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Isolation and cloning of β-2 microglobulin from the teleost fish, Oreochromis nilotica
The Scientific & Social Program: Vth ISDCI Congress
$39
A5 ISOLATION AND CLONING OF J3-2 MICROGLOBULIN FROM THE TELEOST FISH, OREOCHROMIS NILOTICA. B. Dixon*, G. Stuart and B. Pohajdak. Marine Gene Probe Laboratory, Dept. of Biology, Dalhousie University, Halifax, N. S., B3H 4J1. We have synthesized oligonucleotide primers corresponding to two conserved regions of the J3-2 microglobulin gene. These were used as primers in the polymerase chain reaction (PCR) amplification of the ~-2 microglobulin gene from Oreochromis nilotica. A fragment of the expected size 111bp was isolated, subcloned, and sequenced. Sequence homology analysis strongly indicates that this fragment codes for a portion of the !3-2 microglobulin gene (52.2% identity and 54% of remaining amino acids conserved at the amino acid level.) Northern analysis revealed a 0.8 kb transcript similar in size to the J3-2 microglobulin gene of mammalian species. Southern blot analysis has shown that the 111 bp fragment hybridizes to a single copy gene. We have recently cloned a full length cDNA clone and also a genomic clone containing the ~-2 microglobulin gene. Subcloning and sequencing of both of these clones is in progress.
A6 CHARACTERIZATION OF CHANNEL CATFISH ALIA)ANTIGENS AND THEIR ROLE(S) IN 3"HE IMMUNOLOGIC RECOGNITION OF ANTIGEN. A . N . Vallejo*, N . W . Miller & L. W. Clem.
Department of Microbiology, University of Mississippi Medical Center, Jackson, MS 3912.6-4505, U.S.A. Alloantigenic differences among individuals of the same species, including teleosts, have long been recognized. In channel catfLsh, such differences are exemplified by vigorous alloreactivities of lymphocytes in MLR experiments. We have recently described a method for developing long term catfish monocyte lines which not only produce IL-1 but can also serve as effective antigen presenting cells (APC) in the generation of secondary in vitro T-dependent antigen-specific immune responses by autologous responder lymphocytes. However, these APCs elicit only vigorous proliferation (i.e. MLR) but not specific antibody production by allogeneic responders. Studies were therefore conducted to explore further the mechanism of this putative immune restriction of antibody responses as well as alloreactivities among individual fish to the long term monocyte lines. A battery of alloantisera was generated against the cell lines. Immunofluoresence by flow cytometry revealed considerable specificity of each alloantiserum for the immunizing cell line. Further, the addition of homologous but not heterologous antisera to mixed cultures of cell line APC and autologous responders resulted in quantitative decreases in the in vitro proliferative responses. Results of immunoprecipitation experiments indicated that these alloantisera recognized membrane proteins (or complexes thereof) exhibiting relative molecular masses similar to known MHC molecules. Ongoing studies are aimed at characterizin~ these alloantigeus at both the structural and genetic levels. [Supported by NIH Grant 5-R37-AI-19530].
$39
A5 ISOLATION AND CLONING OF J3-2 MICROGLOBULIN FROM THE TELEOST FISH, OREOCHROMIS NILOTICA. B. Dixon*, G. Stuart and B. Pohajdak. Marine Gene Probe Laboratory, Dept. of Biology, Dalhousie University, Halifax, N. S., B3H 4J1. We have synthesized oligonucleotide primers corresponding to two conserved regions of the J3-2 microglobulin gene. These were used as primers in the polymerase chain reaction (PCR) amplification of the ~-2 microglobulin gene from Oreochromis nilotica. A fragment of the expected size 111bp was isolated, subcloned, and sequenced. Sequence homology analysis strongly indicates that this fragment codes for a portion of the !3-2 microglobulin gene (52.2% identity and 54% of remaining amino acids conserved at the amino acid level.) Northern analysis revealed a 0.8 kb transcript similar in size to the J3-2 microglobulin gene of mammalian species. Southern blot analysis has shown that the 111 bp fragment hybridizes to a single copy gene. We have recently cloned a full length cDNA clone and also a genomic clone containing the ~-2 microglobulin gene. Subcloning and sequencing of both of these clones is in progress.
A6 CHARACTERIZATION OF CHANNEL CATFISH ALIA)ANTIGENS AND THEIR ROLE(S) IN 3"HE IMMUNOLOGIC RECOGNITION OF ANTIGEN. A . N . Vallejo*, N . W . Miller & L. W. Clem.
Department of Microbiology, University of Mississippi Medical Center, Jackson, MS 3912.6-4505, U.S.A. Alloantigenic differences among individuals of the same species, including teleosts, have long been recognized. In channel catfLsh, such differences are exemplified by vigorous alloreactivities of lymphocytes in MLR experiments. We have recently described a method for developing long term catfish monocyte lines which not only produce IL-1 but can also serve as effective antigen presenting cells (APC) in the generation of secondary in vitro T-dependent antigen-specific immune responses by autologous responder lymphocytes. However, these APCs elicit only vigorous proliferation (i.e. MLR) but not specific antibody production by allogeneic responders. Studies were therefore conducted to explore further the mechanism of this putative immune restriction of antibody responses as well as alloreactivities among individual fish to the long term monocyte lines. A battery of alloantisera was generated against the cell lines. Immunofluoresence by flow cytometry revealed considerable specificity of each alloantiserum for the immunizing cell line. Further, the addition of homologous but not heterologous antisera to mixed cultures of cell line APC and autologous responders resulted in quantitative decreases in the in vitro proliferative responses. Results of immunoprecipitation experiments indicated that these alloantisera recognized membrane proteins (or complexes thereof) exhibiting relative molecular masses similar to known MHC molecules. Ongoing studies are aimed at characterizin~ these alloantigeus at both the structural and genetic levels. [Supported by NIH Grant 5-R37-AI-19530].