| Research Abstract |
In mammals, the murine major histocompatibility complex (MHC) is the only region where breakpoints of meiotic recombination are systematically studied at the molecular level. In this region, meiotic recombinations do not occur at random but are clustered in limited regions known as hotspots. Thus far, four hotspots have been identified in this region. The presence or absence of a hotspot on a certain DNA segment depends on the MHC haplotypes involved in genetic crosses. For example, the Eb hotspot is observed in crosses between standard laboratory haplotypes. In genetic crosses including cas3 and wm7 haplotypes which were derived from Asian wild mice, most of the recombinations occurred at the Lmp2 hotspot. Lmp2 and Eb hotspots have been well characterized at the molecular level. Sequences around these two hotspots were determined and the fine locations of the breakpoints were analyzed. Comparison of the sequences between Lmp2 and Eb hotspots revealed several molecular motifs commonly
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shared by the two hotspots.
In order to elucidate the roles of these motifs in recombinations at the hotspots and to understand the mechanism by which recombinations are restricted to hotspots, we have characterize other hotspots at the molecular level. Meiotic recombination takes place at a high frequency at a hotspot in the vicinity of the Pb gene, when the wild mouse derived cas4 haplotype is used in the genetic cross. Molecular characterization of this hotspot, designated Pb hotspot, has not yet been done. So far, we have obtained six independent recombinants at the Pb hotspot after screening six hundred mice generated from crosses between cas4 and wm7 haplotypes. The breakpoints of these recombinants were localized to a 15 kb of DNA fragment in the vicinity of the Pb gene. In this study, we attempted to make a more complete map of the breakpoints of the six recombinants. First, we constructed the restriction map of the 15 kb of DNA fragment including the breakpoints. Subsequently, we determined the parental origins of several short DNA segments consisting of the 15 kb of DNA fragment in the six recombinants, by examining polymorphisms between parental DNA segments through PCR-SSCP analysis. At a result, at least five recombinations were found to be confined to a 5 kb of DNA segment located proximal to the 3'end of the Pb gene.
We have determined nucleotide sequence around Pb hotspot. The result clearly indicated that the hotspot is located at 3'end of the Pb gene. Thus, it appeared that all four hotspots characterized in the mouse MHC are located at either 3'end or introns of genes, contrasting to the cases in budding yeast. The result suggests that molecular machinery operating in meiotic recombinations at the mouse hotspots is different from that in budding yeast. Less
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