ISOLATING THE SCID MOUSE DNA REPAIR GENE

描述(改编自申请人的摘要)：本报告的目的 提出的是对老鼠的鉴定和刻画 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 DNA修复系统的罕见的可存活的哺乳动物模型。SCID小鼠是 成熟淋巴细胞由于DNA失效而严重缺乏 抗原受体基因的重组。这些老鼠已经被证明是 对淋巴系统个体发育的研究和动物模型的建立具有非常重要的价值 用于疫苗研发的传染病。详细分析了 SCID基因在疾病和健康中的作用是明确的， 但由于试图分离该基因的努力没有取得成功，这一努力受到了阻碍。 这项建议概述了一种分离SCID基因的方法，即通过定位 克隆。这种方法还没有直接应用于身份识别 但已经成功地用来分离出几个 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谱 基于对近300个SCID基因座区域的分析 回交后代。申请人已经鉴定出三个分子标记 不与SCID表型重新结合，因此必须是 在生理上接近SCID基因。申请人已使用这些标记来 几种小鼠和人酵母人工染色体文库的筛选 并鉴定了多个DNA克隆。 这项提案的第一个目标将是确定一个YAC克隆 含有SCID基因。这将使用替代方案来实现 接近了。申请者有一个可以用来检测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 通过这项检测，在含有SCID的YAC中，其他重叠的YAC将是 分离以增加基因组覆盖率。这些YAC将被用来 生成包含SCID的基因组区域的物理地图 轨迹。该地区的物理地图将被延长，直到重组 SCID轨迹两侧的点是交叉的。一旦实现这一点， 申请者将确信SCID基因座包含在 一组连续的YAC克隆。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基因 Northern blotts，以及对CPG岛屿的分析。同源人类YAC将 用于鉴定人类和小鼠之间的保守序列区域， 它们很可能是候选基因。 一旦分离出SCID基因，将对其进行序列鉴定 与其他蛋白质的同源性及其在正常发育和 对DNA损伤的反应。此外，SCID的同源物将是 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.