MAPPING AND ISOLATION OF THE DB GENE

尽管有强有力的证据表明，中国的肥胖趋势 人类遗传了影响这种神经内分泌的基因 目前尚不清楚患者的易感性。这个项目的目标是 构建一张大比例的物理地图，并最终将 在经典的肥胖小鼠模型中发生突变的单个基因， Db/db鼠标。这是一个广泛的拟议项目，其中 机缘巧合可能会起到重要作用。尽管如此，有几个 可以利用系统的和互补的方法来 有针对性地尝试识别这个重要的基因。 将使用两种载体系统来克隆小鼠染色体 4来自小鼠/仓鼠杂交细胞系的DNA片段。 将构建一个粘粒文库来分离DNA片段 20-50kb大小的酵母人工染色体文库 使用华盛顿大学最近开发的系统 将被构建来分离DNA片段100-700千碱基 尺码。从这些连续的克隆中获得了详细的大规模基因 Db突变周围区域的地图将被组装起来 利用脉冲场凝胶电泳，间接末端标记 作图，并与一组随机寡核苷酸杂交 探测器。随后，对衍生基因进行了系统的筛选 将进行db基因座的序列测定。三种方法 将用于：1)在此克隆区域中搜索小的缺失 小鼠染色体4，2)寻找变异或缺失的mRNA 脑(特别是下丘脑)等组织中的序列 它们是编码的异常数据库的可能的表达位点 基因产物，以及3)从该克隆区域中筛选序列 对于非甲基化CpG二核苷酸簇 发现发生在表达基因的位置。

Despite strong evidence that the tendency towards obesity in humans is inherited the genes contributing to this neuroendocrine predisposition are not known. It is the goal of this project to construct a large scale physical map of and ultimately isolate the single gene that is mutated in a classical mouse model of obesity, the db/db mouse. This is an extensive proposed project in which serendipity may play an important role. Nonetheless, several systematic and complementary approaches can be utilized to make a directed attempt at identifying this important gene. Two vector systems will be employed to clone mouse chromosome 4 DNA fragments derived from a mouse/hamster hybrid cell line. A cosmid library will be constructed to isolate DNA fragments 20-50 kilobases in size and a yeast artificial chromosome library employing a system recently developed at Washington University will be constructed to isolate DNA fragments 100-700 kilobases in size. From these contiguous clones a detailed large scale genetic map of the region surrounding the db mutation will be assembled utilizing pulsed field gel electrophoresis, indirect end-label mapping, and hybridization with a panel of random oligonucleotide probes. Subsequently, a systematic screening of the derived gene sequences for the db locus wll be undertaken. Three approaches will be used: 1) searching for small deletions in this cloned region of mouse chromosome 4, 2) looking for variant or absent mRNA sequences in tissues such as brain (specifically hypothalamus) which are possible sites of expression of an abnormal db encoded gene product, and 3) screening sequences from this cloned region for clusters of non-methylated CpG dinucleotides which have been found to occur at the sites of expressed genes.