CHARACTERISTICS OF A MURINE MHC HOTSPOT OF RECOMBINATION

小鼠的主要组织相容性复合体（MHC）由以下组成： 17号染色体上一系列紧密连锁的基因座， 控制细胞膜和血清糖蛋白参与 免疫系统的重要功能。 终止于 这一领域的大部分并不是以随机的方式发生的，而是在 称为重组"热点"的离散区域。 主要 本建议的重点是研究一个这样的地区的具体地点， 重组，特别是发生在第二内含子中的重组 E-β基因。 这项研究的总体目标是增加 我们对参与的分子机制的了解， 真核生物减数分裂重组 这项拨款申请提出 描述位于E-β内的关键核苷酸序列 重组区，这是必要的交叉事件。 已查明几个涉嫌参与的领域。 这些包括反转录子序列，一段中间重复序列， 与整合/切除序列高度同源的DNA (Ins)小鼠多瘤病毒，以及含有 四聚体重复AGGC。 该项目将由 使用质粒/内含子构建体的体外测定。 两 每个领域的相同模块被怀疑是重要的， 重组过程将被克隆到pUC质粒中， 这种方式允许分子内 重组产生截短的质粒。 的构建体 也将被设计成指示基因 （lacZ）将从重组质粒中切除。 这将 作为一个方便的标记来区分 非重组的构建体和其中重组的过程 发生了分子内重组。 检测试剂盒包含 构建DNA将在睾丸细胞存在下进行 提取物，然后转化和铺板。 的相对 lacZ-至lacZ+菌落的频率将作为 重组效率指标。 这项研究将导致 为了更清楚地了解必要的信号， 位点特异性重组。

The major histocompatibility complex (MHC) of the mouse consists of a series of closely] linked loci on chromosome 17 which control cell membrane and serum glycoproteins involved in important functions of the immune system. Recombination over much of this area does not occur in a random manner, but in discrete areas termed "hotspots" of recombination. The major focus of this proposal is to study one such area of site-specific recombination, specifically that occurring in the second intron of the E-beta gene. The overall goal of this research is to add to our knowledge of the molecular mechanisms involved in eukaryote meiotic recombination. This grant application proposes to describe key nucleotide sequences located within the E-beta recombination area that are necessary for the cross-over event. Several areas of suspected involvement have been identified. These include retroposon sequence, a section of middle repetitive DNA with strong homology to the integration/excision sequence (Ins) of the mouse polyoma virus, and an area containing the tetrameric repeat AGGC. This project will be accomplished by the use of an In Vitro assay using plasmid/intron constructs. Two identical modules of each area suspected of being important in the recombination process will be cloned into pUC plasmids in such a manner allowing for the possibility of intramolecular recombination resulting in truncated plasmids. The constructs will also be designed in such a manner that an indicator gene (lacZ) will be excised out of the recombined plasmid. This will serve as a convenient marker to distinguish between non-recombined constructs and those in which a process of intramolecular recombination has taken place. Assays containing construct DNA will be carried out in the presence of testis cell extracts followed by transformation and plating. The relative frequency of lacZ- to lacZ+ colonies will then serve as an indicator of recombination efficiency. This research will lead to a clearer understanding as to the signals necessary for site-specific recombination.