Molecular mapping of murine I region recombinants. II. Crossing over in the E beta gene is restricted to a 4.5 kb stretch of DNA that excludes the beta 1 exon.

The crossing over in five murine I-region recombinants (Is/Ik) was studied by restriction fragment length polymorphism analysis after Southern blot hybridization by using I-region-specific probes. These recombinants included three recently developed strains, B10.ASR1, B10.ASR11, and B10.ASR12; and two strains B10.S(9R) and B10.HTT derived earlier. Although these recombinants were reciprocal in haplotype orientation to the three recombinants we reported recently, these too crossed over within the same 7 kb stretch of DNA in the E beta gene. This 7 kb stretch of DNA included the 3' half of the first intron, the beta 1 exon, the second intron, and the beta 2 exon. A comparison of the cDNA sequences of the two parental E beta alleles revealed that although the beta 2 exons were identical, there were several nucleotide differences between the two beta 1 exons. This allowed us to determine the parental origin of the beta 1 exon in the recombinants at the level of transcription by using S1 nuclease mapping. Thus we were able to show that in each case the 3' portion of the first intron and the beta 1 exon were upstream from the site of crossover. All eight recombinants involving the k and s haplotypes now can be mapped within a 4.5 kb stretch of DNA, which includes only the beta 1-beta 2 intron and the beta 2 exon of the E beta gene. These findings imply that the I-E molecules expressed in these recombinants will probably have conserved sequences and therefore will exhibit identical I-E-restricted immune responses, despite the fact that crossing over could have occurred at different sites within the beta 1-beta 2 intron.