Quantitative trait loci (QTLs) are chromosomal regions containing genes (polygenes) that influence a quantitative (or complex) trait such as alcohol (ethanol) withdrawal severity. During the last four years of present R01 funding, we have mapped several QTLs that jointly have a major influence on the severity of alcohol withdrawal in populations derived from the C57BL/6 (B6) and DBA/2 (D2) inbred mouse strains. The three largest QTLs (LOD>4, p<10/-5) are on distal chromosome 1, mid chromosome 4 and proximal chromosome 11. Based on mouse-human linkage homology, the human counterparts of the three mouse QTLs map to human chromosomes 1q21- 32, 9p23, and 5q31-35, respectively. Using congenic strains developed to isolate each of the three QTLs against a uniform (inbred) genetic background, we propose to continue these studies toward the eventual identification of the genes that underly each QTL. To accomplish this, we propose the following. (1) Narrow the QTL interval down from our present approximately 20 cM to approximately 1 cM using interval specific congenic strains. (2) Mouse and human gene databases will be searched to find candidate genes that map within our 1 cM QTL interval. In the case of human databases, this will involve regions of known mouse-human linkage homology. (3) Promising candidate genes will be scanned for base pair differences between B6 and D2 genotypes by SSCP and tested for alcohol withdrawal regulated expression differences using Northern blot analysis. (4) When most brain mRNA transcripts for evidence of alcohol withdrawal gene regulation specific to individual QTL regions isolated in a congenic strain. Where such regulation is found, a cDNA reverse transcribed from the differentially regulated mRNA species can be sequenced and serve as a 3' tag for a specific gene or genes. This tag can be used to identify the gene by matching its sequence to those in mouse or human gene/EST databases developed as part of the Human Genome Project, or as a probe for full length cDNA cloning and sequencing. Finally, DD-PCR will also be used to detect differential regulation due to alcohol withdrawal without regard to genotype (QTL), for this will pick up additional genes not polymorphic in our mouse populations.
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