CHARACTERISTICS OF A MURINE MHC HOTSPOT OF RECOMBINATION

小鼠的主要组织相容性复合体(MHC)由 17号染色体上一系列紧密连锁的基因座 调控细胞膜和血清糖蛋白参与 免疫系统的重要功能。重组结束 这一领域的大部分并不是以随机的方式发生的，而是在 被称为重组“热点”的离散区域。少校 这项建议的重点是研究这样一个特定地点的领域 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 据我们所知，涉及到的分子机制 真核细胞减数分裂重组。这项拨款申请提出了 描述位于E-β内的关键核苷酸序列 交叉事件所必需的重组区域。 已经确定了几个涉嫌参与的领域。 这些序列包括逆转录子序列，一段中间重复的 与整合/切除序列有很强同源性的DNA 小鼠多瘤病毒的(INS)和包含 四聚体重复序列AGGC。这个项目将由 使用质粒/内含子构建的体外检测。二 怀疑重要的每个区域的相同模块 重组过程将被克隆到pUC质粒中 这种方式允许分子内 重组导致截短的质粒。这些构造 也将被设计成这样一种方式，即指示基因 (LacZ)将从重组质粒中被切除。这将是 作为一个方便的标记来区分 未重组的构造以及其中的过程 分子内重组已经发生。含有以下成分的化验 构建DNA将在睾丸细胞存在的情况下进行 提取物，然后转化和电镀。相对的 LacZ-to LacZ+菌落的频率将作为 重组效率的指标。这项研究将引领 为了更清楚地了解 特定部位重组。

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.