GENE IDENTIFICATION FOR ALCOHOL RELATED QTLS

描述：(改编自调查人员摘要)这一目标 应用于快速确定小鼠和大鼠QTL的潜在基因 与酒精行为有关。一种高通量的DNA自动测序 我们实验室已经建立了一套设备，并将应用于 努力。与酒精相关的QTL的作图和其他信息将 由合作实验室提供，这些实验室被资助产生这样的 QTL，它们的共同工作涵盖了与酒精相关的广泛范围 表型。QTL区域中的候选基因/EST将使用 快速扩展的人类基因/EST序列和图谱数据库， 小鼠和大鼠，以及这些基因组之间已知的同线关系。 为了识别突变，每个QTL区域内的候选基因/EST将是 使用基于快速聚合酶链式反应的方法从相关的小鼠或大鼠中进行测序 我们的实验室开发、验证并目前正在使用。这个平台 技术将能够执行超过10,000个序列读取每个 每年允许对250个候选基因进行直接序列比较 年(相当于每年调查5个QTL的所有可能的候选基因)。 因为人类、小鼠和大鼠身上新的基因数据非常丰富 由EST/cDNA测序和作图工作提供，这是主要的重点 应用程序将使用这些基于cDNA的数据来调查完整的 候选基因蛋白质编码区。排序的优先顺序为 根据几个标准给予候选基因，包括假定的 生物学作用和可能与酒精作用、MAP位置、组织的相关性 表达数据等。此外，已缩小到 相对较小的大小(例如1-2 cM)将优先于以下QTL 包含相当大的基因组间隔(例如&gt；10 cm)，尽管令人信服 这样大的QTL区域的候选基因也将被优先考虑。至 解决了基因调控中的突变变化的可能性 负责QTL，将对候选基因的表达进行检测 通过Northern杂交或定量聚合酶链式反应的方法。在QTL具有 被缩小到一个非常小的染色体区域，完成了对 将考虑基因组区域。确认一种突变是 对QTL负责的人将通过以下方式获得 相关动物数量较多，例如表型RIS、F2s和遗传 如果可用，并通过对不同的 等位基因。一旦基因差异被证明与一种 QTL，即可获得人类基因。这将为直接 在酗酒人群中检测该基因以探索其潜力 与酗酒有关。

DESCRIPTION: (Adapted from the Investigator's Abstract) The goal of this application is to rapidly identify genes underlying QTLs in mouse and rat related to alcohol action. A high throughput automated DNA sequencing facility has been established in our laboratory and will be applied to this effort. Mapping and other information regarding alcohol related QTLs will be provided by collaborating laboratories who are funded to generate such QTLs, and whose work together encompasses a broad range of alcohol-related phenotypes. Candidate genes/ESTs in QTL regions will be identified using the rapidly expanding gene/EST sequence and mapping databases for human, mouse and rat, and the known syntenic relationships between these genomes. To identify mutations, candidate genes/ESTs within each QTL region will be sequenced from relevant mice or rats using a rapid PCR-based protocol developed, validated and currently in use in our laboratory. This platform technology will be capable of carrying out over 10,000 sequence reads per year permitting the direct sequence comparisons of 250 candidate genes per year (the equivalent of surveying all likely candidate genes for 5 QTLs/yr). Because of the wealth of new gene data in human, mouse and rat that is being provided by EST/cDNA sequencing and mapping efforts, major focus of this application will be to use this cDNA-based data to survey the complete protein coding regions of candidate genes. Priority for sequencing will be given to candidate genes based on several criteria, including putative biological role and likely relevance to alcohol action, map location, tissue expression data, etc. In addition, QTLs that have been narrowed to relatively small sizes (e.g. 1-2 cM) will be given priority over QTLs that encompass rather large genomic intervals (e.g. >10 cM), although compelling candidate genes in such larger QTL regions will also be given priority. To address the possibility that mutational changes in the regulation of a gene are responsible for the QTL, expression of candidate genes will be assayed by Northern blot or quantitative PCR approaches. In cases where a QTL has been narrowed to a very small chromosomal region, complete sequencing of the genomic region will be given consideration. Confirmation that a mutation is responsible for a QTL will be obtained by following the mutation through larger numbers of relevant animals, e.g. phenotyped RIs, F2s and congenics where available, and by functional expression studies of the different alleles. Once a gene difference has been shown to be causally linked to a QTL, the human gene will be obtained. This will set the stage for direct testing of the gene in alcoholic populations to explore its potential relevance to alcoholism.