JP4 Immunoglobulin VH-genes of the Atlantic salmon (salmo salar L.): Definition of eight VH-families

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Abstracts of the 7th Congress of the ISDCI: Session J

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MOLECULAR CHARACTERIZATION OF Q-LIKE Ig-ASSOCIATED MOLECULES IN THE HAGFISH S.vnn Rumfell, Martin Flajnik Department Medicine, Miami, FL 33 136. USA

of Microbiology

and Immunology,

University

AND of Miami

School

of

Immunoglobulin (Ig) and J chains have been clearly identified in all jawed vertebrates as primordial as cartilaginous fish such as the nurse shark. Ancestors of the jawless vertebrates, the hagfish being one of two extant species, predate the jawed species by 100 million years. These primitive, ancient animals have a complement cascade (at least C3); however, to date no hallmark molecules of the adaptive immune system such as Ig have been found. In a continuing search for the evolutionary origins of Ig-like molecules we have identified by Western analysis several hagfish proteins that cross react with antisera specific for the C-terminus of nurse shark IgM and evolutionarily conserved regions of the J chain. These hagfish proteins recognized by the IgMspecific antisera are disulfide linked proteins forming several sizes of multimers with variation between individuals, and correlate approximately to the molecular size of bona fide Ig. Protein and gene identification was undertaken by immunoscreening a hagfish white blood cell and gill cDNA library with these antisera. In addition, PCR screening the same library or cDNA from various tissues using degenerate J chain primers based on regions conserved from sharks to mammals has been undertaken.

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IMMUNOGLOBULIN V,-GENES OF THE ATLANTIC SALMON (Saho salar L.): DEFINITION OF EIGHT V,-FAMILIES. Stein T. Solem’, Darlene L. Middleton’, Gregory W. War?, Jan E. Killie’ and Trend 0. J@rgenxn’ 1) Department of Marine Biochemistry, Norwegian College of Fishery Science, University of Tromse, Norway 2) Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, USA

In order to understand the basic mechanisms of the so-called < of antibody responsesin teleost fish we decided to study transcription of immunoglobulin genes in Atlantic salmon related to this phenomenon, Such studies requires gene-specific probes and we therefore cloned and sequenced genes coding the variable region of the heavy chain of immunoglobulins. The main purpose of the cloning was to define the possible Vu-gene families of the Atlantic salmon and then syntesiseV,-family spesific probes to be used as markers of antigen specificities in studies of antibody resonsesto haptenated antigenes. From the head kidney of Atlantic salmon mRNA were isolated and an oligo-dT primed cDNA library was constructed. The library was screened with a mix of three PCR-generated variable region probes. 100 clones were isolated and sequenced. Based upon sequence analysis and comparisons, these clones were sorted into eight different Vn-families. V,-genes in some of these families were highly related (85-94% similarity) to Vu-families of the rainbow trout, while others demonstrated a much lower homology (63% similarity) to this species. Currently, we are estimating the approximate number of members within each Vu-family. and also analysing the heterogeneity of the third complementarity-determining region (CDR3) in the Atlantic salmon Vregion.