Pilot 10D developed from the ongoing Center grant in which genetic mapping allowed identification of the chromosome location of individual genes (Quantitative Trait Loci, or QTLs) responsible for genetic differences in ethanol (alcohol) responses in mice. QTLs involved in a variety of ethanol responses were mapped to mouse Chromosome 4. These include definitively mapped QTLs for conditioned taste aversion, preference drinking, and Chromosome 4. These include definitively mapped QTLs for conditioned taste aversion, preference drinking, and acute withdrawal (using the criteria of Lander and Kruglyak, 1995), and a suggestive QTL for chronic withdrawal.
The specific aims of Pilot 10D are to identify and test candidate genes for these QTLs as a first step toward elucidating the functional mechanism by which the underlying gene(s) influences one or more of these behavioral responses to ethanol. In the very new future, approximately 99% of all genes will be identified and physically mapped, at least underlying a QTL is rapidly becoming a matter of testing candidate genes (Rikke and Johnson, 1998). Knowing what genetic markers define the QTL interval indicates what genes are in the region (section C.2), and testing candidate genes is the pressing issue. Pilot 10 will perform two kinds of candidate gene tests. First, we will test for coding variation between appropriate animals (e.g., between the C57BL/6J (B6) and DBA/2J (D2) alleles, or between Withdrawal Seizure-Prone (WSP) and -Resistant (WSR) strains for chronic ethanol withdrawal QTL candidate genes). DNA sequencing will be used to identify coding sequence differences between the B6 and D2 alleles (or other comparisons) to determine if sequence differences result in a change in protein sequence of the gene product (many sequence differences may be functionally silent by occurring in the non-coding region or occurring in the coding region but causing no change in amino acid sequence). Second, in situ hybridization will be used to test for expression differences between appropriate animals [e.g., between interval specific congenic strains (ISCS) vs. the appropriate background strain]. Pilot Component 10D directly addresses the major Center theme, to identify specific genes underlying alcohol responses. I anticipate that the data generated in this component will be used to apply for a new RO1 grant in which I will examine whether there is a genetic relationship between a gene difference and a phenotype(s) of interest (e.g., using recombinant inbred), and whether a gene difference is causally linked to a QTL (e.g., using allele substitution in """"""""rescued"""""""" transgenics). Pilot 10D is related to other Components in its emphasis on identifying the genes underlying ethanol response QTLs. Pilot 10D will be active in two years of Center support.
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