INeuron, Vol. 6, 811-823, May, 1991, Copyright
0 1991 by Cell Press
Structure and Expression of a Rat Agrin
Fabio Rupp, Donald G. Payan,* Catherine Magill-Sole, David M. Cowan, and Richard H. Scheller Howard Hughes Medical Institute Department of Molecular and Cellular Beckman Center ‘,tanford University Ctanford, California 94305
Physiology
Summary Agrin is a component of the basal lamina that causes the aggregation of acetylcholine receptors on cultured muscle fibers. An agrin cDNA clone isolated from electromotor neurons of a marine ray was used to characterize the corresponding cDNAs from a rat embryonic spinal cord library. Analysis of a set of clones predicts a 1940 amino acid protein containing 141 cysteine residues. The predicted protein has nine domains homologous to protease inhibitors, a region similar to domain III of laminin, and four epidermal growth factor repeats. The agrin gene is expressed in rat embryonic nervous svstem and muscle. The rat agrin protein is concentrated at synapses, where it may play a role in development and regeneration. Introduction The formation of synapses during development and their reorganization during learning is an important example of localized cell surface differentiation. A widely studied synapse is the neuromuscular junction, the highly specialized site that mediates precise and intricate interactions between motor neurons and muscle fibers (Dennis, 1981). The structural components of the neuromuscular junction are the presynaptic axon terminal, the postsynaptic cell membrane, and the synaptic cleft, occupied by basal lamina. Differentiation of each of these structures during development is thought to be regulated by sets 01 interactions between molecules synthesized by nerve and muscle (Schuetze and Role, 1987). One of the dramatic events that occurs during development of the neuromuscular junction is the localizdtion of acetylcholine receptors (AChRs) at the synapse. Early in development embryonic AChRs are widely distributed across the surface of muscle fibers. However, upon contact of the muscle fiber by the nerve, AChRs begin to accumulate beneath the nerve terminal to form discrete, high density patches of approximately 3 x IO3 receptors per pm* (Anderson et al., 1977; Bevan and Steinbach, 1977). The accumulation of AChRs at the developing neuromuscular junc* Present address: Howard Hughes Medical Institute, Department of Medicine, University of California San Francisco, San FrPncisco, California 94143.
tion has been shown to be a combination of both the lateral migration of preexisting receptors and the local insertion of newly synthesized receptors (ZiskindConhaim et al., 1984; Dubinsky et al., 1989; Anderson and Cohen, 1977). Numerous studies have examined neuron-derived factors that induce the aggregation of AChRs. Extracts of tissues from the central nervous system of the rat have been shown to increase the number of AChRs and the number of AChR patches (But-Caron et al., 1983; Jesse11 et al., 1979; Neugebauer et al., 1985; Olek et al., 1983; Podleski et al., 1978; Salpeter et al., 1982). In addition, several other molecules with AChR-aggregating activity have been identified (Usdin and Fischbach, 1986; reviewed in Schuetze and Role, 1987). These include ascorbic acid (Knaack and Podleski, 1985), the transferrin-like protein sciatin (Markelonis et al., 1982), and calcitonin gene-related peptide (New and Mudge, 1986; Fontaine et al., 1986, 1987). An important common feature of the above molecules is the fact that they principally influence synaptogenesis by increasing the rate of appearance of new AChRs (Role et al., 1985; Harris et al., 1988). In contrast, studies using extracts of basal laminaenriched fractions of marine ray electric organ revealed the presence of molecules that cause aggregation of AChRs that are already present on the surface of myotubes (Godfrey et al., 1984,1988; McMahan and Slater, 1984). This activity has been shown to be caused by the protein agrin (Nitkin et al., 1987). Agrin is believed to cause the formation of AChR patches by a process of lateral migration within the muscle fiber plasma membrane (Fallon et al., 1985; Nitkin et al., 1987; Reist et al., 1987). Thus, agrin is functionally distinct from the above AChR-aggregating molecules. Antibodies raised against agrin recognize a protein concentrated in the synaptic basal lamina at the neuromuscular junction and have been used to immunoprecipitate agrin-like molecules frorn electric organ extracts (Smith et al., 1987). Theobservation that AChR aggregates appear in concert with the formation of the basal lamina has contributed to the proposal that agrin becomes stably incorporated into the basal lamina,where it plays a role in maintaining the integrity of the postsynaptic apparatus (Anglister and McMahan, 1985; McMahan and Wallace, 1989). Proteins associated with the basal lamina may have several additional activities. Laminin promotes axon outgrowth (Edgar et al., 1984; Liesi et al., 1989), in addition to its known tissue support and cell integrative functions (reviewed in Beck et al., 1990). Furthermore, there is increasing evidence for a growing number of related extracellular matrix molecules, such as s-laminin (Hunter et al., 1989a). These molecules may be localized to unique tissue compartments, such as the synapse, where they may function to regulate axon outgrowth and aspects of synapse formation. We report the isolation of a set of overlapping cDNA
1 121
TGCTTATCTCCTACCATGMCCTGGAGGGCTGGGGAGAGGACCTGGTGCTCCCCTAGCCATMCTGGCTAAACTTTGAGATCACAGCTGCATCTGCTGCTCCAGAGCACCCAGCATGGGG GGAGTAGCTCCGMGAGGCCTMGGGCACTCCATAAGAACTCCCACACACACCCAGGAATTGGGCTCGAGCTGTATGTATCATGCCTCCTClGCCACTGGAACACAGACCCAGGCAGGAG HPPLPLEHRPROE
241 14
CCTGGTGCCTCCATGCTGGTTCGATACTTCATGATCCCCTGC~CATCTGCTTGATCCTGTTGGCCACTTCCACATTGGGCTTTGCGGTTCTGCTTTTCCTCAGCAACTAC~CCTGGA PGASHLVRYFMlPCNlCLlLLATSTLGFAVLLFLSNYKPG
361 54
ATCCACTTCACACCAGCGCCTCCTACGCCTCCTGATGTATGCAGGGGMTGTTATGTGGCTTTGGTGCTGTGTGTGMCCTAGTGTTGAGGATCCAGGCCGTGCCTCCTGTGTGTGC~G IHFTPAPPTPPOVCRGMLCGFGAVCEPSVEDPGRASCVCK
481 94
MGMTGCTTGCCCTGCTACGGTGGCTCCTGTGTGTGGCTCAGATGCCTCCACCTATAGCAACGAGTGTGAGCTGCAGCGCGCGCAGTGCAACCAGCAACGGCGCATCCGCCTGCTTCGC KNACPATVAPVCGSDASTYSNECELPRAaCNPPRRlRLLR
601 134
CAAGGGCCATGTGGGTCCCGGGACCCCTGTGCCAACGTGACCTGCAGTTTCGGTAGTACCTGTGTACCTTCGGCTGACGGACAGAClGCCTCATGTCTGTGTCCTACAACCTGClTCGGG OGPCGSRDPCANVTCSFGSTCVPSADGOTASCLCPTTCFG
721 174
GCCCCTGATGGCACAGTGTGTGGCAGTGACGGTGACTACCCTAGTGAATGCCAGCTGCTTAGTCATGCCTGTGCCAGCCAGGAGCACATCTTCAAGAAGTTCAATGGTCCTTGTGAT APDGTVCGSDGVDYPSECOLLStlACASOEHlFKKFNGPCD
841 214
CCCTGCCAGGGCAGCATGTCAGACCTGAACCACATTTGCCGTGTGAACCCACGTACACGGCACCCAGAAATGCTTCTGCGGCCTGAGAACTGCCCTGCCCAGCACACGCCTATCTGTGGA PCOGSNSDLNHlCRVNPRTRHPEMLLRPENCPAOHTPlCG
961 254
GATGATGGGGTCACCTATGAAAACGACTGTGTCATGAGCCGTATAGGTGCAACCCGTGGCCTGCTTCTCCAGAAAGTACGCTCTGGTCAATGCCAAACTCGAGACCAGTGCCCGGAGACC DDGVTYENDCVHSRlGATRGLLLOKVRSGOCOTRDOCPET
1081 294
TGCCAGTTTAACTCCGTATGCCTGTCCCGCCGTGGCCGTCCCCACTGTTCCTGCGATCGTGTCACCTGTGATGGGTCTTACAGGCCTGTGTGTGCCCAAGATGGGCACACATACAACAAT COFNSVCLSRRGRPHCSCDRVTCDGSYRPVCAODGHTYNN
1201 334
GACTGTTGGCGCCAACAGGCTGAGTGTCGACAACAGCGGGCCATTCCTCCCAAGCACCAGGGCCCGTGTGACCAGACCCCGTCCCCATGCCATGGAGTGCAATGTGCATTTGGGGCAGTA DCUROOAECROORAIPPKHOGPCDOTPSPCHGVOCAFGAV
1321 374
TGCACAGTGAAGAACGGGAAAGCCGAGTGCGAGTGCCAGCGGGTGTGCTCCGGCATCTACGATCCTGTGTGCGGCAGTGACGGTGTCACTTACGGCAGTGTGTGCGAGCTGGAATCCATG CTVKNGKAECECORVCSGlYDPVCGSDGVTYGSVCELESM
1441 414
GCCTGTACCCTTGGGCGGGAAATCCAAGTGGCCCGCAGAGGACCCTGTGACCCATGTGGGCAGTGCCGTTTTGGATCCCTGTGCGAGGTGGAAACTGGACGCTGTGTGTGCCCCTCTGAG ACTLGRElOVARRGPCDPCGOCRFGSLCEVETGRCVCPSE
1561 454
TGTGTGGAGTCAGCCCAGCCCGTATGCGGTTCTGATGGACACACATACGCTAGTGAATGTGAGCTGCATGTCCACGCCTGTACGCACCAGATCAGCCTATACGTGGCCTCAGCTGGACAC CVESAOPVCGSDGHTYASECELtiVHACTHOlSLYVASAGH
1681 494
TGCCAGACCTGTGGAGAAAAAGTTTGTACTTTTGGGGCTGTGTGCTCAGCTGGACAGTGTGTATGTCCCCGTTGTGAGCACCCTCCACCTGGCCCTGTGTGTGGCAGTGATGGTGTCACC COTCGEKVCTFGAVCSAGOCVCPRCEHPPPGPVCGSDGVT
1801 534
TACCTCAGTGCCTGTGAGCTACGCGAAGCTGCCTGTCAGCAGCAGGTACAAATTGAGGAGGCCCATGCAGGGCCATGTGAGCCGGCTGAGTGTGGCTCAGGGGGCTCTGGTTCTGGGGAG YLSACELREAACOOOVO1EEAHAGPCEPAECGSGGSGSGE
1921 574
GACGACGAGTGTGAACAGGAGCTATGCCGGCAGCGTGGTGGTATCTGGGACGAGGACTCAGAAGATGGGCCATGTGTCTGTGACTTTAGCTGCCAGAGTGTCCCTAGAAGCCCAGTGTGl DDECEOELCRORGGIUDEDSEOGPCVCDFSCPSVPRSPVCDFSCOSVPRSPVC
2041 614
GGCTCGGATGGAGTCACCTATGGCACCGAGTGTGATCTGAAGAAGGCCAGGTGCGAATCACAGCAAGAACTGTACGTTGCTGCTCAGGGAGCCTGCCGTGGCCCTACCTTGGCTCCACTG GSDGVTYGTECDLKKARCESOOELYVAAOGACRGPTLAPL
2161 654
CTACCTGTGGCCTTCCCACACTGTGCCCAAACCCCCTATGGCTGCTGCCAGGACAATTTCACTGCTGCCCAGGGTGTGGGCTTGGCTGGCTGTCCCAGCACCTGCCATTGCAACCCCCAT LPVAFPHCAOTPYGCCODNFTAAOGVGLAGCPSTCHCNPH
2281 694
GGCTCCTACAGTGGCACTTGTGACCCAGCCACAGGGCAGTGCTCCTGCCGACCAGGTGTAGGAGGCCTCAGGTGTGATCGCTGTGAACCTGGCTTCTGGAACTTCCGTGGCATCGTCACC GSYSGTCDPATGOCSCRPGVGGLRCDRCEPGFUNFRGIVT
2401 734
GATGGACATAGTGGTTGCACTCCCTGCAGCTGCGACCCTCGGGGTGCCGTAAGGGATGACTGTGAACAGATGACTGGATTGTGTTCCTGTAGGCCTGGTGTGGCTGGTCCCAAGTGTGGA DGHSGCTPCSCDPRGAVRDDCEOMTGLCSCRPGVAGPKCG
2521 774
CAGTGTCCAGATGGTCAAGTCCTGGGCCATCTAGGCTGTGAAGCAGATCCCATGACACCTGTGACTTGTGTGGAGATACACTGCGAGTTCGGGGCCTCCTGCGTAGAGAAGGCTGGTTTT OCPDGOVLGHLGCEADPMTPVTCVEKAGF
2641 814
GCCCAATGTATCTGCCCAACCCTCACATGTCCAGAGGCTAACTCTACCAAGGTCTGCGGATCAGACGGTGTGACATATGGCAATGAATGCCAGCTGAAGGCCATTGCCTGCCGCCAGCGT AOCICPTLTCPEANSTKVCGSDGVTYGNECOLKA~ACROR
2761 a54
CTGGACATCTCCACTCAGAGTCTTGGTCCCTGCCAGGAGAGTGTTACTCCTGGGGCTTCCCCAACATCTGCATCTATGACTACCCCAAGGCATATCCTGAGCAAGACGCTGCCATTTCCC LDlSTOSLGPCOESVTPGASPTSASHTTPRHlLSKTLPFP
2aal a94
CACAACAGCCTTCCTCTGTCTCCCGGCAGTACTACCCATGATTGGCCCACCCCATTACCCATATCACCTCACACCACAGTCAGCATCCCCAGAAGCACCGCGTGGCCTGTGCTGACCGTG HNSLPLSPGSTTHDUPTPLPISPHTTVSIPRSTAUPVLTV
3001 934
CCCCCTACAGCAGCAGCCTCTGACGTAACCAGTCTCGCAACATCAATCTTCAGTGAATCCGGCAGCGCCAATGGGAGTGGCGACGAGGAACTGAGTGGAGATGAGGAGGCCAGTGGGGGC PPTAAASDVTSLATSIFSESGSANGSGDEELSGDEEASGG
3121 974
GGGTCTGGGGGACTTGAGCCCCCGGTGGGCAGCATTGTGGTGACCCATGGGCCACCCATCGAGAGGGCTTCCTGCTACAACTCACCTTTGGGCTGCTGCTCAGATGGCAAGACACCCTCA GSGGLEPPVGSIVVTHGPPIERASCYNSPLGCCSDGKTPS
3241 1014
CTGGACTCCGAAGGCTCCAACTGTCCTGCTACCAAGGCATTCCAGGGCGTGCTGGAGCTTGAGGGGGTCGAGGGACAGGAACTGTTCTACACACCTGAGATGGCTGACCCCAAGTCAGAG LDSEGSNCPATKAFOGVLELEGVEGOELFYTPEMADPKSE
3361 1054
TTGTTTGGGGAGACCGCAAGGAGCATTGAAAGCACGCTGGACGACCTGTTCCGGAATTCAGACGTCAAGAAGGACTTCTGGAGTGTCCGCCTACGGGAACTGGGGCCTGGCAAGTTGGTC LFGETARSIESTLDDLFRNSDVKKDFUSVRLRELGPGKLV
3481 1094
CGCGCCATTGTGGACGTTCACTTTGACCCTACCACAGCCTTCCAGGCATCAGATGTGGGTCAGGCCTTGCTCCGACAGATCCAGGTATCTAGGCCATGGGCCCTGGCAGTGAGGAGGCCT RAIVDVHFDPTTAFOASDVGOALLROIOVSRPUALAVRRP
0 1991 by Cell Press
Structure and Expression of a Rat Agrin
Fabio Rupp, Donald G. Payan,* Catherine Magill-Sole, David M. Cowan, and Richard H. Scheller Howard Hughes Medical Institute Department of Molecular and Cellular Beckman Center ‘,tanford University Ctanford, California 94305
Physiology
Summary Agrin is a component of the basal lamina that causes the aggregation of acetylcholine receptors on cultured muscle fibers. An agrin cDNA clone isolated from electromotor neurons of a marine ray was used to characterize the corresponding cDNAs from a rat embryonic spinal cord library. Analysis of a set of clones predicts a 1940 amino acid protein containing 141 cysteine residues. The predicted protein has nine domains homologous to protease inhibitors, a region similar to domain III of laminin, and four epidermal growth factor repeats. The agrin gene is expressed in rat embryonic nervous svstem and muscle. The rat agrin protein is concentrated at synapses, where it may play a role in development and regeneration. Introduction The formation of synapses during development and their reorganization during learning is an important example of localized cell surface differentiation. A widely studied synapse is the neuromuscular junction, the highly specialized site that mediates precise and intricate interactions between motor neurons and muscle fibers (Dennis, 1981). The structural components of the neuromuscular junction are the presynaptic axon terminal, the postsynaptic cell membrane, and the synaptic cleft, occupied by basal lamina. Differentiation of each of these structures during development is thought to be regulated by sets 01 interactions between molecules synthesized by nerve and muscle (Schuetze and Role, 1987). One of the dramatic events that occurs during development of the neuromuscular junction is the localizdtion of acetylcholine receptors (AChRs) at the synapse. Early in development embryonic AChRs are widely distributed across the surface of muscle fibers. However, upon contact of the muscle fiber by the nerve, AChRs begin to accumulate beneath the nerve terminal to form discrete, high density patches of approximately 3 x IO3 receptors per pm* (Anderson et al., 1977; Bevan and Steinbach, 1977). The accumulation of AChRs at the developing neuromuscular junc* Present address: Howard Hughes Medical Institute, Department of Medicine, University of California San Francisco, San FrPncisco, California 94143.
tion has been shown to be a combination of both the lateral migration of preexisting receptors and the local insertion of newly synthesized receptors (ZiskindConhaim et al., 1984; Dubinsky et al., 1989; Anderson and Cohen, 1977). Numerous studies have examined neuron-derived factors that induce the aggregation of AChRs. Extracts of tissues from the central nervous system of the rat have been shown to increase the number of AChRs and the number of AChR patches (But-Caron et al., 1983; Jesse11 et al., 1979; Neugebauer et al., 1985; Olek et al., 1983; Podleski et al., 1978; Salpeter et al., 1982). In addition, several other molecules with AChR-aggregating activity have been identified (Usdin and Fischbach, 1986; reviewed in Schuetze and Role, 1987). These include ascorbic acid (Knaack and Podleski, 1985), the transferrin-like protein sciatin (Markelonis et al., 1982), and calcitonin gene-related peptide (New and Mudge, 1986; Fontaine et al., 1986, 1987). An important common feature of the above molecules is the fact that they principally influence synaptogenesis by increasing the rate of appearance of new AChRs (Role et al., 1985; Harris et al., 1988). In contrast, studies using extracts of basal laminaenriched fractions of marine ray electric organ revealed the presence of molecules that cause aggregation of AChRs that are already present on the surface of myotubes (Godfrey et al., 1984,1988; McMahan and Slater, 1984). This activity has been shown to be caused by the protein agrin (Nitkin et al., 1987). Agrin is believed to cause the formation of AChR patches by a process of lateral migration within the muscle fiber plasma membrane (Fallon et al., 1985; Nitkin et al., 1987; Reist et al., 1987). Thus, agrin is functionally distinct from the above AChR-aggregating molecules. Antibodies raised against agrin recognize a protein concentrated in the synaptic basal lamina at the neuromuscular junction and have been used to immunoprecipitate agrin-like molecules frorn electric organ extracts (Smith et al., 1987). Theobservation that AChR aggregates appear in concert with the formation of the basal lamina has contributed to the proposal that agrin becomes stably incorporated into the basal lamina,where it plays a role in maintaining the integrity of the postsynaptic apparatus (Anglister and McMahan, 1985; McMahan and Wallace, 1989). Proteins associated with the basal lamina may have several additional activities. Laminin promotes axon outgrowth (Edgar et al., 1984; Liesi et al., 1989), in addition to its known tissue support and cell integrative functions (reviewed in Beck et al., 1990). Furthermore, there is increasing evidence for a growing number of related extracellular matrix molecules, such as s-laminin (Hunter et al., 1989a). These molecules may be localized to unique tissue compartments, such as the synapse, where they may function to regulate axon outgrowth and aspects of synapse formation. We report the isolation of a set of overlapping cDNA
1 121
TGCTTATCTCCTACCATGMCCTGGAGGGCTGGGGAGAGGACCTGGTGCTCCCCTAGCCATMCTGGCTAAACTTTGAGATCACAGCTGCATCTGCTGCTCCAGAGCACCCAGCATGGGG GGAGTAGCTCCGMGAGGCCTMGGGCACTCCATAAGAACTCCCACACACACCCAGGAATTGGGCTCGAGCTGTATGTATCATGCCTCCTClGCCACTGGAACACAGACCCAGGCAGGAG HPPLPLEHRPROE
241 14
CCTGGTGCCTCCATGCTGGTTCGATACTTCATGATCCCCTGC~CATCTGCTTGATCCTGTTGGCCACTTCCACATTGGGCTTTGCGGTTCTGCTTTTCCTCAGCAACTAC~CCTGGA PGASHLVRYFMlPCNlCLlLLATSTLGFAVLLFLSNYKPG
361 54
ATCCACTTCACACCAGCGCCTCCTACGCCTCCTGATGTATGCAGGGGMTGTTATGTGGCTTTGGTGCTGTGTGTGMCCTAGTGTTGAGGATCCAGGCCGTGCCTCCTGTGTGTGC~G IHFTPAPPTPPOVCRGMLCGFGAVCEPSVEDPGRASCVCK
481 94
MGMTGCTTGCCCTGCTACGGTGGCTCCTGTGTGTGGCTCAGATGCCTCCACCTATAGCAACGAGTGTGAGCTGCAGCGCGCGCAGTGCAACCAGCAACGGCGCATCCGCCTGCTTCGC KNACPATVAPVCGSDASTYSNECELPRAaCNPPRRlRLLR
601 134
CAAGGGCCATGTGGGTCCCGGGACCCCTGTGCCAACGTGACCTGCAGTTTCGGTAGTACCTGTGTACCTTCGGCTGACGGACAGAClGCCTCATGTCTGTGTCCTACAACCTGClTCGGG OGPCGSRDPCANVTCSFGSTCVPSADGOTASCLCPTTCFG
721 174
GCCCCTGATGGCACAGTGTGTGGCAGTGACGGTGACTACCCTAGTGAATGCCAGCTGCTTAGTCATGCCTGTGCCAGCCAGGAGCACATCTTCAAGAAGTTCAATGGTCCTTGTGAT APDGTVCGSDGVDYPSECOLLStlACASOEHlFKKFNGPCD
841 214
CCCTGCCAGGGCAGCATGTCAGACCTGAACCACATTTGCCGTGTGAACCCACGTACACGGCACCCAGAAATGCTTCTGCGGCCTGAGAACTGCCCTGCCCAGCACACGCCTATCTGTGGA PCOGSNSDLNHlCRVNPRTRHPEMLLRPENCPAOHTPlCG
961 254
GATGATGGGGTCACCTATGAAAACGACTGTGTCATGAGCCGTATAGGTGCAACCCGTGGCCTGCTTCTCCAGAAAGTACGCTCTGGTCAATGCCAAACTCGAGACCAGTGCCCGGAGACC DDGVTYENDCVHSRlGATRGLLLOKVRSGOCOTRDOCPET
1081 294
TGCCAGTTTAACTCCGTATGCCTGTCCCGCCGTGGCCGTCCCCACTGTTCCTGCGATCGTGTCACCTGTGATGGGTCTTACAGGCCTGTGTGTGCCCAAGATGGGCACACATACAACAAT COFNSVCLSRRGRPHCSCDRVTCDGSYRPVCAODGHTYNN
1201 334
GACTGTTGGCGCCAACAGGCTGAGTGTCGACAACAGCGGGCCATTCCTCCCAAGCACCAGGGCCCGTGTGACCAGACCCCGTCCCCATGCCATGGAGTGCAATGTGCATTTGGGGCAGTA DCUROOAECROORAIPPKHOGPCDOTPSPCHGVOCAFGAV
1321 374
TGCACAGTGAAGAACGGGAAAGCCGAGTGCGAGTGCCAGCGGGTGTGCTCCGGCATCTACGATCCTGTGTGCGGCAGTGACGGTGTCACTTACGGCAGTGTGTGCGAGCTGGAATCCATG CTVKNGKAECECORVCSGlYDPVCGSDGVTYGSVCELESM
1441 414
GCCTGTACCCTTGGGCGGGAAATCCAAGTGGCCCGCAGAGGACCCTGTGACCCATGTGGGCAGTGCCGTTTTGGATCCCTGTGCGAGGTGGAAACTGGACGCTGTGTGTGCCCCTCTGAG ACTLGRElOVARRGPCDPCGOCRFGSLCEVETGRCVCPSE
1561 454
TGTGTGGAGTCAGCCCAGCCCGTATGCGGTTCTGATGGACACACATACGCTAGTGAATGTGAGCTGCATGTCCACGCCTGTACGCACCAGATCAGCCTATACGTGGCCTCAGCTGGACAC CVESAOPVCGSDGHTYASECELtiVHACTHOlSLYVASAGH
1681 494
TGCCAGACCTGTGGAGAAAAAGTTTGTACTTTTGGGGCTGTGTGCTCAGCTGGACAGTGTGTATGTCCCCGTTGTGAGCACCCTCCACCTGGCCCTGTGTGTGGCAGTGATGGTGTCACC COTCGEKVCTFGAVCSAGOCVCPRCEHPPPGPVCGSDGVT
1801 534
TACCTCAGTGCCTGTGAGCTACGCGAAGCTGCCTGTCAGCAGCAGGTACAAATTGAGGAGGCCCATGCAGGGCCATGTGAGCCGGCTGAGTGTGGCTCAGGGGGCTCTGGTTCTGGGGAG YLSACELREAACOOOVO1EEAHAGPCEPAECGSGGSGSGE
1921 574
GACGACGAGTGTGAACAGGAGCTATGCCGGCAGCGTGGTGGTATCTGGGACGAGGACTCAGAAGATGGGCCATGTGTCTGTGACTTTAGCTGCCAGAGTGTCCCTAGAAGCCCAGTGTGl DDECEOELCRORGGIUDEDSEOGPCVCDFSCPSVPRSPVCDFSCOSVPRSPVC
2041 614
GGCTCGGATGGAGTCACCTATGGCACCGAGTGTGATCTGAAGAAGGCCAGGTGCGAATCACAGCAAGAACTGTACGTTGCTGCTCAGGGAGCCTGCCGTGGCCCTACCTTGGCTCCACTG GSDGVTYGTECDLKKARCESOOELYVAAOGACRGPTLAPL
2161 654
CTACCTGTGGCCTTCCCACACTGTGCCCAAACCCCCTATGGCTGCTGCCAGGACAATTTCACTGCTGCCCAGGGTGTGGGCTTGGCTGGCTGTCCCAGCACCTGCCATTGCAACCCCCAT LPVAFPHCAOTPYGCCODNFTAAOGVGLAGCPSTCHCNPH
2281 694
GGCTCCTACAGTGGCACTTGTGACCCAGCCACAGGGCAGTGCTCCTGCCGACCAGGTGTAGGAGGCCTCAGGTGTGATCGCTGTGAACCTGGCTTCTGGAACTTCCGTGGCATCGTCACC GSYSGTCDPATGOCSCRPGVGGLRCDRCEPGFUNFRGIVT
2401 734
GATGGACATAGTGGTTGCACTCCCTGCAGCTGCGACCCTCGGGGTGCCGTAAGGGATGACTGTGAACAGATGACTGGATTGTGTTCCTGTAGGCCTGGTGTGGCTGGTCCCAAGTGTGGA DGHSGCTPCSCDPRGAVRDDCEOMTGLCSCRPGVAGPKCG
2521 774
CAGTGTCCAGATGGTCAAGTCCTGGGCCATCTAGGCTGTGAAGCAGATCCCATGACACCTGTGACTTGTGTGGAGATACACTGCGAGTTCGGGGCCTCCTGCGTAGAGAAGGCTGGTTTT OCPDGOVLGHLGCEADPMTPVTCVEKAGF
2641 814
GCCCAATGTATCTGCCCAACCCTCACATGTCCAGAGGCTAACTCTACCAAGGTCTGCGGATCAGACGGTGTGACATATGGCAATGAATGCCAGCTGAAGGCCATTGCCTGCCGCCAGCGT AOCICPTLTCPEANSTKVCGSDGVTYGNECOLKA~ACROR
2761 a54
CTGGACATCTCCACTCAGAGTCTTGGTCCCTGCCAGGAGAGTGTTACTCCTGGGGCTTCCCCAACATCTGCATCTATGACTACCCCAAGGCATATCCTGAGCAAGACGCTGCCATTTCCC LDlSTOSLGPCOESVTPGASPTSASHTTPRHlLSKTLPFP
2aal a94
CACAACAGCCTTCCTCTGTCTCCCGGCAGTACTACCCATGATTGGCCCACCCCATTACCCATATCACCTCACACCACAGTCAGCATCCCCAGAAGCACCGCGTGGCCTGTGCTGACCGTG HNSLPLSPGSTTHDUPTPLPISPHTTVSIPRSTAUPVLTV
3001 934
CCCCCTACAGCAGCAGCCTCTGACGTAACCAGTCTCGCAACATCAATCTTCAGTGAATCCGGCAGCGCCAATGGGAGTGGCGACGAGGAACTGAGTGGAGATGAGGAGGCCAGTGGGGGC PPTAAASDVTSLATSIFSESGSANGSGDEELSGDEEASGG
3121 974
GGGTCTGGGGGACTTGAGCCCCCGGTGGGCAGCATTGTGGTGACCCATGGGCCACCCATCGAGAGGGCTTCCTGCTACAACTCACCTTTGGGCTGCTGCTCAGATGGCAAGACACCCTCA GSGGLEPPVGSIVVTHGPPIERASCYNSPLGCCSDGKTPS
3241 1014
CTGGACTCCGAAGGCTCCAACTGTCCTGCTACCAAGGCATTCCAGGGCGTGCTGGAGCTTGAGGGGGTCGAGGGACAGGAACTGTTCTACACACCTGAGATGGCTGACCCCAAGTCAGAG LDSEGSNCPATKAFOGVLELEGVEGOELFYTPEMADPKSE
3361 1054
TTGTTTGGGGAGACCGCAAGGAGCATTGAAAGCACGCTGGACGACCTGTTCCGGAATTCAGACGTCAAGAAGGACTTCTGGAGTGTCCGCCTACGGGAACTGGGGCCTGGCAAGTTGGTC LFGETARSIESTLDDLFRNSDVKKDFUSVRLRELGPGKLV
3481 1094
CGCGCCATTGTGGACGTTCACTTTGACCCTACCACAGCCTTCCAGGCATCAGATGTGGGTCAGGCCTTGCTCCGACAGATCCAGGTATCTAGGCCATGGGCCCTGGCAGTGAGGAGGCCT RAIVDVHFDPTTAFOASDVGOALLROIOVSRPUALAVRRP
