Transgenic mice, constructed by microinjection of specifically tailored Igl-1 (Lambda 1) genes, will be utilized to 1) characterize the defect in Lambda 1 expression exhibited by SJL (and SJA) mice, and to analyze the effect that the expression of an already rearranged Lamba 1 gene has on rearrangement and expression of endogenous light chain isotypes. The coding region of the Igl-1 of SJL differs from that of BALB/c by one nucleotide in codon 155. This difference (GGT in BALB/c and GTT in SJL) results in the replacement of glycine by valine at this position in the Lambda 1 light chain. Genetic mapping has shown that the cis-acting element controlling the expression of Igl-1 in these strains is closely linked to Igl-1 locus. Experiments to directly test whether or not the cis acting element is the coding difference, gly to val, are planned. Vectors will be constructed which contain rearranged Igl-1 genes isolated from both RALB/c and SJA hybridomas. These clones will be altered specifically at codon 155 by oligonucleotide-directed mutagenesis. Transgenic mice will be constructed by microinjection of the RALB/c wild type (gly-155), BALb/c mutant (val-155), and SJA mutant (gly-155) vectors. Expression of these genes will be compared among transgenic recipients. Expression of a histone gene incorporated into the three injected clones will be used as a standard for evaluation of tissue-specific expression and integration-site-related differences. If the replacement of gly by valo is found to affect the expression of Igl-1, the physiological consequences of thes replacement will be analyzed. If expression is independent of this amino acid replacement, experiments will be initiated to reveal the cis-acting regulatory element. The rules governing allelic and isotype exclusion, in particular the nature of the STOP signal which precludes further rearrangements, will be clarified by studying isotype expression in individual B-cells from these transgenic mice. B-cell hybridomas will be made from BALB/c (gly-155) transgenic mice which express Lambda 1, and analyzed for both DNA rearrangements at the Ig loci and isotype expression (mRNA and protein). The question of whether Kappa rearrangements much precede expression of Lambda will also be answered.
