MAPPING AND STRUCTURAL ANALYSIS OF MOUSE OBESITY GENES

尽管有证据表明人类患肥胖症的趋势是 遗传并喜欢在本质上是多基因的个体基因 对这种神经内分泌易感性的影响在很大程度上是未知的。 肥胖表型的几个突变代表靶标 所谓的“反向遗传”方法，不需要任何知识 或关于缺陷的生物学性质的假设，并寻求 最终，仅根据其染色体位置来识别基因。 这个项目的具体目标是(1)隔离更多的匿名用户 与db基因座紧密连锁的dna标记，(2)提供更详细的 小鼠db基因座遗传连锁图谱的解析 4号染色体，(3)获得db基因座的物理图谱，(4)到 检查db基因座和另外两个常染色体突变基因座 在小鼠中寻找结构基因组DNA的证据 异常，以及(5)开始研究，最终分离出这些 肥胖基因。明确的方法将包括隔离 来自噬菌体Lambda基因组文库的匿名DNA标记 以小鼠4号染色体为唯一小鼠来源的杂交细胞系 一组遗传物质的缺失和作图分析 辐射减量杂交细胞系的种间RFLP分析 和重组近交系小鼠杂交，并进行了分离分析 脉冲场凝胶电泳(PFGE)检测大分子DNA 吸墨水。基因组DNA制备(包括琼脂糖塞中的样品 适合于PFGE分析)将从小鼠组织和细胞系中 与世隔绝。传统DNA Southern杂交与遗传连锁 将进行实验，以提供更多的探测和映射 旨在缩小对小鼠4号染色体的关注以识别 Db基因。PGFE实验的印迹转移将与 已知的各种小鼠和人类DNA探针与 小鼠的DB突变和其他肥胖突变。对…的考试 这些大规模的南方斑点将集中在(1)提供物理上的 Db基因座附近突变复合体的遗传图谱和(2) 从db/db小鼠身上发现潜在的DNA结构异常， 小鼠4号染色体db位点附近的突变定位等 定位到不同但可接近的小鼠的单基因肥胖突变 染色体基因座。长期目标是最终确定 导致这些遗传性常染色体肥胖综合征的基因。 这项拨款提案具有医学意义，因为任何见解 获得这些小鼠突变可能有助于识别小鼠和 人类中潜在的同源基因导致遗传倾向 患肥胖症。

Despite evidence that the tendency to develop obesity in humans is inherited and like to be polygenic in nature, the individual genes contributing to this neuroendocrine predisposition are largely unknown. Several mutations in exhibiting obesity phenotypes represent targets for the so-called "reverse genetic" approach which requires no knowledge or assumptions concerning the biological nature of the defect and seeks ultimately to identify genes based solely on their chromosomal location. The specific aims of this project are(1) to isolate additional anonymous DNA markers closely linked to the db locus, (2) provide more detailed resolution to the genetic linkage map of the db gene locus on mouse chromosome 4, (3) to derive the physical map of the db locus, (4) to examine the db locus and two other autosomal mutant gene loci that lead to obesity in the mouse for evidence of structural genomic DNA abnormalities, and (5) to begin studies ultimately to isolate these obesity genes. The explicit approaches will include the isolation of anonymous DNA markers from a phage lambda genomic library created from a hybrid cell line containing mouse chromosome 4 as its only mouse-derived genetic material, the deletion and mapping analysis of a panel of irradiation-reduced hybrid cell lines, RFLP analysis of interspecific and recombinant inbred mouse crosses, and separating and analyzing very large DNA molecules by pulsed field gel electrophoresis (PFGE) and blotting. Genomic DNA preparation (including samples in agarose plugs suitable for PFGE analysis) from mouse tissues and cell lines will be isolated. Coventional DNA Southern blots and genetic linkage experiments will be performed to provide more probes and mapping resources intended to narrow the focus on mouse chromosome 4 to identify the db gene. Blot transfers of PGFE experiments will be hybridized with various mouse and human DNA probes which are known to map closely to the db mutation and the other obesity mutations in mice. The examination of these large scale Southern blots will focus on (1) providing a physical genetic map for a complex of mutations near the db locus and (2) identifying potential structural abnormalities in DNA from db/db mice, mutants mapping near the db locus on mouse chromosome 4, and other single gene obesity mutants mapping to different but accessible mouse chromosomal loci. The long term objective are ultimately to identify the genes responsible for these genetic autosomal obesity syndromes. This grant proposal is of medical significance because any insights gained into these mouse mutations may help identify genes in mice and potential homologues in man contributing to the inherited tendency to develop obesity.