| Summary: | Currently little is known about the mechanisms and locations of
lymphocyte development in teleosts. In this study several aspects of the
underlying factors which govern B lymphocyte development in trout
were investigated which included: the isolation and characterization of
immunoglobulin heavy chain (IgH) genes, the recombination activating
genes 1 and 2 (RAG1 and RAG2) and the use of cellular markers to
identify tissues harboring precursor B-cells.
Immunoglobulin heavy chains are part of the structural
components which make up antibody molecules produced by B-cells.
We isolated various full-length IgH cDNA clones, some of which
contained the secreted while others contained the membrane bound
form of IgH. Upon characterization of the membrane bound forms,
typical features common to all IgH cDNAs were found including a
leader peptide, a variable region and constant domain containing
transmembrane (TM) segments as well. Further sequence analysis of
this region revealed that the TM domains were spliced directly to the
CH3 domains which results in the loss of the entire CH4 region. Our
results support previous observations of unusual splicing events in fish
IgH genes.
RAG1 and -2 in mammals have been shown to be essential for
carrying out V (D) J recombination of lymphocyte receptors and are
found to be expressed within primary lymphoid tissues and precursor
lymphocytes. We isolated the RAG locus from a rainbow trout genomic
library and characterized their conservation and expression.
Overall the complete amino acid sequences of RAG1 and RAG2
displayed 78% and 75% similarity when compared to RAG genes from
higher vertebrates thus demonstrating the highly conserved nature of
these genes. Tissue specific expression of both genes was primarily
associated with the thymus and pronephros in both juvenile and adult
trout. Based upon these observation we conclude that the thymus and
pronephros likely serve as the tissue sites for V (D) J recombination in
trout and are thus primary lymphoid organs.
Finally we addressed the question as to where B-cell
lymphopoiesis occurs in trout. Our results using both
immunofluorescence and confocal microscopy putatively demonstrate
that the thymus harbors precursor B-cells and thus alludes to a dual
function for both B and T-cell development in trout. === Graduation date: 1996
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