Construction of a partial rabbit spleen cDNA library and identification of immunoglobulin clones.

Abstract

A partial cDNA library was constructed from total poly A(+)-RNA isolated from the spleen of a rabbit (kappa allotype b5; heavy chain allotypes a3d12e15) that had been hyperimmunized with Streptococcus pneumoniae (type III). In spite of the absence of either specific DNA probes for rabbit immunoglobulin (Ig) sequences or cross-hybridizing mouse Ig DNA probes, recombinant clones containing cDNA sequences of rabbit gamma heavy chain and kappa light chains were identified by a combination of screening techniques: (a) colony hybridization using labeled mRNA; (b) mRNA hybridization selection and translation and (c) hybridization to electrophoretically fractionated poly A(+)-RNA ("Northern" analysis). Sequencing of three kappa light chain recombinant DNA sequences, including part of the 3' untranslated (UT) region, has confirmed the fact that recombinant DNA for kappa light chain mRNA has been identified. An unexpectedly high degree of homology between the 3' UT region sequence of this DNA from a rabbit of b5 allotype and the published 3' UT sequence from a b4 rabbit was found. It appears that 3' UT sequences from b4 and b5 alleles have diverged less than the coding sequences for the constant regions. The functional significance of this conservation of 3' UT sequences remains to be elucidated.