Patent Application: US-47953795-A

Abstract:
a pharmaceutical composition which comprises a recombinant vaccinia virus containing a dna fragment encoding for a polypeptide recognized by a particular antibody h23 , which recognizes a particular tumor antigen expressed on breast cancer cells is provided . the antibody specifically binds to an epitope comprising a tandem repeat sequence of 20 amino acids comprised in a transmembrane form as well as a secreted form of the polypeptides specifically bound by antibody h23 .

Description:
complementary and genomic dna fragments coding for portions of a polypeptide that specifically binds h23 are isolated according to the procedure described in wreschner et al ., eur . j . biochem , ( 1990 ) 189 : 463 . these fragments are thereafter used to reconstruct a dna fragment coding for the complete h23 - eta antigen in its secreted or transmembrane form . the plasmid constructions are described below , reference being made to fig1 . a . preparation of a vaccinia virus capable of promoting the synthesis of the secreted form of h23 - eta . an ecori - pvuii complementary dna fragment ( no . 1 ) is introduced between the ecori and pvuii sites of the multiple insertion region of the vector ppolyii described in lathe et al ., gene ( 1987 ) 57 : 193 to give plasmid peta - 5 &# 39 ;. a pvuii genomic dna fragment ( no . 2 ), containing 4 repeat units , is introduced into the pvuii site of the multiple insertion region of peta - 5 &# 39 ;, downstream of the fragment no . 1 and in the appropriate orientation . in the repeat units a , b , c and d , the codons xxx 1 and xxx 2 are , respectively , cca ( pro ) and ccc ( pro ), cca and ccc , gca ( ala ) and gcc , cca and gcc . likewise , the codon yyy is acc ( thr ) in the repeat units a , b and c ; the codon yyy is aac ( asn ) in the unit d . a bamhi - sali fragment coding for the complete secreted form of h23 - eta is excised from the plasmid finally obtained . this fragment is then inserted between the bamhi and sali sites of the transfer vector ptg194 - poly described in kieny et al ., bio / technology , ( 1986 ) 4 : 790 , downstream of the vaccinia virus promoter e7 . 5k and inside the vaccinia virus gene coding for thymidine kinase . the transfer vector obtained in the above paragraph is thereafter used to transfer the block for expression of the secreted form of h23 - eta into the genome of vaccinia virus , copenhagen strain , according to the method described in kieny et al ., nature ( 1984 ) 312 : 163 . the vaccinia virus vv - eta - s is thereby obtained . b . preparation of a vaccinia virus capable of promoting the synthesis of the transmembrane form of h23 - eta . a pvuii - psti genomic dna fragment ( no . 3 ), containing 4 repeat units , is introduced between the pvuii and psti sites of the multiple insertion region of peta - 5 &# 39 ;, downstream of the fragment no . 1 and in the appropriate orientation . in the repeat units a , b , c and d , the codons xxx 1 and xxx 2 are , respectively , cca ( pro ) and ccc ( pro ), cca and ccc , gca ( ala ) and gcc , cca and gcc . likewise , the codon yyy is acc ( thr ) in the repeat units a , b and c ; the codon yyy is aac ( asn ) in the unit d . an ecori - psti fragment corresponding to the cloned fragments is excised from the final plasmid obtained . the ecori cohesive end is converted to a blunt end by treatment with klenow polymerase . this fragment is then introduced between the xhoi site , treated beforehand with klenow polymerase , and the psti site of the multiple insertion region of the vector ppolyii - sfi / not - 14 described in lathe et al ., supra , to give plasmid peta - t - 5 &# 39 ;. a psti - bali complementary dna fragment ( no . 4 ) is introduced between the psti and bali sites of peta - t - 5 &# 39 ;. a bali - bali complementary dna fragment ( no . 5 ) is then inserted into the bali site of the plasmid finally obtained . a bglii - ssti fragment coding for the complete transmembrane form of h23 - eta is excised from the plasmid obtained in the above paragraph ; it is then introduced between the bamhi and ssti sites of the transfer vector ptg186 - poly described in kieny et al ., ( 1986 ), supra , downstream of the vaccinia virus promoter e7 . 5k and inside the vaccinia virus gene coding for thymidine kinase . the transfer vector obtained in the above paragraph is thereafter used to transfer the block for expression of the transmembrane form of h23 - eta into the genome of vaccinia virus , copenhagen strain ( vv - o ), according to the method described in kieny et al ., ( 1984 ), supra . the vaccinia virus vv - eta - t is thereby obtained . stocks of purified viruses are prepared using bhk - 21 cells . bhk - 21 cells are infected with the recombinant viruses vv - eta - s and vv - eta - t ( 0 . 1 pfu / cell ) for 48 hours . after this time , the cultures are frozen at - 20 ° c . and then thawed at room temperature . after destruction of the cell walls by 3 successive treatments with a &# 34 ; potter &# 34 ; in a hypotonic buffer , the soluble proteins of the supernatant are loaded onto a cushion of 36 % ( w / v ) sucrose and centrifuged ( beckman sw 28 , 1h , 14k ). the pellet containing the virus is taken up in solution in 10 mm tris - hcl ph 8 and placed on a linear ( 20 - 40 %) sucrose gradient . after centrifugation ( sw 28 , 40 min , 14k ), the opalescent band containing the virus is withdrawn using a syringe and concentrated by centrifugation ( sw 28 , 20k , 1 h ). the virus is lastly taken up in a small volume of 10 mm tris - hcl ph 8 so as to obtain a viral stock assaying at approximately 10 10 pfu / ml . a bamhi - sali dna fragment coding for the secreted form of h23 - eta is excised from the plasmid obtained in example 1a , first paragraph . it is then reintroduced between the bamhi and sali sites of the multiple insertion region of plasmid phmg described in gautier et al ., nucl . acid res ., ( 1989 ) 17 ( 20 ): 83 , so as to be placed under the control of the promoter of the 3 - hydroxy - 3 - methylglutarylcoenzyme a reductase ( hmgcr ) gene , downstream of the signal sequence of sv40 polya . plasmid phmg - eta - s is thereby obtained . likewise , plasmid phmg - eta - t is constructed in a similar manner by insertion of a bamhi - ecorv dna fragment derived from the plasmid obtained in example 1b , paragraph 2 . cells of the tumor cell line fr3t3 - ras - 1 , obtained from fisher rat fibroblasts by matriceau et al ., embo j . ( 1985 ) 4 : 1435 , and cells of the mouse mammary carcinoma line mm5t , derived from c3h mice , are cotransfected ( i ) with phmg - eta - s and plasmid pag60 described in colbere - garapin et al ., j . mol . biol . ( 1981 ) 150 : 1 which contains a gene for resistance to geneticin ( g418 ) or ( ii ) with phmg - eta - t and pag60 . to accomplish the transfection , the calcium phosphate precipitation method of graham et al ., virology ( 1973 ) 52 : 456 modified by wigler et al ., cell ( 1978 ) 14 : 725 is used . the transfected clones are selected in the presence of 500 μl / ml of g418 and are thereafter cultured . selection of the clones expressing h23 - eta is accomplished by labelling the cells with peroxidase after reaction with antibody h23 . cell lines in the pure state are obtained by the limiting dilution method , and the expression of h23 - eta is monitored . fisher iops line male and female rats and c3h line female mice aged 4 to 5 weeks are immunized in the following manner : a purified viral preparation of vv - eta - s , vv - eta - t or vv - o is administered to the animals , by scarification of the tail , in a volume of 10 μl corresponding to approximately 2 × 10 7 pfu . this treatment is repeated 10 days later . the f - s , f - t , f - c , m - s , m - t and m - c tumor lines are cultured in modified dulbecco medium ( gibco ) supplemented with 10 % of fetal calf serum , 100 units of penicillin and 100 μg / ml of streptomycin . the cultures are then treated with trypsin , washed and suspended in pbs ( phosphate buffered saline ) buffer . 14 days after the first stage of immunization , 2 × 10 4 f - c cells , 4 × 10 4 f - s cells , 1 . 5 × 10 5 f - t cells or 2 × 10 6 m - c , m - s or m - t cells are injected subcutaneously into an animal in a volume of 100 μl . the appearance of the subcutaneous tumors is monitored daily . the diameter of the tumors is measured in two dimensions . the complete data for the experiment and the results are presented in table i below : table i__________________________________________________________________________ number of meaaured average animals having diameter of the a tumor nodule tumor nodules ( in percentage relative to the mm ) × days after of animals tumor total number of injection of the free fromanimal virus cells animals treated cells tumors__________________________________________________________________________ f - c 4 / 4 31 ( 20 days ) 0fisher f - s 3 / 4 25 ( 25 days ) 25line f - t 3 / 6 25 ( 30 days ) 50male vv - eta - s f - c 8 / 8 40 ( 20 days ) 0rats f - s 3 / 8 7 . 5 ( 25 days ) 62 . 5 f - t 1 / 8 0 . 87 ( 30 days ) 87 . 5 vv - eta - t f - c 8 / 8 32 ( 20 days ) 0 f - s 1 / 8 0 . 38 ( 25 days ) 87 . 5 f - t 0 / 8 0 ( 30 days ) 100 f - s 10 / 10 11 . 2 ( 20 days ) 0 f - t 10 / 10 25 ( 20 days ) 0 vv - eta - s f - s 9 / 10 16 ( 20 days ) 10 f - t 9 / 10 30 ( 20 days ) 10 vv - eta - t f - s 5 / 10 1 . 7 ( 20 days ) 50 f - t 5 / 10 2 . 8 ( 20 days ) 50fisher vv - o f - s 10 / 10 19 . 8 ( 20 days ) 0line f - t 10 / 10 28 ( 20 days ) 0female vv - eta - s f - s 8 / 10 10 . 6 ( 20 days ) 20rats f - t 9 / 9 33 . 8 ( 20 days ) 0 vv - eta - t f - s 5 / 10 0 . 1 ( 25 days ) 50 f - t 1 / 10 90__________________________________________________________________________ table i shows that , when the animals are subjected to infection with f - s or f - t , the incidence of appearance of tumors in a group of animals treated beforehand using the vaccinia virus vv - eta - s or vv - eta - t is lower than in the groups of untreated animals or animals treated with a vv - o vaccinia virus . moreover , the size of the tumor nodules which appear in animals treated beforehand with vv - eta - s or vv - eta - t is much smaller than that of the tumor nodules observed in the untreated animals or animals treated with vv - o . immunization using vv - eta - s or vv - eta - t is effective only in the case of tumors induced with cells expressing the secreted or transmembrane form of h23 - eta . the vaccinal effect of the viruses is hence very specific . lastly , the vaccinal effect of vv - eta - t appears to be superior to that of vv - eta - s , irrespective of the form of h23 - eta expressed by the cells inducing the tumors . fisher line rats are infected with tumor cells as described in example 4 . as soon as tumors have appeared ( 10 to 15 days later ), treatment is carried out using the viral preparations , as described in example 4 . the data and results of the experiment are presented in table ii below : table ii______________________________________ number of animals having a tumor nodule measured relative to the total average diameter number of animals of the tumors treated ( in mm ) 25 days 50 days 25 days 50 days tumor after after after aftervirus cells injection injection injection injection______________________________________vv - o f - s 10 / 10 10 / 10 27 . 8 all dead f - t 10 / 10 10 / 10 27 . 7 all deadvv - eta - s f - s 10 / 10 10 / 10 31 . 5 all dead f - t 9 / 10 7 / 10 15 . 5 8 . 5vv - eta - t f - s 9 / 10 10 / 10 26 . 8 50 . 2 f - t 7 / 10 7 / 10 11 . 6 9 . 4______________________________________ table ii shows that the treatment of an infection with vv - eta - s or vv - eta - t has a favorable effect on the incidence of appearance and the size of the tumors relative to the control test . moreover , vv - eta - t appears to be more effective than vv - eta - s . __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 5 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 6192 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( ix ) feature :( a ) name / key : sig . sub .-- peptide ( b ) location : 58 .. 120 ( ix ) feature :( a ) name / key : repeat . sub .-- region ( b ) location : 439 .. 5239 ( d ) other information : / note = &# 34 ; the nucleotides spanning439 - 5239 constitute a repeated region wherein the repeatis 60 nucleotides and encodes 20 amino acids , 17 ofwhich are fixed . the number of such repeats varies from1 to 80 .&# 34 ;( ix ) feature :( a ) name / key : mat . sub .-- peptide ( b ) location : 121 .. 6166 ( ix ) feature :( a ) name / key : repeat . sub .-- region ( b ) location : 457 ( d ) other information : / note = &# 34 ; nucleotide 457 is x1 = nnnwhich is the codon for pro or ala wherein pro = cct , ccc , cca , or ccg ; and ala = gct , gcc , gca , or gcg .&# 34 ;( ix ) feature :( a ) name / key : repeat . sub .-- region ( b ) location : 487 ( d ) other information : / note = &# 34 ; nucleotide 487 is y = nnnwhich is the codon for thr or asn wherein thr = act , acc , aca , or acg ; and asn = aat or aac .&# 34 ;( ix ) feature :( a ) name / key : repeat . sub .-- region ( b ) location : 496 ( d ) other information : / note = &# 34 ; nucleotide 496 is x2 = nnnwhich is the codon for pro or ala wherein pro = cct , ccc , cca , or ccg ; and ala = gct , gcc , gca , or gcg .&# 34 ;( xi ) sequence description : seq id no : 1 : gaattccctggctgcttgaatctgttctgccccctccccacccatttcaccaccaccatg60acaccgggcacccagtctcctttcttcctgctgctgctcctcacagtgcttacagttgtt120acaggttctggtcatgcaagctctaccccaggtggagaaaaggagacttcggctacccag180agaagttcagtgcccagctctactgagaagaatgctgtgagtatgaccagcagcgtactc240tccagccacagccccggttcaggctcctccaccactcagggacaggatgtcactctggcc300ccggccacggaaccagcttcaggttcagctgccacctggggacaggatgtcacctcggtc360ccagtcaccaggccagccctgggctccaccaccccgccagcccacgatgtcacctcagcc420ccggacaacaagccagccccgggctccaccgcccccnnngcccacggtgtcacctcggcc480ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc540ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc600ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc660ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc720ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc780ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc840ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc900ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc960ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc1020ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc1080ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc1140ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc1200ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc1260ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc1320ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc1380ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc1440ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc1500ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc1560ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc1620ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc1680ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc1740ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc1800ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc1860ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc1920ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc1980ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc2040ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc2100ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc2160ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc2220ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc2280ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc2340ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc2400ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc2460ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc2520ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc2580ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc2640ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc2700ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc2760ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc2820ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc2880ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc2940ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc3000ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc3060ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc3120ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc3180ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc3240ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc3300ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc3360ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc3420ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc3480ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc3540ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc3600ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc3660ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc3720ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc3780ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc3840ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc3900ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc3960ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc4020ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc4080ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc4140ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc4200ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc4260ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc4320ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc4380ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc4440ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc4500ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc4560ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc4620ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc4680ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc4740ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc4800ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc4860ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc4920ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc4980ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc5040ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc5100ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc5160ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc5220ccggacnnnaggccgnnnttgggctccaccgcccctccagtccacaatgtcacctcggcc5280tcaggctctgcatcaggctcagcttctactctggtgcacaacggcacctctgccagggct5340accacaaccccagccagcaagagcactccacccagcattcccagccaccactctgatact5400cctaccacccttgccagccatagcaccaagactgatgccagtagcactcaccatagcacg5460gtacctcctctcacctcctccaatcacagcacttctccccagttgtctactggggtctct5520ttctttttcctgtcttttcacatttcaaacctccagtttaattcctctctggaagatccc5580agcaccgactactaccaagagctgcagagagacatttctgaaatgtttttgcagaattat5640aaacaagggggttttctgggcctctccaatattaagttcaggccagaatctgtggtggta5700caattgactctggccttccgagaaggtaccatcaatgtccacgacgtggagacacagttc5760aatcagtataaaacggaagcagcctctcgatataacctgacgatctcagacgtcagcgtg5820agtcatgtgccatttcctttctctgcccagtctggggctggggtgccaggctggggcatc5880gcgctgctggtgctggtctgtgttctggttgcgctggccattgtctatctcattgccttg5940gctgtctgtcagtgccgccgaaagaactacgggcagctggacatctttccagcccgggat6000acctaccatcctatgagcgagtaccccacctaccacacccatgggcgctatgtgccccct6060agcagtaccgatcgtagcccctatgagaaggtttctgcaggtaatggtggcagcagcctc6120tcttacacaaacccagcagtggcagccacttctgccaacttgtaggggcacgtcgccctc6180tgagctgagtgg6192 ( 2 ) information for seq id no : 2 :( i ) sequence characteristics :( a ) length : 2035 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( ix ) feature :( a ) name / key : peptide ( b ) location : 128 .. 1899 ( d ) other information : / note = &# 34 ; the amino acids spanning128 to 1899 constitute a repeated region wherein therepeat is 20 amino acids , 17 of which are fixed . thenumber of such repeats varies from 1 to 40 .&# 34 ;( ix ) feature :( a ) name / key : peptide ( b ) location : 134 ( d ) other information : / note = &# 34 ; amino acid 134 is x1 = xaaxaa xaa which is the codon for pro or ala whereinpro = cct , ccc , cca , or ccg ; and ala = gct , gcc , gca , or gcg .&# 34 ;( ix ) feature :( a ) name / key : peptide ( b ) location : 144 ( d ) other information : / note = &# 34 ; amino acid 144 is y = xaawhich is the codon for thr or asn wherein thr = act , acc , aca , or acg ; and asn = aat or aac .&# 34 ;( ix ) feature :( a ) name / key : peptide ( b ) location : 147 ( d ) other information : / note = &# 34 ; amino acid 147 is x2 = xaawhich is the codon for pro or ala wherein pro = cct , ccc , cca , or ccg ; and ala = gct , gcc , gca , or gcg .&# 34 ;( ix ) feature :( a ) name / key : peptide ( b ) location : 1 .. 21 ( d ) other information : / note = &# 34 ; amino acids 1 to 21 are a21 amino acid precursor sequence .&# 34 ;( xi ) sequence description : seq id no : 2 : 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( 2 ) information for seq id no : 3 :( i ) sequence characteristics :( a ) length : 20 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( ix ) feature :( a ) name / key : peptide ( b ) location : 7 .. 20 ( d ) other information : / note = &# 34 ; xaa at positions 7 and 20is x which is pro or ala .&# 34 ;( ix ) feature :( a ) name / key : peptide ( b ) location : 12 ( d ) other information : / note = &# 34 ; xaa at position 12 is ywhich is thr or asn .&# 34 ;( xi ) sequence description : seq id no : 3 : proglyserthralaproxaaalahisglyvalthrseralaproasp151015xaaargproxaa20 ( 2 ) information for seq id no : 4 :( i ) sequence characteristics :( a ) length : 6449 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( ix ) feature :( a ) name / key : sig . sub .-- peptide ( b ) location : 58 .. 120 ( ix ) feature :( a ) name / key : repeat . sub .-- region ( b ) location : 439 .. 5239 ( d ) other information : / note = &# 34 ; the nucleotides spanning439 - 5239 constitute a repeated region wherein the repeatis 60 nucleotides and encodes 20 amino acids , 17 of whichare fixed . the number of such repeats varies from 1 to 80 ( ix ) feature :( a ) name / key : mat . sub .-- peptide ( b ) location : 121 .. 5661 ( ix ) feature :( a ) name / key : repeat . sub .-- region ( b ) location : 457 ( d ) other information : / note = &# 34 ; nucleotide 457 is x1 = nnnwhich is the codon for pro or ala wherein pro = cct , ccc , cca , or ccg ; and ala = gct , gcc , gca , or gcg .&# 34 ;( ix ) feature :( a ) name / key : repeat . sub .-- region ( b ) location : 487 ( d ) other information : / note = &# 34 ; nucleotide 487 is y = nnnwhich is the codon for thr or asn wherein thr = act , acc , aca , or acg ; and asn = aat or aac .&# 34 ;( ix ) feature :( a ) name / key : repeat . sub .-- region ( b ) location : 496 ( d ) other information : / note = &# 34 ; nucleotide 496 is x2 = nnnwhich is the codon for pro or ala wherein pro = cct , ccc , cca , or ccg ; and ala = gct , gcc , gca , or gcg .&# 34 ;( xi ) sequence description : seq id no : 4 : gaattccctggctgcttgaatctgttctgccccctccccacccatttcaccaccaccatg60acaccgggcacccagtctcctttcttcctgctgctgctcctcacagtgcttacagttgtt120acaggttctggtcatgcaagctctaccccaggtggagaaaaggagacttcggctacccag180agaagttcagtgcccagctctactgagaagaatgctgtgagtatgaccagcagcgtactc240tccagccacagccccggttcaggctcctccaccactcagggacaggatgtcactctggcc300ccggccacggaaccagcttcaggttcagctgccacctggggacaggatgtcacctcggtc360ccagtcaccaggccagccctgggctccaccaccccgccagcccacgatgtcacctcagcc420ccggacaacaagccagccccgggctccaccgcccccnnngcccacggtgtcacctcggcc480ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc540ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc600ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc660ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc720ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc780ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc840ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc900ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc960ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc1020ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc1080ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc1140ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc1200ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc1260ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc1320ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc1380ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc1440ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc1500ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc1560ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc1620ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc1680ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc1740ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc1800ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc1860ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc1920ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc1980ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc2040ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc2100ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc2160ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc2220ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc2280ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc2340ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc2400ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc2460ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc2520ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc2580ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc2640ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc2700ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc2760ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc2820ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc2880ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc2940ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc3000ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc3060ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc3120ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc3180ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc3240ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc3300ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc3360ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc3420ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc3480ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc3540ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc3600ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc3660ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc3720ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc3780ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc3840ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc3900ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc3960ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc4020ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc4080ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc4140ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc4200ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc4260ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc4320ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc4380ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc4440ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc4500ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc4560ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc4620ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc4680ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc4740ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc4800ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc4860ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc4920ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc4980ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc5040ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc5100ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc5160ccggacnnnaggccgnnnccgggctccaccgcccccnnngcccacggtgtcacctcggcc5220ccggacnnnaggccgnnnttgggctccaccgcccctccagtccacaatgtcacctcggcc5280tcaggctctgcatcaggctcagcttctactctggtgcacaacggcacctctgccagggct5340accacaaccccagccagcaagagcactccattctcaattcccagccaccactctgatact5400cctaccacccttgccagccatagcaccaagactgatgccagtagcactcaccatagcacg5460gtacctcctctcacctcctccaatcacagcacttctccccagttgtctactggggtctct5520ttctttttcctgtcttttcacatttcaaacctccagtttaattcctctctggaagatccc5580agcaccgactactaccaagagctgcagagagacatttctgaaatggtgagtatcggcctt5640tccttccccatgctcccctgaagcagccatcagaactgtccacaccctttgcatcaagcc5700tgagtcctttccctctcaccccagtttttgcagatttataaacaagggggttttctgggc5760ctctccaatattaagttcaggtacagttctgggtgtggacccagtgtggtggttggaggg5820ttgggtggtggtcatgaccgtaggagggactggtcgcacttaaggttgggggaagagtcg5880tgagccagagctgggacccgtggctgaagtgcccatttccctgtgaccaggccaggatct5940gtggtggtacaattgactctggccttccgagaaggtaccatcaatgtccacgacgtggag6000acacagttcaatcagtataaaacggaagcagcctctcgatataacctgacgatctcagac6060gtcagcggtgaggctacttccctggctgcagcccagcaccatgccggggccctctccttc6120cagtgcctgggtccccgctctttccttagtgctggcagcgggaggggcgcctcctctggg6180agactgccctgaccactgcttttccttttagtgagtcatgtgccatttcctttctctgcc6240cagtctggggctggggtgccaggctggggcatcgcgctgctggtgctggtctgtgttctg6300gttgcgctggccattgtctatctcattgccttggtgagtgcagtccctggccctgatcag6360agccccccgttagaaggcactccatggcctgccataacctcctatctccccaggctgtct6420gtcagtgccgccgaaagaactacgggcag6449 ( 2 ) information for seq id no : 5 :( i ) sequence characteristics :( a ) length : 1867 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( ix ) feature :( a ) name / key : peptide ( b ) location : 128 .. 1727 ( d ) other information : / note = &# 34 ; the amino acids spanning128 to 1727 constitute a repeated region wherein therepeat is 20 amino acids , 17 of which are fixed . thenumber of such repeats varies from 1 to 40 .&# 34 ;( ix ) feature :( a ) name / key : peptide ( b ) location : 134 ( d ) other information : / note = &# 34 ; amino acid 134 is x1 = xaawhich is the codon for pro or ala wherein pro = cct , ccc , cca , or ccg ; and ala = gct , gcc , gca , or gcg .&# 34 ;( ix ) feature :( a ) name / key : peptide ( b ) location : 144 ( d ) other information : / note = &# 34 ; amino acid 144 is y = xaawhich is the codon for thr or asn wherein thr = act , acc , aca , or acg ; and asn = aat or aac .&# 34 ;( ix ) feature :( a ) name / key : peptide ( b ) location : 147 ( d ) other information : / note = &# 34 ; amino acid 147 is x2 = xaawhich is the codon for pro or ala wherein pro = cct , ccc , cca , or ccg ; and ala = gct , gcc , gca , or gcg .&# 34 ;( ix ) feature :( a ) name / key : peptide ( b ) location : 1 .. 21 ( d ) other information : / note = &# 34 ; amino acids 1 to 21 are a21 amino acid precursor sequence .&# 34 ;( xi ) sequence description : seq id no : 5 : 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