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.
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