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A.P4 Characterization of beta-2-microglobulin expression from two teleost species
The Scientific and Social Program of the V1'h ISDCI Congress
$47
A.P3 THE MOLECULAR
ONTOGENY
OF 7.8S AND 5.7S IgY IN THE DUCK
(ANAS
PLATYRHYNCHOS) Yuki Bando l, Kathy E. Magor 2, Gregory W. Warr 2, David A. Higgins 1 IDepartment of Pathology, University of Hong Kong, Queen Mary Hospital, Hong Kong, and 2Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425-2211, USA The duck produces two secreted forms of IgY: a larger form (7.8S, containing 4 heavy chain constant region domains) and a truncated form (5.7S, containing only CH1 and CH2 domains) structurally and antigenically resembling an F(ab')2 fragment of the larger. The two heavy chains are produced by alternate splicing of the transcript from a single gene to give rise to two separate RNA messages. During the immune response 7.8S IgY antibodies precede the 5.7S lgY, which subsequently becomes the major antibody produced but which lacks the effector functions associated with the Fc. We have studied, by Northern blot analysis of mRNA, the production of 7.8S and 5.7S IgY in the lymphoid organs of ducks during embryonic and post-hatching ontogeny and, in immunologically mature birds, their occurrence following antigenic stimulation. The results show that all lymphoid organs produce both forms of IgY but, since the relative amounts produced vary greatly from organ to organ, their production is either not interlinked, or is subjected to different control mechanisms in the different organs.
A.P4 CHARACTERIZATION TELEOST SPECIES
OF BETA-2-MICROGLOBULIN
EXPRESSION FROM TWO
Bill Pohajdak 1, Brian Dixon 1, Saskia H.M. van Erp 2, Pedro N.S. Rodrigues 2, Ren6 J.M. Stet2 ~Dept. of Biology, Dalhousie University, Halifax, Nova Scotia B3H 4J1, Canada, and 2Dept. of Experimental Animal Morphology and Cell Biology, Wageningen Agricultural University, Wageningen, The Netherlands Using degenerate PCR primers based on published beta-2-microglobulin (b2m) sequences we were able to clone a portion of the gene from tilapia (Oreochromisniloticus L., Orni-B2m). These primers were used to clone a full length b2m cDNA from carp (Cyprinus carpio L., Cyca-B2m). The carp sequence showed 50% identity (17 of 43 substitutions conserved) to rabbit b2m. Both sequences showed strong similarities to all previously published vertebrate b2m sequences. Results from Northern analysis indicated messages of approximately 800-1000 bases long, which corresponds to the previously published lengths of b2m mRNAs. Southern blotting indicated that both Cyca-B2m and Orni-B2m were encoded by a single copy gene. Phylogenetic analysis indicated that both sequences were related to the b2m of higher vertebrates, but they grouped together in an ancestral position. A 20 aa peptide synthesized to match Orni-B2m and a 73 aa recombinant protein produced in E. coli, using the Cyca-B2m cDNA clone in the pRSET vector, were used to inoculate rabbits. The resultant sera recognized cell surface proteins of peripheral blood lymphocytes, erythrocytes, and cells from thymus, spleen and head kidney in a FACS analysis. There was, however, no cross reactivity between the sera. Western analysis and immunoprecipitations using these sera is being carried out.
$47
A.P3 THE MOLECULAR
ONTOGENY
OF 7.8S AND 5.7S IgY IN THE DUCK
(ANAS
PLATYRHYNCHOS) Yuki Bando l, Kathy E. Magor 2, Gregory W. Warr 2, David A. Higgins 1 IDepartment of Pathology, University of Hong Kong, Queen Mary Hospital, Hong Kong, and 2Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425-2211, USA The duck produces two secreted forms of IgY: a larger form (7.8S, containing 4 heavy chain constant region domains) and a truncated form (5.7S, containing only CH1 and CH2 domains) structurally and antigenically resembling an F(ab')2 fragment of the larger. The two heavy chains are produced by alternate splicing of the transcript from a single gene to give rise to two separate RNA messages. During the immune response 7.8S IgY antibodies precede the 5.7S lgY, which subsequently becomes the major antibody produced but which lacks the effector functions associated with the Fc. We have studied, by Northern blot analysis of mRNA, the production of 7.8S and 5.7S IgY in the lymphoid organs of ducks during embryonic and post-hatching ontogeny and, in immunologically mature birds, their occurrence following antigenic stimulation. The results show that all lymphoid organs produce both forms of IgY but, since the relative amounts produced vary greatly from organ to organ, their production is either not interlinked, or is subjected to different control mechanisms in the different organs.
A.P4 CHARACTERIZATION TELEOST SPECIES
OF BETA-2-MICROGLOBULIN
EXPRESSION FROM TWO
Bill Pohajdak 1, Brian Dixon 1, Saskia H.M. van Erp 2, Pedro N.S. Rodrigues 2, Ren6 J.M. Stet2 ~Dept. of Biology, Dalhousie University, Halifax, Nova Scotia B3H 4J1, Canada, and 2Dept. of Experimental Animal Morphology and Cell Biology, Wageningen Agricultural University, Wageningen, The Netherlands Using degenerate PCR primers based on published beta-2-microglobulin (b2m) sequences we were able to clone a portion of the gene from tilapia (Oreochromisniloticus L., Orni-B2m). These primers were used to clone a full length b2m cDNA from carp (Cyprinus carpio L., Cyca-B2m). The carp sequence showed 50% identity (17 of 43 substitutions conserved) to rabbit b2m. Both sequences showed strong similarities to all previously published vertebrate b2m sequences. Results from Northern analysis indicated messages of approximately 800-1000 bases long, which corresponds to the previously published lengths of b2m mRNAs. Southern blotting indicated that both Cyca-B2m and Orni-B2m were encoded by a single copy gene. Phylogenetic analysis indicated that both sequences were related to the b2m of higher vertebrates, but they grouped together in an ancestral position. A 20 aa peptide synthesized to match Orni-B2m and a 73 aa recombinant protein produced in E. coli, using the Cyca-B2m cDNA clone in the pRSET vector, were used to inoculate rabbits. The resultant sera recognized cell surface proteins of peripheral blood lymphocytes, erythrocytes, and cells from thymus, spleen and head kidney in a FACS analysis. There was, however, no cross reactivity between the sera. Western analysis and immunoprecipitations using these sera is being carried out.