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A.9 The isolation of putative major histocompatibility complex gene fragments from dogfish and nurse shark
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The Scientific and Social Program of the W h ISDCI Congress
A.9 THE ISOLATION OF PUTATIVE MAJOR HISTOCOMPATIBILTY GENE FRAGMENTS FROM DOGFISH AND NURSE SHARK
COMPLEX
Simona Bartl, Irving L. Weissman University of North Carolina, Department of Biological Sciences, Wilmington, NC 28403, USA, and Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA We designed three highly-degenerate oligonucleotides to the membrane proximal domains of class I and class II genes to use as primers in the polymerase chain reaction (PCR). We isolated ten PCR products containing open-reading frames through both primers and exhibiting similarity to MHC genes. Surprisingly, no two identical clones were found. When nucleotide sequences of several clones were used to search GenBank, the sequences with greatest similarity encoded MHC molecules. Since we found eight nurse shark clones which potentially contained MHC gene fragments, it is possible that the nurse shark may have several MHC pseudogenes. The other two PCR products may represent non-MHC immunoglobulin gene superfamily members. Since MHC class I and dass llc~ and /3 homologues have been isolated from shark, it remains to be determined which MHC molecules are encoded by these gene fragments. One of them may represent a ~2microglobulin gene fragment. We are presently using these PCR products to screen a shark cDNA library.
A.10 ISOLATION, GENETICS, HEAVY CHAIN AND
AND STRUCTURAL B2-MICROBLOBULIN
ANALYSIS OF MHC FROM RAINBOW
CLASS I TROUT
(ONCORHYNCHUS MYKISS) Benny P. Shum, Kaoru Azumi, Peter R. Parham Departments of Cell Biology and Microbiology & Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA Here we report the isolation of cDNA encoding Major Histocompatibility (MHC) class 1 heavy chains and B~-microglobulin (B2M) from rainbow trout. Using primers derived from sequences conserved in other species, rainbow trout spleen cDNA was successfully amplified by polymerase chain reaction (PCR) to give DNA fragments homologous to the MHC classs I heavy chain t~3 domain and to I'~-microglobulin (fl2M). The deduced amino-acid sequence of a 199 bp product is 33% identical to that of a salmon class I heavy chain cDNA (Grimholt et al., Immunogenetics 37: 469-473, 1993). As a probe, this 199 bp product detected a - 1 . 4 Kb band on a Northern blot of spleen poly-A mRNA. Upon restriction fragment length polymorphism analysis (RFLP), this probe hybridized to 3 - 5 Hind III- or Taq I-digested genomic DNA fragments from each of 10 outbred individuals examined, and all bands appeared polymorphic within this population. Our preliminary data suggest that the rainbow trout genome contains a smaller classical class I heavy chain gene family than its mammalian counterpart, with around two polymorphic loci per haploid MHC. The deduced amino-acid sequence of a 118 bp PCR product is 68% identical with that of a zebrafish ll2M cDNA (Ono et al., Immunogenetics 38: 1-10, 1993). With these PCR-amplified products as probes, we have isolated two potentially full-length class I heavy chain cDNA clones ( - 1.3 Kb) and three 1~2M clones ( - 1 Kb) from a spleen cDNA library. We are sequencing these clones to identify conserved features of the MHC class I molecule throughout evolution and the nature of class I polymorphism in rainbow trout.
The Scientific and Social Program of the W h ISDCI Congress
A.9 THE ISOLATION OF PUTATIVE MAJOR HISTOCOMPATIBILTY GENE FRAGMENTS FROM DOGFISH AND NURSE SHARK
COMPLEX
Simona Bartl, Irving L. Weissman University of North Carolina, Department of Biological Sciences, Wilmington, NC 28403, USA, and Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA We designed three highly-degenerate oligonucleotides to the membrane proximal domains of class I and class II genes to use as primers in the polymerase chain reaction (PCR). We isolated ten PCR products containing open-reading frames through both primers and exhibiting similarity to MHC genes. Surprisingly, no two identical clones were found. When nucleotide sequences of several clones were used to search GenBank, the sequences with greatest similarity encoded MHC molecules. Since we found eight nurse shark clones which potentially contained MHC gene fragments, it is possible that the nurse shark may have several MHC pseudogenes. The other two PCR products may represent non-MHC immunoglobulin gene superfamily members. Since MHC class I and dass llc~ and /3 homologues have been isolated from shark, it remains to be determined which MHC molecules are encoded by these gene fragments. One of them may represent a ~2microglobulin gene fragment. We are presently using these PCR products to screen a shark cDNA library.
A.10 ISOLATION, GENETICS, HEAVY CHAIN AND
AND STRUCTURAL B2-MICROBLOBULIN
ANALYSIS OF MHC FROM RAINBOW
CLASS I TROUT
(ONCORHYNCHUS MYKISS) Benny P. Shum, Kaoru Azumi, Peter R. Parham Departments of Cell Biology and Microbiology & Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA Here we report the isolation of cDNA encoding Major Histocompatibility (MHC) class 1 heavy chains and B~-microglobulin (B2M) from rainbow trout. Using primers derived from sequences conserved in other species, rainbow trout spleen cDNA was successfully amplified by polymerase chain reaction (PCR) to give DNA fragments homologous to the MHC classs I heavy chain t~3 domain and to I'~-microglobulin (fl2M). The deduced amino-acid sequence of a 199 bp product is 33% identical to that of a salmon class I heavy chain cDNA (Grimholt et al., Immunogenetics 37: 469-473, 1993). As a probe, this 199 bp product detected a - 1 . 4 Kb band on a Northern blot of spleen poly-A mRNA. Upon restriction fragment length polymorphism analysis (RFLP), this probe hybridized to 3 - 5 Hind III- or Taq I-digested genomic DNA fragments from each of 10 outbred individuals examined, and all bands appeared polymorphic within this population. Our preliminary data suggest that the rainbow trout genome contains a smaller classical class I heavy chain gene family than its mammalian counterpart, with around two polymorphic loci per haploid MHC. The deduced amino-acid sequence of a 118 bp PCR product is 68% identical with that of a zebrafish ll2M cDNA (Ono et al., Immunogenetics 38: 1-10, 1993). With these PCR-amplified products as probes, we have isolated two potentially full-length class I heavy chain cDNA clones ( - 1.3 Kb) and three 1~2M clones ( - 1 Kb) from a spleen cDNA library. We are sequencing these clones to identify conserved features of the MHC class I molecule throughout evolution and the nature of class I polymorphism in rainbow trout.