MAPPING AND STRUCTURAL ANALYSIS OF MOUSE OBESITY GENES

小鼠肥胖基因的定位和结构分析

• 批准号: 3245560
• 负责人: FREDERICK T FIEDOREK
• 金额: $ 12.56万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3245560
• 关键词: DNA; chromosome walking; chromosomes; disease /disorder model; gene deletion mutation; gene mutation; gene rearrangement; genetic disorder; genetic library; genetic mapping; genetic markers; human genetic material tag; hybrid cells; laboratory mouse; linkage mapping; molecular cloning; nucleic acid probes; nucleic acid structure; obesity; phenotype; pulsed field gel electrophoresis; restriction fragment length polymorphism; southern blotting

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.

尽管有证据表明，人类肥胖的趋势是 遗传和喜欢是多基因的性质，个别基因， 导致这种神经内分泌倾向的原因在很大程度上是未知的。 表现出肥胖表型的几个突变代表了目标 对于所谓的“反向遗传”方法， 或有关缺陷和寻求的生物学性质的假设 最终仅根据染色体位置来识别基因。 该项目的具体目标是（1）隔离额外的匿名 与db基因座紧密连锁的DNA标记，（2）提供更详细的 小鼠db基因位点遗传连锁图的解析 4号染色体，（3）推导db基因座的物理图谱，（4） 检查db基因座和其他两个常染色体突变基因座， 肥胖小鼠的基因组DNA结构的证据 异常，和（5）开始研究，最终分离这些 肥胖基因 明确的方法将包括隔离 来自噬菌体λ基因组文库的匿名DNA标记物，所述噬菌体λ基因组文库是从 含有小鼠4号染色体作为其唯一小鼠来源的杂交细胞系 遗传物质，删除和作图分析的小组， 辐射减少的杂交细胞系，种间限制性片段长度多态性分析 和重组近交系小鼠杂交，并分离和分析非常 脉冲场凝胶电泳（PFGE）检测大DNA分子， 印迹 基因组DNA制备（包括琼脂糖塞中的样品 适合于PFGE分析）的小鼠组织和细胞系的细胞。 与世隔绝 传统DNA Southern印迹和遗传连锁 将进行实验以提供更多的探针和映射 旨在缩小对小鼠4号染色体的关注， DB基因 PGFE实验的印迹转移将与 已知各种小鼠和人类DNA探针与 db突变和其他肥胖突变。 审查 这些大规模的Southern印迹将集中在（1）提供一个物理 db基因座附近突变复合体的遗传图谱，以及（2） 鉴定来自db/db小鼠的DNA中的潜在结构异常， 在小鼠4号染色体上的db基因座附近定位的突变体，以及其他 单基因肥胖突变体映射到不同但可接近的小鼠 染色体位点 长期目标是最终确定 导致这些遗传性常染色体肥胖综合症的基因。 这项拨款申请具有医学意义，因为任何见解 获得这些小鼠突变可能有助于识别小鼠中的基因， 人类中的潜在同源物有助于遗传倾向， 发展成肥胖症。