ISOLATING THE SCID MOUSE DNA REPAIR GENE

描述（改编自申请人的摘要）：本申请的目的 建议是对小鼠重症的鉴定和表征 联合免疫缺陷（SCID）基因。 携带scid突变的小鼠， SCID小鼠缺乏双链DNA修复，代表了 罕见的DNA修复系统哺乳动物模型。 SCID小鼠 由于DNA缺失，成熟淋巴细胞严重缺乏 抗原受体基因的重组。 这些老鼠被证明是 对研究淋巴细胞的个体发育和建立淋巴细胞模型有重要价值。 传染病疫苗研发。 仔细的分析 SCID基因在疾病和健康中的功能是明确的， 但由于未能成功分离出该基因而受到阻碍。 该建议概述了一种通过定位分离scid基因的方法。 克隆。 这一方法尚未直接应用于识别 scid基因，但已成功地用于分离几个 重要的人类疾病基因。 申请人已经建立了基因组的高分辨率连锁图， 基于对近300个包含scid基因座的区域的分析， 回交后代 申请人已经确定了三个分子标记 不与scid表型重组，因此， 与scid基因相近 申请人已使用这些标记物， 筛选小鼠和人酵母人工染色体（YAC）文库 并鉴定了多个DNA克隆 这项建议的第一个目标是确定一个YAC克隆， 含有scid基因。 这将通过使用替代 接近。 申请人有一个可以测定YAC的系统 直接用于它们在体外补充SCID缺陷的能力。 如果没有 通过该测定，在含有scid的YAC中， 分离以增加基因组覆盖率。 这些YAC将用于 生成包含scid的基因组区域的物理图谱 基因座 该地区的物理地图将被延长，直到重组 交叉位于SCID基因座两侧的点。 一旦实现了这一点， 申请人将确信scid基因座包含在 YAC克隆的连续集合。 必须包含scid基因座的YAC将 用于通过与cDNA文库杂交来鉴定表达的基因， 北方印迹和CpG岛分析。 人类YAC将 用于鉴定人和小鼠之间保守序列的区域， 很可能是候选基因。 一旦scid基因被分离出来， 与其他蛋白质的同源性以及在正常发育和 DNA损伤的反应。 此外，scid的同系物将 从其他物种中分离出来进行比较和进化分析。

DESCRIPTION (Adapted from the applicant's abstract): The aim of this proposal is the identification and characterization of the mouse severe combined immunodeficiency (scid) gene. Mice carrying the scid mutation, SCID mice, are deficient in double strand DNA repair, representing one of the rare viable mammalian models of a DNA repair system. SCID mice are profoundly deficient in mature lymphocytes due to failure of DNA recombination of the antigen receptor genes. The mice have proven to be invaluable for studies of lymphoid ontogeny and establishment of models of infectious diseases for vaccine development. Detailed analysis of the function of the scid gene in disease and in health is clearly warranted, but has been hindered by the lack of success trying to isolate the gene. This proposal outlines an approach to isolate the scid gene by positional cloning. This approach has not been directly applied to the identification of the scid gene, but has been used successfully to isolate several important human disease genes. The applicant has established a high resolution linkage map of the genomic region containing the scid locus based on the analysis of nearly 300 backcross progeny. The applicant has identified three molecular markers that do not recombine with the scid phenotype and, therefore, must be physically close to the scid gene. The applicant has used these markers to screen several mouse and human Yeast Artificial Chromosome (YAC) libraries and has identified multiple DNA clones. The first goal of this proposal will be to identify a YAC clone that contains the scid gene. This will be achieved using alternative approaches. The applicant has a system by which YACs can be assayed directly for their ability to complement the scid defect in vitro. If none of the YACs contain scid by this assay, additional overlapping YACs will be isolated to increase the genomic coverage. These YACs will be used to generate a physical map of the region of the genome containing the scid locus. The physical map of the region will be extended until recombination points, which flank the scid locus, are crossed. Once this is achieved, the applicant will be confident that the scid locus is contained within the contiguous set of YAC clones. YACs that must contain the scid locus will be used to identify expressed genes by hybridization to cDNA libraries and Northern blots, and analysis for CpG islands. Homologous human YACs will be used to identify regions of conserved sequence between human and mouse, which are likely to be candidate genes. Once the scid gene is isolated, it will be characterized for sequence homology to other proteins and the expression in normal development and in response to DNA damage. In addition, the homologues of scid will be isolated from other species for comparative and evolutionary analysis.