630 

cca gga gaa cca ggc cct ccg gga cag cag gga aac cat ggg tcc cag     2033 
Pro Gly Glu Pro Gly Pro Pro Gly Gln Gln Gly Asn His Gly Ser Gln 
    635                 640                 645 

gga ctc ccc ggt ccc cag gga ctc att ggc act cct ggg gag aag ggt     2081 
Gly Leu Pro Gly Pro Gln Gly Leu Ile Gly Thr Pro Gly Glu Lys Gly 
650                 655                 660                 665 

ccc cct gga aac cca gga att cca ggc ctc cca gga tcc gat ggc cct     2129 
Pro Pro Gly Asn Pro Gly Ile Pro Gly Leu Pro Gly Ser Asp Gly Pro 
                670                 675                 680 

ctg ggt cac cca gga cat gag ggc ccc acg gga gag aaa ggg gct cag     2177 
Leu Gly His Pro Gly His Glu Gly Pro Thr Gly Glu Lys Gly Ala Gln 
            685                 690                 695 

ggt cca cca ggg tcg gca ggc cct ccg ggc tat cct gga cct cgg gga     2225 
Gly Pro Pro Gly Ser Ala Gly Pro Pro Gly Tyr Pro Gly Pro Arg Gly 
        700                 705                 710 

gtg aag ggc act tca ggc aac cgg ggc ctc cag ggg gag aaa ggc gag     2273 
Val Lys Gly Thr Ser Gly Asn Arg Gly Leu Gln Gly Glu Lys Gly Glu 
    715                 720                 725 

aag gga gag gac ggc ttc cca ggc ttc aag ggc gat gtg ggg ctc aaa     2321 
Lys Gly Glu Asp Gly Phe Pro Gly Phe Lys Gly Asp Val Gly Leu Lys 
730                 735                 740                 745 

ggt gat cag ggg aaa ccc gga gct cca ggt ccc cgg gga gag gat ggt     2369 
Gly Asp Gln Gly Lys Pro Gly Ala Pro Gly Pro Arg Gly Glu Asp Gly 
                750                 755                 760 

cct gag ggg ccg aag ggg cag gcg ggg cag gct ggc gag gag ggg ccc     2417 
Pro Glu Gly Pro Lys Gly Gln Ala Gly Gln Ala Gly Glu Glu Gly Pro 
            765                 770                 775 

cca ggc tca gct ggg gag aag ggc aag ctt ggg gtg cca ggc ctc cca     2465 
Pro Gly Ser Ala Gly Glu Lys Gly Lys Leu Gly Val Pro Gly Leu Pro 
        780                 785                 790 

ggt tat cca gga cgc cct gga cct aag gga tct att gga ttt ccc ggt     2513 
Gly Tyr Pro Gly Arg Pro Gly Pro Lys Gly Ser Ile Gly Phe Pro Gly 
    795                 800                 805 

ccc ctg gga ccc ata gga gag aaa ggg aag tcg gga aag aca ggg cag     2561 
Pro Leu Gly Pro Ile Gly Glu Lys Gly Lys Ser Gly Lys Thr Gly Gln 
810                 815                 820                 825 

cca ggc ctg gaa gga gag cgg gga cca cca ggt tcc cgt gga gag agg     2609 
Pro Gly Leu Glu Gly Glu Arg Gly Pro Pro Gly Ser Arg Gly Glu Arg 
                830                 835                 840 

ggg caa ccg ggt gcc aca ggg caa cca ggc ccc aag ggc gat gtg ggc     2657 
Gly Gln Pro Gly Ala Thr Gly Gln Pro Gly Pro Lys Gly Asp Val Gly 
            845                 850                 855 

cag gat gga gcc cct ggg atc cct gga gaa aag ggc ctc cct ggt ctg     2705 
Gln Asp Gly Ala Pro Gly Ile Pro Gly Glu Lys Gly Leu Pro Gly Leu 
        860                 865                 870 

caa ggc cct cca gga ttc cct ggg cca aag ggc ccc cct ggt cac caa     2753 
Gln Gly Pro Pro Gly Phe Pro Gly Pro Lys Gly Pro Pro Gly His Gln 
    875                 880                 885 

ggt aaa gat ggg cga cca ggg cac cct gga cag aga gga gaa ctg ggc     2801 
Gly Lys Asp Gly Arg Pro Gly His Pro Gly Gln Arg Gly Glu Leu Gly 
890                 895                 900                 905 

ttc caa ggt cag aca ggc ccg cct gga cca gct ggt gtc tta ggc cct     2849 
Phe Gln Gly Gln Thr Gly Pro Pro Gly Pro Ala Gly Val Leu Gly Pro 
                910                 915                 920 

cag gga aag aca gga gaa gtg gga cct cta ggt gaa agg ggg cct cca     2897 
Gln Gly Lys Thr Gly Glu Val Gly Pro Leu Gly Glu Arg Gly Pro Pro 
            925                 930                 935 

ggc ccc cct gga cct cct ggt gaa caa ggt ctt cct ggc ctg gaa ggc     2945 
Gly Pro Pro Gly Pro Pro Gly Glu Gln Gly Leu Pro Gly Leu Glu Gly 
        940                 945                 950 

aga gag ggg gcc aag ggg gaa ctg gga cca cca gga ccc ctt ggg aaa     2993 
Arg Glu Gly Ala Lys Gly Glu Leu Gly Pro Pro Gly Pro Leu Gly Lys 
    955                 960                 965 

gaa ggg cca gct gga ctc agg ggc ttt ccc ggc ccc aaa ggg ggc cct     3041 
Glu Gly Pro Ala Gly Leu Arg Gly Phe Pro Gly Pro Lys Gly Gly Pro 
970                 975                 980                 985 

ggg gac ccg gga cct act ggc tta aag ggt gat aag ggc ccc cca ggg     3089 
Gly Asp Pro Gly Pro Thr Gly Leu Lys Gly Asp Lys Gly Pro Pro Gly 
                990                 995                1000 

cct gtg ggg gcc aat ggc tcc cct ggt gag cgc ggt cct ttg ggc cca     3137 
Pro Val Gly Ala Asn Gly Ser Pro Gly Glu Arg Gly Pro Leu Gly Pro 
           1005                1010                1015 

gca gga ggc att gga ctt cct ggc caa agt ggc agc gaa ggc ccc gtt     3185 
Ala Gly Gly Ile Gly Leu Pro Gly Gln Ser Gly Ser Glu Gly Pro Val 
       1020                1025                1030 

ggc cct gca ggc aag aag ggg tcc cgg gga gaa cgt ggc ccc cct ggc     3233 
Gly Pro Ala Gly Lys Lys Gly Ser Arg Gly Glu Arg Gly Pro Pro Gly 
   1035                1040                1045 

ccc act ggc aaa gat ggg atc cca ggg ccc ctg ggg cct ctg gga ccc     3281 
Pro Thr Gly Lys Asp Gly Ile Pro Gly Pro Leu Gly Pro Leu Gly Pro 
1050               1055                1060                1065 

cct gga gct gct ggg cct tct ggc gag gaa ggg gac aag ggg gat gtg     3329 
Pro Gly Ala Ala Gly Pro Ser Gly Glu Glu Gly Asp Lys Gly Asp Val 
               1070                1075                1080 

ggt gcc ccc gga cac aag ggg agt aaa ggc gat aaa gga gac gcg ggc     3377 
Gly Ala Pro Gly His Lys Gly Ser Lys Gly Asp Lys Gly Asp Ala Gly 
           1085                1090                1095 

cca cct gga caa cca ggg ata cgg ggt cct gca gga cac cca ggt ccc     3425 
Pro Pro Gly Gln Pro Gly Ile Arg Gly Pro Ala Gly His Pro Gly Pro 
       1100                1105                1110 

ccg gga gca gac ggg gct cag ggg cgc cgg gga ccc cca ggc ctc ttt     3473 
Pro Gly Ala Asp Gly Ala Gln Gly Arg Arg Gly Pro Pro Gly Leu Phe 
   1115                1120                1125 

ggg cag aaa gga gat gac gga gtc aga ggc ttt gtg ggg gtg att ggc     3521 
Gly Gln Lys Gly Asp Asp Gly Val Arg Gly Phe Val Gly Val Ile Gly 
1130               1135                1140                1145 

cct cct gga ctg cag ggg ctg cca ggc cct ccg gga gag aaa ggg gag     3569 
Pro Pro Gly Leu Gln Gly Leu Pro Gly Pro Pro Gly Glu Lys Gly Glu 
               1150                1155                1160 

gtc gga gac gtc ggg tcc atg ggt ccc cat gga gct cca ggt cct cgg     3617 
Val Gly Asp Val Gly Ser Met Gly Pro His Gly Ala Pro Gly Pro Arg 
           1165                1170                1175 

ggt ccc caa ggc ccc act gga tca gag ggc act cca ggg ctg cct gga     3665 
Gly Pro Gln Gly Pro Thr Gly Ser Glu Gly Thr Pro Gly Leu Pro Gly 
       1180                1185                1190 

gga gtt ggt cag cca ggc gcc gtg ggt gag aag ggt gag cga ggg gac     3713 
Gly Val Gly Gln Pro Gly Ala Val Gly Glu Lys Gly Glu Arg Gly Asp 
   1195                1200                1205 

gct gga gac cca ggg cct cca gga gcc cca ggc atc ccg ggg ccc aag     3761 
Ala Gly Asp Pro Gly Pro Pro Gly Ala Pro Gly Ile Pro Gly Pro Lys 
1210               1215                1220                1225 

gga gac att ggt gaa aag ggg gac tca ggc cca tct gga gct gct gga     3809 
Gly Asp Ile Gly Glu Lys Gly Asp Ser Gly Pro Ser Gly Ala Ala Gly 
               1230                1235                1240 

ccc cca ggc aag aaa ggt ccc cct gga gag gat gga gcc aaa ggg agc     3857 
Pro Pro Gly Lys Lys Gly Pro Pro Gly Glu Asp Gly Ala Lys Gly Ser 
           1245                1250                1255 

gtg ggc ccc acg ggg ctg ccc gga gat cta ggg ccc cca gga gac cct     3905 
Val Gly Pro Thr Gly Leu Pro Gly Asp Leu Gly Pro Pro Gly Asp Pro 
       1260                1265                1270 

gga gtt tca ggc ata gat ggt tcc cca ggg gag aag gga gac cct ggt     3953 
Gly Val Ser Gly Ile Asp Gly Ser Pro Gly Glu Lys Gly Asp Pro Gly 
   1275                1280                1285 

gat gtt ggg gga ccg ggt ccg cct gga gct tct ggg gag ccc ggc gcc     4001 
Asp Val Gly Gly Pro Gly Pro Pro Gly Ala Ser Gly Glu Pro Gly Ala 
1290               1295                1300                1305 

ccc ggg ccc ccc ggc aag agg ggt cct tca ggc cac atg ggt cga gaa     4049 
Pro Gly Pro Pro Gly Lys Arg Gly Pro Ser Gly His Met Gly Arg Glu 
               1310                1315                1320 

ggc aga gaa ggg gag aaa ggt gcc aag ggg gag cca ggt cct gat ggg     4097 
Gly Arg Glu Gly Glu Lys Gly Ala Lys Gly Glu Pro Gly Pro Asp Gly 
           1325                1330                1335 

ccc cca ggg agg acg ggt cca atg ggg gct aga ggg ccc cct gga cgt     4145 
Pro Pro Gly Arg Thr Gly Pro Met Gly Ala Arg Gly Pro Pro Gly Arg 
       1340                1345                1350 

gtg ggg cct gag ggt ctt cga ggg atc cct ggc cct gtg ggt gaa cca     4193 
Val Gly Pro Glu Gly Leu Arg Gly Ile Pro Gly Pro Val Gly Glu Pro 
   1355                1360                1365 

ggc ctc ctg gga gcc cct gga cag atg ggc cct cct ggc ccc ctg ggg     4241 
Gly Leu Leu Gly Ala Pro Gly Gln Met Gly Pro Pro Gly Pro Leu Gly 
1370               1375                1380                1385 

ccc tct ggc ctc cca ggg ctg aag gga gac act ggc ccc aag ggg gaa     4289 
Pro Ser Gly Leu Pro Gly Leu Lys Gly Asp Thr Gly Pro Lys Gly Glu 
               1390                1395                1400 

aag ggc cac att gga ttg atc ggt ctc att ggc ccc ccg gga gaa gct     4337 
Lys Gly His Ile Gly Leu Ile Gly Leu Ile Gly Pro Pro Gly Glu Ala 
           1405                1410                1415 

ggt gag aaa gga gat cag ggg ttg cca ggc gtg cag gga ccc cct ggt     4385 
Gly Glu Lys Gly Asp Gln Gly Leu Pro Gly Val Gln Gly Pro Pro Gly 
       1420                1425                1430 

ccc aag gga gac cct ggt ccc cct ggt ccc att ggc tct ctg ggc cac     4433 
Pro Lys Gly Asp Pro Gly Pro Pro Gly Pro Ile Gly Ser Leu Gly His 
   1435                1440                1445 

cct ggg ccc cca ggt gtg gcg ggc cct cta gga cag aaa ggc tca aaa     4481 
Pro Gly Pro Pro Gly Val Ala Gly Pro Leu Gly Gln Lys Gly Ser Lys 
1450               1455                1460                1465 

ggg tct ccg ggg tcc atg ggc ccc cgt gga gac act gga cct gca ggc     4529 
Gly Ser Pro Gly Ser Met Gly Pro Arg Gly Asp Thr Gly Pro Ala Gly 
               1470                1475                1480 

cca cca ggc ccc ccg ggt gcc cct gcc gag ctg cat ggg ctg cgc agg     4577 
Pro Pro Gly Pro Pro Gly Ala Pro Ala Glu Leu His Gly Leu Arg Arg 
           1485                1490                1495 

cgc cgg cgc ttc gtc cca gtc ccg ctt cca gtc gtg gag ggc ggc ctg     4625 
Arg Arg Arg Phe Val Pro Val Pro Leu Pro Val Val Glu Gly Gly Leu 
       1500                1505                1510 

gag gag gtg ctg gcc tcg ctc aca tcg ctg agc ttg gag ctg gag cag     4673 
Glu Glu Val Leu Ala Ser Leu Thr Ser Leu Ser Leu Glu Leu Glu Gln 
   1515                1520                1525 

ctg cgg cgt cct ccc ggc act gcg gag cgc ccg ggc ctc gtg tgc cac     4721 
Leu Arg Arg Pro Pro Gly Thr Ala Glu Arg Pro Gly Leu Val Cys His 
1530               1535                1540                1545 

gag ctg cac cgc aac cac ccg cac ctg cct gat ggg gaa tac tgg att     4769 
Glu Leu His Arg Asn His Pro His Leu Pro Asp Gly Glu Tyr Trp Ile 
               1550                1555                1560 

gac ccc aac cag ggc tgc gcg cgg gac tcg ttc agg gtt ttt tgc aac     4817 
Asp Pro Asn Gln Gly Cys Ala Arg Asp Ser Phe Arg Val Phe Cys Asn 
           1565                1570                1575 

ttc acg gcg gga gga gag acc tgc ctc tat ccc gac aag aag ttt gag     4865 
Phe Thr Ala Gly Gly Glu Thr Cys Leu Tyr Pro Asp Lys Lys Phe Glu 
       1580                1585                1590 

atc gtg aaa ttg gcc tcc tgg tcc aag gaa aag cct gga ggc tgg tat     4913 
Ile Val Lys Leu Ala Ser Trp Ser Lys Glu Lys Pro Gly Gly Trp Tyr 
   1595                1600                1605 

agc aca ttc cgt cga ggg aag aag ttc tcc tac gtg gac gcc gac ggg     4961 
Ser Thr Phe Arg Arg Gly Lys Lys Phe Ser Tyr Val Asp Ala Asp Gly 
1610               1615                1620                1625 

tcc cca gtg aat gtc gtg cag ctg aac ttc ctg aaa ctg ctg agt gcc     5009 
Ser Pro Val Asn Val Val Gln Leu Asn Phe Leu Lys Leu Leu Ser Ala 
               1630                1635                1640 

aca gct cgc cag aac ttc acc tac tcc tgc cag aat gca gct gcc tgg     5057 
Thr Ala Arg Gln Asn Phe Thr Tyr Ser Cys Gln Asn Ala Ala Ala Trp 
           1645                1650                1655 

ctg gac gaa gcc acg ggt gac tac agc cac tcc gcc cgc ttc ctt ggc     5105 
Leu Asp Glu Ala Thr Gly Asp Tyr Ser His Ser Ala Arg Phe Leu Gly 
       1660                1665                1670 

acc aat gga gag gag ctg tct ttc aac cag acg aca gca acc act gtc     5153 
Thr Asn Gly Glu Glu Leu Ser Phe Asn Gln Thr Thr Ala Thr Thr Val 
   1675                1680                1685 

agc gtc ccc cag gat ggc tgc cgg ctc cgg aaa gga cag acg aag acc     5201 
Ser Val Pro Gln Asp Gly Cys Arg Leu Arg Lys Gly Gln Thr Lys Thr 
1690               1695                1700                1705 

ctt ttc gaa ttc agc tct tct cga gcg gga ttt ctg ccc ctg tgg gat     5249 
Leu Phe Glu Phe Ser Ser Ser Arg Ala Gly Phe Leu Pro Leu Trp Asp 
               1710                1715                1720 

gtg gcg gcc act gac ttt ggc cag acg aac caa aag ttt ggg ttt gaa     5297 
Val Ala Ala Thr Asp Phe Gly Gln Thr Asn Gln Lys Phe Gly Phe Glu 
           1725                1730                1735 

ctg ggc ccc gtc tgc ttc agc agc tgagagtgtc cggggtggga gggaccgtga    5351 
Leu Gly Pro Val Cys Phe Ser Ser 
       1740                1745 

gggagcccca gaatggggtg catttggtgc tgaggctttg aagccaccgt atttttcgtt   5411 

acctgtgact atggagccaa tgggatgtga cttcgctcat cacggtcagt cattccttct   5471 

cctttccagg gtgctggggg ctggggttcc ctggcccaag ggtccagcct cctctcaccc   5531 

cattccaggt ggcatactgc agtctggctc tttctcccct ccctccccac ccaagcctca   5591 

cctccccacc ccttgaaccc ccatgcaatg agcttctaac tcagagctga tgaacaaaag   5651 

cccccccacc cccaatgcct gcctcctcac tcctccgtcg ctgcccttca caccttttgg   5711 

tgctacccct ccccagagtt aagcactgga tgtctcctga tcccaggctg ggacccctac   5771 

ccccaccccc tttgatcctt tctacttcca cggtgaaagg actgaggtcg gactacagag   5831 

ggaagaggga cttcccttga ctgggttgtg tttcttttcc tgcctcagcc cagctctgca   5891 

aatcccctcc ccctgcccct cacctcccca ggctcacctt gccatgccag gtggtttggg   5951 

gaccaagatg ttgggggggt gaatcaggat cctaatggtg ctgccctatt tatacctggg   6011 

tctgtattaa aagggaaagt cccccctgtt gtagatttca tctgcttcct ccttagggaa   6071 

ggctgggata tgatgagaga ttccagccca agcccggccc cccaccgcca ggccataggg   6131 

cataatttgc atctcaaatc tgagaataaa ctgatgaact gtggaaaaaa aaaaaaaaaa   6191 

aaaaaaaaa                                                           6200 

 
           
             4  
             1745  
             PRT  
             Homo sapiens  
           
            4 

Met Gly Asn Arg Arg Asp Leu Gly Gln Pro Arg Ala Gly Leu Cys Leu 
  1               5                  10                  15 

Leu Leu Ala Ala Leu Gln Leu Leu Pro Gly Thr Gln Ala Asp Pro Val 
             20                  25                  30 

Asp Val Leu Lys Ala Leu Gly Val Gln Gly Gly Gln Ala Gly Val Pro 
         35                  40                  45 

Glu Gly Pro Gly Phe Cys Pro Gln Arg Thr Pro Glu Gly Asp Arg Ala 
     50                  55                  60 

Phe Arg Ile Gly Gln Ala Ser Thr Leu Gly Ile Pro Thr Trp Glu Leu 
 65                  70                  75                  80 

Phe Pro Glu Gly His Phe Pro Glu Asn Phe Ser Leu Leu Ile Thr Leu 
                 85                  90                  95 

Arg Gly Gln Pro Ala Asn Gln Ser Val Leu Leu Ser Ile Tyr Asp Glu 
            100                 105                 110 

Arg Gly Ala Arg Gln Leu Gly Leu Ala Leu Gly Pro Ala Leu Gly Leu 
        115                 120                 125 

Leu Gly Asp Pro Phe Arg Pro Leu Pro Gln Gln Val Asn Leu Thr Asp 
    130                 135                 140 

Gly Arg Trp His Arg Val Ala Val Ser Ile Asp Gly Glu Met Val Thr 
145                 150                 155                 160 

Leu Val Ala Asp Cys Glu Ala Gln Pro Pro Val Leu Gly His Gly Pro 
                165                 170                 175 

Arg Phe Ile Ser Ile Ala Gly Leu Thr Val Leu Gly Thr Gln Asp Leu 
            180                 185                 190 

Gly Glu Lys Thr Phe Glu Gly Asp Ile Gln Glu Leu Leu Ile Ser Pro 
        195                 200                 205 

Asp Pro Gln Ala Ala Phe Gln Ala Cys Glu Arg Tyr Leu Pro Asp Cys 
    210                 215                 220 

Asp Asn Leu Ala Pro Ala Ala Thr Val Ala Pro Gln Gly Glu Pro Glu 
225                 230                 235                 240 

Thr Pro Arg Pro Arg Arg Lys Gly Lys Gly Lys Gly Arg Lys Lys Gly 
                245                 250                 255 

Arg Gly Arg Lys Gly Lys Gly Arg Lys Lys Asn Lys Glu Ile Trp Thr 
            260                 265                 270 

Ser Ser Pro Pro Pro Asp Ser Ala Glu Asn Gln Thr Ser Thr Asp Ile 
        275                 280                 285 

Pro Lys Thr Glu Thr Pro Ala Pro Asn Leu Pro Pro Thr Pro Thr Pro 
    290                 295                 300 

Leu Val Val Thr Ser Thr Val Thr Thr Gly Leu Asn Ala Thr Ile Leu 
305                 310                 315                 320 

Glu Gly Ser Leu Asp Pro Asp Ser Gly Thr Glu Leu Gly Thr Leu Glu 
                325                 330                 335 

Thr Lys Ala Ala Arg Glu Asp Glu Glu Gly Asp Asp Ser Thr Met Gly 
            340                 345                 350 

Pro Asp Phe Arg Ala Ala Glu Tyr Pro Ser Arg Thr Gln Phe Gln Ile 
        355                 360                 365 

Phe Pro Gly Ala Gly Glu Lys Gly Ala Lys Gly Glu Pro Ala Val Ile 
    370                 375                 380 

Glu Lys Gly Gln Gln Phe Glu Gly Pro Pro Gly Ala Pro Gly Pro Gln 
385                 390                 395                 400 

Gly Val Val Gly Pro Ser Gly Pro Pro Gly Pro Pro Gly Phe Pro Gly 
                405                 410                 415 

Asp Pro Gly Pro Pro Gly Pro Ala Gly Leu Pro Gly Ile Pro Gly Ile 
            420                 425                 430 

Asp Gly Ile Arg Gly Pro Pro Gly Thr Val Ile Met Met Pro Phe Gln 
        435                 440                 445 

Phe Ala Gly Gly Ser Phe Lys Gly Pro Pro Val Ser Phe Gln Gln Ala 
    450                 455                 460 

Gln Ala Gln Ala Val Leu Gln Gln Thr Gln Leu Ser Met Lys Gly Pro 
465                 470                 475                 480 

Pro Gly Pro Val Gly Leu Thr Gly Arg Pro Gly Pro Val Gly Leu Pro 
                485                 490                 495 

Gly His Pro Gly Leu Lys Gly Glu Glu Gly Ala Glu Gly Pro Gln Gly 
            500                 505                 510 

Pro Arg Gly Leu Gln Gly Pro His Gly Pro Pro Gly Arg Val Gly Lys 
        515                 520                 525 

Met Gly Arg Pro Gly Ala Asp Gly Ala Arg Gly Leu Pro Gly Asp Thr 
    530                 535                 540 

Gly Pro Lys Gly Asp Arg Gly Phe Asp Gly Leu Pro Gly Leu Pro Gly 
545                 550                 555                 560 

Glu Lys Gly Gln Arg Gly Asp Phe Gly His Val Gly Gln Pro Gly Pro 
                565                 570                 575 

Pro Gly Glu Asp Gly Glu Arg Gly Ala Glu Gly Pro Pro Gly Pro Thr 
            580                 585                 590 

Gly Gln Ala Gly Glu Pro Gly Pro Arg Gly Leu Leu Gly Pro Arg Gly 
        595                 600                 605 

Ser Pro Gly Pro Thr Gly Arg Pro Gly Val Thr Gly Ile Asp Gly Ala 
    610                 615                 620 

Pro Gly Ala Lys Gly Asn Val Gly Pro Pro Gly Glu Pro Gly Pro Pro 
625                 630                 635                 640 

Gly Gln Gln Gly Asn His Gly Ser Gln Gly Leu Pro Gly Pro Gln Gly 
                645                 650                 655 

Leu Ile Gly Thr Pro Gly Glu Lys Gly Pro Pro Gly Asn Pro Gly Ile 
            660                 665                 670 

Pro Gly Leu Pro Gly Ser Asp Gly Pro Leu Gly His Pro Gly His Glu 
        675                 680                 685 

Gly Pro Thr Gly Glu Lys Gly Ala Gln Gly Pro Pro Gly Ser Ala Gly 
    690                 695                 700 

Pro Pro Gly Tyr Pro Gly Pro Arg Gly Val Lys Gly Thr Ser Gly Asn 
705                 710                 715                 720 

Arg Gly Leu Gln Gly Glu Lys Gly Glu Lys Gly Glu Asp Gly Phe Pro 
                725                 730                 735 

Gly Phe Lys Gly Asp Val Gly Leu Lys Gly Asp Gln Gly Lys Pro Gly 
            740                 745                 750 

Ala Pro Gly Pro Arg Gly Glu Asp Gly Pro Glu Gly Pro Lys Gly Gln 
        755                 760                 765 

Ala Gly Gln Ala Gly Glu Glu Gly Pro Pro Gly Ser Ala Gly Glu Lys 
    770                 775                 780 

Gly Lys Leu Gly Val Pro Gly Leu Pro Gly Tyr Pro Gly Arg Pro Gly 
785                 790                 795                 800 

Pro Lys Gly Ser Ile Gly Phe Pro Gly Pro Leu Gly Pro Ile Gly Glu 
                805                 810                 815 

Lys Gly Lys Ser Gly Lys Thr Gly Gln Pro Gly Leu Glu Gly Glu Arg 
            820                 825                 830 

Gly Pro Pro Gly Ser Arg Gly Glu Arg Gly Gln Pro Gly Ala Thr Gly 
        835                 840                 845 

Gln Pro Gly Pro Lys Gly Asp Val Gly Gln Asp Gly Ala Pro Gly Ile 
    850                 855                 860 

Pro Gly Glu Lys Gly Leu Pro Gly Leu Gln Gly Pro Pro Gly Phe Pro 
865                 870                 875                 880 

Gly Pro Lys Gly Pro Pro Gly His Gln Gly Lys Asp Gly Arg Pro Gly 
                885                 890                 895 

His Pro Gly Gln Arg Gly Glu Leu Gly Phe Gln Gly Gln Thr Gly Pro 
            900                 905                 910 

Pro Gly Pro Ala Gly Val Leu Gly Pro Gln Gly Lys Thr Gly Glu Val 
        915                 920                 925 

Gly Pro Leu Gly Glu Arg Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly 
    930                 935                 940 

Glu Gln Gly Leu Pro Gly Leu Glu Gly Arg Glu Gly Ala Lys Gly Glu 
945                 950                 955                 960 

Leu Gly Pro Pro Gly Pro Leu Gly Lys Glu Gly Pro Ala Gly Leu Arg 
                965                 970                 975 

Gly Phe Pro Gly Pro Lys Gly Gly Pro Gly Asp Pro Gly Pro Thr Gly 
            980                 985                 990 

Leu Lys Gly Asp Lys Gly Pro Pro Gly Pro Val Gly Ala Asn Gly Ser 
        995                1000                1005 

Pro Gly Glu Arg Gly Pro Leu Gly Pro Ala Gly Gly Ile Gly Leu Pro 
   1010                1015                1020 

Gly Gln Ser Gly Ser Glu Gly Pro Val Gly Pro Ala Gly Lys Lys Gly 
1025               1030                1035                1040 

Ser Arg Gly Glu Arg Gly Pro Pro Gly Pro Thr Gly Lys Asp Gly Ile 
               1045                1050                1055 

Pro Gly Pro Leu Gly Pro Leu Gly Pro Pro Gly Ala Ala Gly Pro Ser 
           1060                1065                1070 

Gly Glu Glu Gly Asp Lys Gly Asp Val Gly Ala Pro Gly His Lys Gly 
       1075                1080                1085 

Ser Lys Gly Asp Lys Gly Asp Ala Gly Pro Pro Gly Gln Pro Gly Ile 
   1090                1095                1100 

Arg Gly Pro Ala Gly His Pro Gly Pro Pro Gly Ala Asp Gly Ala Gln 
1105               1110                1115                1120 

Gly Arg Arg Gly Pro Pro Gly Leu Phe Gly Gln Lys Gly Asp Asp Gly 
               1125                1130                1135 

Val Arg Gly Phe Val Gly Val Ile Gly Pro Pro Gly Leu Gln Gly Leu 
           1140                1145                1150 

Pro Gly Pro Pro Gly Glu Lys Gly Glu Val Gly Asp Val Gly Ser Met 
       1155                1160                1165 

Gly Pro His Gly Ala Pro Gly Pro Arg Gly Pro Gln Gly Pro Thr Gly 
   1170                1175                1180 

Ser Glu Gly Thr Pro Gly Leu Pro Gly Gly Val Gly Gln Pro Gly Ala 
1185               1190                1195                1200 

Val Gly Glu Lys Gly Glu Arg Gly Asp Ala Gly Asp Pro Gly Pro Pro 
               1205                1210                1215 

Gly Ala Pro Gly Ile Pro Gly Pro Lys Gly Asp Ile Gly Glu Lys Gly 
           1220                1225                1230 

Asp Ser Gly Pro Ser Gly Ala Ala Gly Pro Pro Gly Lys Lys Gly Pro 
       1235                1240                1245 

Pro Gly Glu Asp Gly Ala Lys Gly Ser Val Gly Pro Thr Gly Leu Pro 
   1250                1255                1260 

Gly Asp Leu Gly Pro Pro Gly Asp Pro Gly Val Ser Gly Ile Asp Gly 
1265               1270                1275                1280 

Ser Pro Gly Glu Lys Gly Asp Pro Gly Asp Val Gly Gly Pro Gly Pro 
               1285                1290                1295 

Pro Gly Ala Ser Gly Glu Pro Gly Ala Pro Gly Pro Pro Gly Lys Arg 
           1300                1305                1310 

Gly Pro Ser Gly His Met Gly Arg Glu Gly Arg Glu Gly Glu Lys Gly 
       1315                1320                1325 

Ala Lys Gly Glu Pro Gly Pro Asp Gly Pro Pro Gly Arg Thr Gly Pro 
   1330                1335                1340 

Met Gly Ala Arg Gly Pro Pro Gly Arg Val Gly Pro Glu Gly Leu Arg 
1345               1350                1355                1360 

Gly Ile Pro Gly Pro Val Gly Glu Pro Gly Leu Leu Gly Ala Pro Gly 
               1365                1370                1375 

Gln Met Gly Pro Pro Gly Pro Leu Gly Pro Ser Gly Leu Pro Gly Leu 
           1380                1385                1390 

Lys Gly Asp Thr Gly Pro Lys Gly Glu Lys Gly His Ile Gly Leu Ile 
       1395                1400                1405 

Gly Leu Ile Gly Pro Pro Gly Glu Ala Gly Glu Lys Gly Asp Gln Gly 
   1410                1415                1420 

Leu Pro Gly Val Gln Gly Pro Pro Gly Pro Lys Gly Asp Pro Gly Pro 
1425               1430                1435                1440 

Pro Gly Pro Ile Gly Ser Leu Gly His Pro Gly Pro Pro Gly Val Ala 
               1445                1450                1455 

Gly Pro Leu Gly Gln Lys Gly Ser Lys Gly Ser Pro Gly Ser Met Gly 
           1460                1465                1470 

Pro Arg Gly Asp Thr Gly Pro Ala Gly Pro Pro Gly Pro Pro Gly Ala 
       1475                1480                1485 

Pro Ala Glu Leu His Gly Leu Arg Arg Arg Arg Arg Phe Val Pro Val 
   1490                1495                1500 

Pro Leu Pro Val Val Glu Gly Gly Leu Glu Glu Val Leu Ala Ser Leu 
1505               1510                1515                1520 

Thr Ser Leu Ser Leu Glu Leu Glu Gln Leu Arg Arg Pro Pro Gly Thr 
               1525                1530                1535 

Ala Glu Arg Pro Gly Leu Val Cys His Glu Leu His Arg Asn His Pro 
           1540                1545                1550 

His Leu Pro Asp Gly Glu Tyr Trp Ile Asp Pro Asn Gln Gly Cys Ala 
       1555                1560                1565 

Arg Asp Ser Phe Arg Val Phe Cys Asn Phe Thr Ala Gly Gly Glu Thr 
   1570                1575                1580 

Cys Leu Tyr Pro Asp Lys Lys Phe Glu Ile Val Lys Leu Ala Ser Trp 
1585               1590                1595                1600 

Ser Lys Glu Lys Pro Gly Gly Trp Tyr Ser Thr Phe Arg Arg Gly Lys 
               1605                1610                1615 

Lys Phe Ser Tyr Val Asp Ala Asp Gly Ser Pro Val Asn Val Val Gln 
           1620                1625                1630 

Leu Asn Phe Leu Lys Leu Leu Ser Ala Thr Ala Arg Gln Asn Phe Thr 
       1635                1640                1645 

Tyr Ser Cys Gln Asn Ala Ala Ala Trp Leu Asp Glu Ala Thr Gly Asp 
   1650                1655                1660 

Tyr Ser His Ser Ala Arg Phe Leu Gly Thr Asn Gly Glu Glu Leu Ser 
1665               1670                1675                1680 

Phe Asn Gln Thr Thr Ala Thr Thr Val Ser Val Pro Gln Asp Gly Cys 
               1685                1690                1695 

Arg Leu Arg Lys Gly Gln Thr Lys Thr Leu Phe Glu Phe Ser Ser Ser 
           1700                1705                1710 

Arg Ala Gly Phe Leu Pro Leu Trp Asp Val Ala Ala Thr Asp Phe Gly 
       1715                1720                1725 

Gln Thr Asn Gln Lys Phe Gly Phe Glu Leu Gly Pro Val Cys Phe Ser 
   1730                1735                1740 

Ser 
1745 

 
           
             5  
             27  
             DNA  
             Bacteriophage lambda  
           
            5 

tccccacctt ttgagcaagt tcagcct                                         27 

 
           
             6  
             28  
             DNA  
             Bacteriophage lambda  
           
            6 

agattggggg taaataacag aggtggct                                        28 

 
           
             7  
             24  
             DNA  
             Homo sapiens  
           
            7 

tgatcctaac caaggttgct cagg                                            24 

 
           
             8  
             24  
             DNA  
             Homo sapiens  
           
            8 

gagtcagcgg aattcaggga cacg                                            24 

 
           
             9  
             23  
             DNA  
             Homo sapiens  
           
            9 

aggcgaggtg atccagccac tgc                                             23 

 
           
             10  
             24  
             DNA  
             Homo sapiens  
           
            10 

gctctctaac gggtaacagg ctcc                                            24 

 
           
             11  
             23  
             DNA  
             Homo sapiens  
           
            11 

atgcaggaag atgaggccat acc                                             23 

 
           
             12  
             24  
             DNA  
             Homo sapiens  
           
            12 

gctctctaac gggtaacagg ctcc                                            24 

 
           
             13  
             21  
             DNA  
             Mus musculus  
           
            13 

ggagagctac gtggattatg c                                               21 

 
           
             14  
             21  
             DNA  
             Mus musculus  
           
            14 

ccatcggaaa ggcacgtgtg g                                               21 

 
           
             15  
             22  
             DNA  
             Mus musculus  
           
            15 

tgagcccacc ggtctccaga gc                                              22 

 
           
             16  
             21  
             DNA  
             Mus musculus  
           
            16 

ccatcggaaa ggcacgtgtg g                                               21 

 
           
             17  
             23  
             DNA  
             Mus musculus  
           
            17 

cttcaagaca cctgctctaa gcg                                             23 

 
           
             18  
             24  
             DNA  
             Mus musculus  
           
            18 

acatacccca tcatgtaagc tacc                                            24 

 
           
             19  
             24  
             DNA  
             Mus musculus  
           
            19 

gtttggattt gaagtcggtc cagc                                            24 

 
           
             20  
             24  
             DNA  
             Mus musculus  
           
            20 

tggcattact gaagcacgct gagg                                            24 

 
           
             21  
             22  
             DNA  
             Mus musculus  
           
            21 

atgtggctta ccgtgtggca cg                                              22 

 
           
             22  
             24  
             DNA  
             Mus musculus  
           
            22 

gctctgtggc ttatgaagtc ttgc                                            24 

 
           
             23  
             27  
             DNA  
             Mus musculus  
           
            23 

cctggcaaga gggtgagtgg tcttcca                                         27 

 
           
             24  
             28  
             DNA  
             Mus musculus  
           
            24 

gcatccaggt ttatgtcaag agtgggct                                        28