Patent Application: US-79506101-A

Abstract:
mammalian α3 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 .

Description:
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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 ). 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 . 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 . 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 . 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 . 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 ). 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 . sequences of clone me7 overlapped those of the est clone and contained an additional 422 - bp at the 5 ′- end . 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 . 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 . 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 ). 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 ). 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 ). 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 ). 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 ). α1 ( v ): a 1815 - bp probe containing mainly triple helical and c - propeptide sequences was an ecori fragment of cdna clone cw32 ( 27 ). α2 ( v ): a a 564 - bp probe corresponding to c - propeptide sequences was an ecori - hindiii fragment of cdna clone pbsl18 ( 43 ). 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 ). 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . probes were prepared as follows using a 17 dpc mouse embryo marathon cdna template : 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 . 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 ). 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 . 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 ). 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 . 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 . 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 . 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 . 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 . all citations mentioned in this patent application are incorporated herein by reference in their entirety as if set forth in fall . 1 . birk , d . e ., fitch , j . m ., babiarz , j . p ., and linsenmayer , t . g . 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( 1984 ) nucleic acids res . 12 , 9383 - 9394 ggtgcaccag gccccgctgg g atg aga agc tgc cgg aga ctg gat cag ctt 111 cag gcc ggc ctc tgc ctg ctc ctg gcc tcc ctg cag ctc gtg tcc tgg 159 acg ctg gct gca gaa cct gtg gac gta ctg gaa gcc tgg ggt gtg cat 207 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 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 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 ttt tct gtg ctg ctc aca ctg agg gcc cag cca gcc aat cag tct gtc 399 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 ctg ggg cca gct ctg ggc ctc ctt ggt gac tcc ttc agg ccc ctc ccc 495 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 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 cca aca ttt ggt cag ggg cct cgg ttt ata agt aca gct gga ctc act 639 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 cag gag ctg ctg tta att cca gac cct cag gct gcc ttc cag gcc tgt 735 gag agc tac ctc cct ggt tgt gaa acc ctc gat tcc aca acc aca ggg 783 gcc ccc aaa gac gat gaa cca gaa acc cct gcc cct cgt cgt cga aag 831 ggc aaa ggg aag aaa aaa ggg cgg ggt cga aag ggc aag gga aga aag 879 aaa aac aag gag acc tca gag ctg agt ccg acc cct ggt gcc cct gag 927 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 ctg cct ctg act ccc aca cct ctg gcc atc acc acc act gtc acg att 1023 gga caa aat gcc aca gtc tcg cag ggg ttg gac tcc ggt act gaa acc 1071 gag cag acg act cca gag gtg gac tct act gag gag ggt gaa gga ggt 1119 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 act gag ttc cag atc ttt cct ggt gct gga gaa aag gga gcg aaa gga 1215 gag cct gcg aca gta gag cag gga cag cag ttt gag ggg cct gca gga 1263 gct cca gga ccc cgg gga ata tct ggt cct tca ggc cct cct ggg cct 1311 ccg ggc ttc cct ggg gac cgt ggt cta ccg ggt cct gcc ggc ctc cca 1359 gga atc cca ggc atc gat gga gcc cgg ggc ctg ccg ggc aca gtg att 1407 atg atg ccg ttc cat ttt gca agc agc tcg atg aag gga ccc cca gtg 1455 tcc ttc cag cag gcc cag gcc cag gca gta ttg caa cag gct cag ctg 1503 tcc atg aaa ggg ccc cct ggt cca gta ggg ctc act ggg cgc cca ggc 1551 cct gtg ggc ctc cct gga tat cca ggt ctg aaa ggt gaa ctg gga gaa 1599 gtg ggg cca cag ggc ccc cga gga tta cag ggc cct cct ggg cct cct 1647 gga cgg gaa ggc aag aca ggc cga gct gga gca gat ggg gct cgg ggg 1695 ctc ccg gga gac aca gga cct aag ggt gac agg ggc ttt gat ggc ctg 1743 ccc ggg ctg cct ggt gag aag ggc caa agg ggt gac ttt gga cga gta 1791 ggg caa cct ggt ccc cca gga gag gat ggt gta aag ggc ctg cag gga 1839 cct cca ggg ccc act ggc cag gct gga gag ccg ggt ccc cga ggt ctg 1887 att ggc ccc aga ggt ctc cca ggt ccc cta gga cgc ccg ggt gtg aca 1935 ggg agt gat ggc gca cca ggg gcc aaa ggc aac gtg ggt cct cct gga 1983 gaa cca gga ccc cca gga cag caa gga aac cac ggc tcc cag gga att 2031 cca ggc ccc cag ggg ccc att ggc act ccc ggg gaa aag ggt ccc cct 2079 gga aac ccc gga att cca ggt gtc cca gga tct gag ggc ccc ccg ggc 2127 cac cca ggc cac gag ggt ccc aca gga gaa aaa ggg gct cag ggc cca 2175 cca gga tca gca ggc cct cgg ggc tat cct gga ctt cgt ggt gtg aag 2223 ggt acc tct ggt aac cgg ggt ctc caa ggc gag aaa gga gaa agg gga 2271 gag gat ggc ttt cct ggc ttc aag ggt gat gag gga cca aaa ggc gac 2319 cgg gga aac ccc gga ccc cca ggt ccc aga gga gag gat ggt cca gaa 2367 gga caa aag ggg cct ggg gga ctg cct ggt gat gag ggt cct cca gga 2415 gca gca ggg gag aag ggc aag ctt ggg gtg cca ggt ctc cca ggt tat 2463 cca gga cgc cca gga cct aag gga tct att gga ttt cct gga ccc ttg 2511 gga cca ctg ggg gag aaa ggc aag cgg ggc aaa gca gga cag cca gga 2559 gag gaa gga gaa cgc ggc aca ccg ggc acc cga gga gac agg gga cag 2607 ccg ggg gcc aca ggc cag cct ggc ccc aag ggt gac gtg ggc cag aat 2655 ggg tct cct ggg ccc cct ggg gaa aag ggt cta ccc ggt ctt caa ggc 2703 cca cca gga ttc ccc gga cca aaa ggc ccc ccg ggt cct cag ggg aaa 2751 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 ggt ctg aca ggc ccc cct gga cca gct ggc gtc ctt ggt cct cag gga 2847 aag gta ggg gac gtg ggg cct cta ggc gag aga ggc ccc cca ggg cct 2895 cct gga cct cct ggt gaa caa ggt ctg cca ggc ata gaa ggc aga gaa 2943 ggg gcc aag ggt gag cta gga ccc ctg ggg tcc gtc ggg aag gag ggg 2991 cca cct ggg ccc agg ggc ttc cct ggc ccc caa gga gcc ccc gga gac 3039 cca gga ccc att ggt ttg aag ggt gac aaa ggt ccc cca ggc cct gtt 3087 ggg gca aat ggc tcc ccg gga gag cgt ggt cct gta ggc ccc tct ggt 3135 ggc att ggg ctt cct ggc cag agt gga ggg caa ggc cct att ggt cct 3183 gct ggc gag aag ggg tcc ccg gga gaa cgg ggt act cct ggt cct act 3231 ggc aaa gat ggt att cca gga ccc ccg ggg ctt cag ggc ccc tct gga 3279 gct gcg ggg cct tct ggg gaa gaa gga gac aag ggg gaa gta ggg atg 3327 cct ggt cac aaa gga agc aaa ggg gat aaa gga gat gca ggc cca cct 3375 gga cca aca gga ata aga ggt cca gca ggc cat tca ggc ctc ccg ggt 3423 gct gat ggc gct cag ggt cgc cgg gga ccc cct ggc ctc ttc ggg cag 3471 aag ggg gat gac gga gtt cga ggc ttt gta ggt gta att ggt cct cca 3519 ggt ctg cag ggg ctg ccg ggt cct ccg ggg gag aag ggc gag gtt gga 3567 gac gta gga tcc atg ggt cca cat gga gct cca ggc cct cgg ggt ccc 3615 cct ggg ccc agt gga tca gag ggc ccc cca ggt ctg cct gga gga gta 3663 gga cag cct ggt gct gtg ggc gag aag ggt gag cca ggg gat gct gga 3711 gac gcc gga ccc cca gga att ccc ggc atc cct ggg ccc aaa ggt gaa 3759 att ggt gaa aag ggg gat tcg ggt cca tca ggg gct gct ggt ccc cca 3807 ggc aag aaa gga ccc cca gga gag gac ggc tct aag ggg aac atg ggt 3855 ccc aca gga ctc cct gga gat cta ggg ccc cca gga gac cct gga gtt 3903 ccg ggt att gat ggc atc cca ggg gag aag gga aat gct ggt gat att 3951 ggg gga ccg ggg cca cct gga gct tcc ggg gaa cct ggt gcc cgt ggc 3999 ctc cct ggc aag agg ggt tcc cct ggc cgc atg ggt cca gaa gga aga 4047 gag ggc gag aaa ggc gcc aag gga gat gct ggt cct gat gga ccc cca 4095 ggc agg aca ggc ccc att ggg gct cga ggg ccc cct gga cga att ggg 4143 cct gat ggt ctt cca ggg atc cct ggt cct gtg ggt gaa cca ggt ctc 4191 ctg gga cct cct ggg cta atc ggc cct cca ggg ccc ctg ggc cca cct 4239 ggc ctc cct ggc ctg aag gga gat gct ggc ccc aag ggg gag aag ggc 4287 cac att ggg cta ata ggc ctc att ggt ccc cca ggg gag gcc ggt gag 4335 aaa ggc gat cag ggg ttg cca ggt gtg cag ggc ccc cca ggc ctt cag 4383 gga gac cct ggt ctc cct ggt cct gtt ggc tcg tta ggt cac cct ggg 4431 ccc cca ggt gtg gtg ggc cct ctg gga cag aag ggc tcc aaa ggg tcc 4479 ccg gga tct ctt ggt cct cgt gga gac cct gga cca gcg ggt cct cct 4527 ggt ccc ccg ggt tct ccg gct gag gtg cat ggc ctg cgc agg cgc cga 4575 tct gtg acg gac acc ctg gaa ggt ggc ctg gag gag gtg atg gcc tca 4623 ctg aat tca ctg agc ttg gag ctg cag cag ttg cag aga cct ctg ggc 4671 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 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 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 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 tgg tcc cga gag aag cct gga ggc tgg tac agc acc ttc cgc cga ggg 4911 aag aag ttc tcc tat gtg gat gct gat ggc tcc ccg gtg aat gtg gtc 4959 cag ttg acc ttc ctg aag ttg ttg agt gct gca gcc cat cag agg ttc 5007 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 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 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 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 tct gtg ggt ttc ctg cct ctg tgg gat gtg gct gcc tct gac ttt ggt 5247 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 ala glu gly pro gly phe cys pro leu arg ile pro gln gly asp arg ala phe arg val gly lys ser ser leu leu ser val pro thr trp gln leu phe pro asp gly his phe pro glu asn phe ser val leu leu thr leu arg ala gln pro ala asn gln ser val leu leu ser ile tyr asp leu leu gly asp ser phe arg pro leu pro lys gln val asn ile met asp gly arg trp his arg val ala val ser ile ser gly asn lys val pro arg phe ile ser thr ala gly leu thr val met gly thr gln asp pro asp pro gln ala ala phe gln ala cys glu ser tyr leu pro gly gly ala arg gly leu pro gly thr val ile met met pro phe his phe glu tyr trp ile asp pro asn gln gly cys ala arg asp ala phe lys val phe cys asn phe thr ala gly gly glu thr cys leu tyr pro asp lys lys phe glu thr val lys leu ala ser trp ser arg glu lys pro asp ala asp gly ser pro val asn val val gln leu thr phe leu lys leu leu ser ala ala ala his gln arg phe thr tyr ile cys gln asn ser val ala trp leu asp glu ala ala gly asp his arg his ser ile ala ala thr ile lys val ser his asp gly cys arg val arg lys gly gln ala lys thr leu phe glu phe ser ser ser val gly phe leu pro leu trp asp val ala ala ser asp phe gly gln thr asn gln lys phe ccgccttgcc cgtgggcccc gccggg atg ggg aac cgc cgg gac ctg ggc cag 113 ccg cgg gcc ggt ctc tgc ctg ctc ctg gcc gcg ctg cag ctt ctg ccg 161 ggg acg cag gcc gat cct gtg gat gtc ctg aag gcc ctg ggt gtg cag 209 gga ggc cag gct ggg gtc ccc gag ggg cct ggc ttc tgt ccc cag agg 257 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 ggc atc ccc acg tgg gaa ctc ttt cca gaa ggc cac ttt cct gag aac 353 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 ctg ctg tcc att tat gat gaa agg ggt gcc cgg cag ttg ggc ctg gca 449 ctg ggg cca gcg ctg ggt ctc cta ggt gac ccc ttc cgc ccc ctc ccc 497 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 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 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 gtg ctg ggg acc cag gac ctt ggg gaa aag act ttc gag gga gac att 689 cag gag ctg ctg ata agc cca gat cct cag gct gcc ttc cag gct tgt 737 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 gct ccc cag ggt gaa cca gaa acc cct cgt cct cgg cgg aag ggg aag 833 gga aaa ggg agg aag aaa ggg cga ggt cgc aag ggg aag ggc agg aaa 881 aag aac aag gaa att tgg acc tca agt cca cct cct gac tcc gca gag 929 aac cag acc tcc act gac atc ccc aag aca gag act cca gct cca aat 977 ctg cct ccg acc ccc acg cct ttg gtc gtc acc tcc act gtg act act 1025 gga ctc aat gcc acg atc cta gag ggg agc ttg gac cct gac agt gga 1073 acc gag ctg ggg acc ctg gag acc aag gca gcc agg gag gat gaa gaa 1121 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 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 aaa gga gag ccc gca gtg att gaa aag ggg cag cag ttt gag gga cct 1265 cca gga gcc cca gga ccc caa ggg gtg gtt ggc ccc tca ggc cct ccc 1313 ggc ccc cca gga ttc cct ggc gac cct ggt cca ccg ggc cct gct ggc 1361 ctc cca gga atc ccc ggc att gat ggg atc cga ggc cca ccg ggc act 1409 gtg atc atg atg ccg ttc cag ttt gca ggc ggc tcc ttt aaa ggc ccc 1457 cca gtc tca ttc cag cag gcc cag gct cag gca gtt ctg cag cag act 1505 cag ctc tct atg aaa ggc ccc cct ggt cca gtg ggg ctc act ggg cgc 1553 cca ggc cct gtg ggt ctc ccc ggg cat cca ggt ctg aaa gga gag gag 1601 gga gca gaa ggg cca cag ggt ccc cga ggc ctg cag gga cct cat gga 1649 ccc cct ggc cga gtg ggc aag atg ggc cgc cct gga gca gat gga gct 1697 cgg ggc ctc cca ggg gac act gga cct aag ggt gat cgt ggc ttc gat 1745 ggc ctc cct ggg ctg cct ggt gag aag ggc caa agg ggt gac ttt ggc 1793 cat gtg ggg caa ccc ggt ccc cca gga gag gat ggt gag agg gga gca 1841 gag gga cct cca ggg ccc act ggc cag gct ggg gag ccg ggt cca cga 1889 gga ctg ctt ggc ccc aga ggc tct cct ggc ccc acg ggt cgc ccg ggt 1937 gtg act gga att gat ggt gct cct ggt gcc aaa ggc aat gtg ggt cct 1985 cca gga gaa cca ggc cct ccg gga cag cag gga aac cat ggg tcc cag 2033 gga ctc ccc ggt ccc cag gga ctc att ggc act cct ggg gag aag ggt 2081 ccc cct gga aac cca gga att cca ggc ctc cca gga tcc gat ggc cct 2129 ctg ggt cac cca gga cat gag ggc ccc acg gga gag aaa ggg gct cag 2177 ggt cca cca ggg tcg gca ggc cct ccg ggc tat cct gga cct cgg gga 2225 gtg aag ggc act tca ggc aac cgg ggc ctc cag ggg gag aaa ggc gag 2273 aag gga gag gac ggc ttc cca ggc ttc aag ggc gat gtg ggg ctc aaa 2321 ggt gat cag ggg aaa ccc gga gct cca ggt ccc cgg gga gag gat ggt 2369 cct gag ggg ccg aag ggg cag gcg ggg cag gct ggc gag gag ggg ccc 2417 cca ggc tca gct ggg gag aag ggc aag ctt ggg gtg cca ggc ctc cca 2465 ggt tat cca gga cgc cct gga cct aag gga tct att gga ttt ccc ggt 2513 ccc ctg gga ccc ata gga gag aaa ggg aag tcg gga aag aca ggg cag 2561 cca ggc ctg gaa gga gag cgg gga cca cca ggt tcc cgt gga gag agg 2609 ggg caa ccg ggt gcc aca ggg caa cca ggc ccc aag ggc gat gtg ggc 2657 cag gat gga gcc cct ggg atc cct gga gaa aag ggc ctc cct ggt ctg 2705 caa ggc cct cca gga ttc cct ggg cca aag ggc ccc cct ggt cac caa 2753 ggt aaa gat ggg cga cca ggg cac cct gga cag aga gga gaa ctg ggc 2801 ttc caa ggt cag aca ggc ccg cct gga cca gct ggt gtc tta ggc cct 2849 cag gga aag aca gga gaa gtg gga cct cta ggt gaa agg ggg cct cca 2897 ggc ccc cct gga cct cct ggt gaa caa ggt ctt cct ggc ctg gaa ggc 2945 aga gag ggg gcc aag ggg gaa ctg gga cca cca gga ccc ctt ggg aaa 2993 gaa ggg cca gct gga ctc agg ggc ttt ccc ggc ccc aaa ggg ggc cct 3041 ggg gac ccg gga cct act ggc tta aag ggt gat aag ggc ccc cca ggg 3089 cct gtg ggg gcc aat ggc tcc cct ggt gag cgc ggt cct ttg ggc cca 3137 gca gga ggc att gga ctt cct ggc caa agt ggc agc gaa ggc ccc gtt 3185 ggc cct gca ggc aag aag ggg tcc cgg gga gaa cgt ggc ccc cct ggc 3233 ccc act ggc aaa gat ggg atc cca ggg ccc ctg ggg cct ctg gga ccc 3281 cct gga gct gct ggg cct tct ggc gag gaa ggg gac aag ggg gat gtg 3329 ggt gcc ccc gga cac aag ggg agt aaa ggc gat aaa gga gac gcg ggc 3377 cca cct gga caa cca ggg ata cgg ggt cct gca gga cac cca ggt ccc 3425 ccg gga gca gac ggg gct cag ggg cgc cgg gga ccc cca ggc ctc ttt 3473 ggg cag aaa gga gat gac gga gtc aga ggc ttt gtg ggg gtg att ggc 3521 cct cct gga ctg cag ggg ctg cca ggc cct ccg gga gag aaa ggg gag 3569 gtc gga gac gtc ggg tcc atg ggt ccc cat gga gct cca ggt cct cgg 3617 ggt ccc caa ggc ccc act gga tca gag ggc act cca ggg ctg cct gga 3665 gga gtt ggt cag cca ggc gcc gtg ggt gag aag ggt gag cga ggg gac 3713 gct gga gac cca ggg cct cca gga gcc cca ggc atc ccg ggg ccc aag 3761 gga gac att ggt gaa aag ggg gac tca ggc cca tct gga gct gct gga 3809 ccc cca ggc aag aaa ggt ccc cct gga gag gat gga gcc aaa ggg agc 3857 gtg ggc ccc acg ggg ctg ccc gga gat cta ggg ccc cca gga gac cct 3905 gga gtt tca ggc ata gat ggt tcc cca ggg gag aag gga gac cct ggt 3953 gat gtt ggg gga ccg ggt ccg cct gga gct tct ggg gag ccc ggc gcc 4001 ccc ggg ccc ccc ggc aag agg ggt cct tca ggc cac atg ggt cga gaa 4049 ggc aga gaa ggg gag aaa ggt gcc aag ggg gag cca ggt cct gat ggg 4097 ccc cca ggg agg acg ggt cca atg ggg gct aga ggg ccc cct gga cgt 4145 gtg ggg cct gag ggt ctt cga ggg atc cct ggc cct gtg ggt gaa cca 4193 ggc ctc ctg gga gcc cct gga cag atg ggc cct cct ggc ccc ctg ggg 4241 ccc tct ggc ctc cca ggg ctg aag gga gac act ggc ccc aag ggg gaa 4289 aag ggc cac att gga ttg atc ggt ctc att ggc ccc ccg gga gaa gct 4337 ggt gag aaa gga gat cag ggg ttg cca ggc gtg cag gga ccc cct ggt 4385 ccc aag gga gac cct ggt ccc cct ggt ccc att ggc tct ctg ggc cac 4433 cct ggg ccc cca ggt gtg gcg ggc cct cta gga cag aaa ggc tca aaa 4481 ggg tct ccg ggg tcc atg ggc ccc cgt gga gac act gga cct gca ggc 4529 cca cca ggc ccc ccg ggt gcc cct gcc gag ctg cat ggg ctg cgc agg 4577 cgc cgg cgc ttc gtc cca gtc ccg ctt cca gtc gtg gag ggc ggc ctg 4625 gag gag gtg ctg gcc tcg ctc aca tcg ctg agc ttg gag ctg gag cag 4673 ctg cgg cgt cct ccc ggc act gcg gag cgc ccg ggc ctc gtg tgc cac 4721 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 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 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 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 agc aca ttc cgt cga ggg aag aag ttc tcc tac gtg gac gcc gac ggg 4961 tcc cca gtg aat gtc gtg cag ctg aac ttc ctg aaa ctg ctg agt gcc 5009 aca gct cgc cag aac ttc acc tac tcc tgc cag aat gca gct gcc tgg 5057 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 acc aat gga gag gag ctg tct ttc aac cag acg aca gca acc act gtc 5153 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 ctt ttc gaa ttc agc tct tct cga gcg gga ttt ctg ccc ctg tgg gat 5249 gtg gcg gcc act gac ttt ggc cag acg aac caa aag ttt ggg ttt gaa 5297 ctg ggc ccc gtc tgc ttc agc agc tgagagtgtc cggggtggga gggaccgtga 5351 phe arg ile gly gln ala ser thr leu gly ile pro thr trp glu leu arg gly gln pro ala asn gln ser val leu leu ser ile tyr asp glu gly arg trp his arg val ala val ser ile asp gly glu met val thr arg phe ile ser ile ala gly leu thr val leu gly thr gln asp leu gly glu lys thr phe glu gly asp ile gln glu leu leu ile ser pro pro asp phe arg ala ala glu tyr pro ser arg thr gln phe gln ile his leu pro asp gly glu tyr trp ile asp pro asn gln gly cys ala arg asp ser phe arg val phe cys asn phe thr ala gly gly glu thr cys leu tyr pro asp lys lys phe glu ile val lys leu ala ser trp lys phe ser tyr val 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