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Cloning and characterization of the MHC Class I and II genes from atlantic salmon (Salmo salar)
S38
The Scientific & Social Program: Vth ISDCI Congress
A3 C L O N I N G AND C H A R A C T E R I Z A T I O N OF THE MHC CLASS I AND GENES F R O M A T L A N T I C S A L M O N (SALMO SALAR). V.M. Fossel, 2, I. H o r d v i k 2, O. B e r g e r s e n I, C. E n d r e s e n 2 & O.Lie I. iNorwegian College of V e r t e r i n a r y Medicine, Oslo, Norway; L a b o r a t o r y of B i o t e c h n o l o g y , U n i v e r s i t y of Bergen, Norway.
II
2
U n t i l r e c e n t l y there was no d i r e c t e v i d e n c e for the p r e s e n c e of the major histocompatibility complex (MHC) in t e l e o s t s . We h a v e now i s o l a t e d a n d c h a r a c t e r i z e d c D N A c l o n e s of the MHC Class I and II genes f r o m A t l a n t i c salmon. Initially, " M H C - c D N A " was s y n t h e s i z e d from t o t a l R N A p r e p a r e d f r o m salmon lymphocytes. The i n t e r n a l prim e r u s e d was b a s e d on a h i g h l y c o n s e r v e d m o t i f in the m e m b r a n e p r o x i m a l d o m a i n of the v e r t e b r a t e M H C - m o l e c u l e s . S u b s e q u e n t l y , the M H C - p r o b e (120bp) was p r e p a r e d by PCR, u s i n g the same a n t i - s e n s e p r i m e r in c o m b i n a t i o n w i t h a s e n s e - p r i m e r h o m o l o g o u s to a n o t h e r c o n s e r v e d motif. The 120bp P C R p r o d u c t was u s e d as probe to s c r e e n a eDNA library from salmon lymphocytes. Several positive clones were isolated, s u b c l o n e d and sequenced. One of the p o s i t i v e clones (pVMFI8) h a d high s e q u e n c e s i m i l a r i t y w i t h the MHC C l a s s I I 5-gene, e s p e c i a l l y the carp TLA-II gene. A n o t h e r clone (pVMF23) was shown to have h i g h s e q u e n c e s i m i l a r i t y with a MHC Class I gene. The 1.2 kb insert of the Class II cDNA was l a b e l l e d and e m p l o y e d as a probe to s c r e e n a g e n o m i c salmon l i b r a r y (EMBL3). Several p o s i t i v e clones were i s o l a t e d and two of these have b e e n d i a g n o s e d as MHC Class II clones and are c u r r e n t l y b e i n g s u b j e c t e d to d e t a i l e d analyses.
A4 POLYMORPHISM OF MhcCyca GENES 1N THE COMMON CARP, Cyprim~xcarpio L. "R.J.M. 5let, A.G. Regelink, G.T. Hermsen, E.M. van Schothorst, S.H.M. van Erp & E. Egbcrts Department of Experimental Animal Morphology and Cell Biology, Agricultural University, P.O. Box 338, 6700 AH Wageningen, The Netherlands Recently, two DNA sequences from carp representing a putative MHC class la genc (Cyca-Z) and a class lib gene (Cyca-YB) have been described (Hashimoto et al., 1990, PNAS 87: 6863). In this study, the KI and Kll probes, homologous to the Cyca-Z exon 3 and a Cyca-YB exon 2 DNA sequence, respectively, have been used to investigate the degree of restriction fragment length polymorphism (RFLP) in lines of carp of different geographical origin. High relative mass DNA samples were obtained from Dutch and lsraelian second generation meiotic gynogenetic families (W and A, respectively), Polish (R3) and Hungarian (R8) inbred lines. mitotic gynogenetic carp clones (E), and an outbred population (WK) of Dutch origin. So far, Southern blot analy~cs of six individuals from the A, W, E, R3 and R8 lines have revealed 15 Kll-positive Pst I fragments, of which 3 are shared by all carp investigated. Hybridization of Eco RI digesled DNA with the KIt probe identified a total of 9 fragments one of which is shared. Differences in RFLPs were observed between each of the lines investigated, but not within the lines, with the exception of the outbrcd carp line WK where several distinct RFLPs could be identified. Serologically and/or cellularly defined MHC haplotypes arc ncccessary to assess the validity of molecular genotyping. While limited data indicate that within the gynogenetic lines mixed leucocyte rcactivities cannot bc observed, in agreement with the absence of polymorphic fragments identified by KIt, positive responses have been obtained in the R3 and R8 inbred lines with individuals not showing KII-RFLPs. With respect to class I polymorphism, as defined by Kl-specific RFLPs, serological typing of carp siblings with alloantisera identifying allelic specificities of a histocompatibility locus might provide an additional means of establishing a correlation with molecular typing. Finally, a carp Agt11 eDNA expression library has been screened with the KII probe to identify the Cyca polymorphism at the protein level. At least one clone (HKII-3B) expressed a 13-galactosidase fusion protein with a relative molecular weight compatible to the length of a Cyca-YB chain.
The Scientific & Social Program: Vth ISDCI Congress
A3 C L O N I N G AND C H A R A C T E R I Z A T I O N OF THE MHC CLASS I AND GENES F R O M A T L A N T I C S A L M O N (SALMO SALAR). V.M. Fossel, 2, I. H o r d v i k 2, O. B e r g e r s e n I, C. E n d r e s e n 2 & O.Lie I. iNorwegian College of V e r t e r i n a r y Medicine, Oslo, Norway; L a b o r a t o r y of B i o t e c h n o l o g y , U n i v e r s i t y of Bergen, Norway.
II
2
U n t i l r e c e n t l y there was no d i r e c t e v i d e n c e for the p r e s e n c e of the major histocompatibility complex (MHC) in t e l e o s t s . We h a v e now i s o l a t e d a n d c h a r a c t e r i z e d c D N A c l o n e s of the MHC Class I and II genes f r o m A t l a n t i c salmon. Initially, " M H C - c D N A " was s y n t h e s i z e d from t o t a l R N A p r e p a r e d f r o m salmon lymphocytes. The i n t e r n a l prim e r u s e d was b a s e d on a h i g h l y c o n s e r v e d m o t i f in the m e m b r a n e p r o x i m a l d o m a i n of the v e r t e b r a t e M H C - m o l e c u l e s . S u b s e q u e n t l y , the M H C - p r o b e (120bp) was p r e p a r e d by PCR, u s i n g the same a n t i - s e n s e p r i m e r in c o m b i n a t i o n w i t h a s e n s e - p r i m e r h o m o l o g o u s to a n o t h e r c o n s e r v e d motif. The 120bp P C R p r o d u c t was u s e d as probe to s c r e e n a eDNA library from salmon lymphocytes. Several positive clones were isolated, s u b c l o n e d and sequenced. One of the p o s i t i v e clones (pVMFI8) h a d high s e q u e n c e s i m i l a r i t y w i t h the MHC C l a s s I I 5-gene, e s p e c i a l l y the carp TLA-II gene. A n o t h e r clone (pVMF23) was shown to have h i g h s e q u e n c e s i m i l a r i t y with a MHC Class I gene. The 1.2 kb insert of the Class II cDNA was l a b e l l e d and e m p l o y e d as a probe to s c r e e n a g e n o m i c salmon l i b r a r y (EMBL3). Several p o s i t i v e clones were i s o l a t e d and two of these have b e e n d i a g n o s e d as MHC Class II clones and are c u r r e n t l y b e i n g s u b j e c t e d to d e t a i l e d analyses.
A4 POLYMORPHISM OF MhcCyca GENES 1N THE COMMON CARP, Cyprim~xcarpio L. "R.J.M. 5let, A.G. Regelink, G.T. Hermsen, E.M. van Schothorst, S.H.M. van Erp & E. Egbcrts Department of Experimental Animal Morphology and Cell Biology, Agricultural University, P.O. Box 338, 6700 AH Wageningen, The Netherlands Recently, two DNA sequences from carp representing a putative MHC class la genc (Cyca-Z) and a class lib gene (Cyca-YB) have been described (Hashimoto et al., 1990, PNAS 87: 6863). In this study, the KI and Kll probes, homologous to the Cyca-Z exon 3 and a Cyca-YB exon 2 DNA sequence, respectively, have been used to investigate the degree of restriction fragment length polymorphism (RFLP) in lines of carp of different geographical origin. High relative mass DNA samples were obtained from Dutch and lsraelian second generation meiotic gynogenetic families (W and A, respectively), Polish (R3) and Hungarian (R8) inbred lines. mitotic gynogenetic carp clones (E), and an outbred population (WK) of Dutch origin. So far, Southern blot analy~cs of six individuals from the A, W, E, R3 and R8 lines have revealed 15 Kll-positive Pst I fragments, of which 3 are shared by all carp investigated. Hybridization of Eco RI digesled DNA with the KIt probe identified a total of 9 fragments one of which is shared. Differences in RFLPs were observed between each of the lines investigated, but not within the lines, with the exception of the outbrcd carp line WK where several distinct RFLPs could be identified. Serologically and/or cellularly defined MHC haplotypes arc ncccessary to assess the validity of molecular genotyping. While limited data indicate that within the gynogenetic lines mixed leucocyte rcactivities cannot bc observed, in agreement with the absence of polymorphic fragments identified by KIt, positive responses have been obtained in the R3 and R8 inbred lines with individuals not showing KII-RFLPs. With respect to class I polymorphism, as defined by Kl-specific RFLPs, serological typing of carp siblings with alloantisera identifying allelic specificities of a histocompatibility locus might provide an additional means of establishing a correlation with molecular typing. Finally, a carp Agt11 eDNA expression library has been screened with the KII probe to identify the Cyca polymorphism at the protein level. At least one clone (HKII-3B) expressed a 13-galactosidase fusion protein with a relative molecular weight compatible to the length of a Cyca-YB chain.