MAPPING AND ISOLATION OF THE DB GENE

尽管有强有力的证据表明， 人类是遗传的基因有助于这种神经内分泌 易感性尚不清楚。 本项目的目标是 构建一个大规模的物理地图， 在经典的肥胖小鼠模型中突变的单个基因， DB/DB鼠标 这是一个广泛的拟议项目，其中 偶然发现可能起着重要作用。 尽管如此， 可以利用系统和互补的方法， 做一个有针对性的尝试来鉴定这个重要的基因。 本研究采用两种载体系统克隆小鼠染色体 4来自小鼠/仓鼠杂交细胞系的DNA片段。 将构建粘粒文库以分离DNA片段 大小为20-50个酶和酵母人工染色体文库 采用华盛顿大学最近开发的一种系统 将构建分离DNA片段100-700个酶， 尺寸 从这些连续的克隆中， 将组装db突变周围区域的图谱 利用脉冲场凝胶电泳，间接末端标记 作图和与一组随机寡核苷酸杂交 probes. 随后，对衍生基因进行系统筛选， 将进行DB基因座的测序。 三种方法 将使用：1）在该克隆区域中搜索小缺失 小鼠4号染色体，2）寻找变异或缺失的mRNA 组织中的序列，如大脑（特别是下丘脑） 其是编码的异常DB的可能表达位点 基因产物，和3）从该克隆区域筛选序列 对于非甲基化的CpG二核苷酸簇， 发现发生在表达基因的位点。

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.