IDENTIFICATION OF GENES INDUCED OR REPRESSED BY CHRONIC ALCOHOL

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The proposed project takes a novel approach to identifying the genes of importance in modulating the severity of alcohol withdrawal in mice by identifying mRNAs expressed and regulated during chronic ethanol exposure. The underlying hypothesis is that changes in gene expression are likely to occur in the brain after chronic exposure to ethanol, and that any gene whose expression is affected by chronic ethanol is a good candidate for having a functionally important role in ethanol neuroadaptation. Genes regulated by chronic ethanol exposure might promote withdrawal risk, or expert protection against withdrawal. We propose to identify novel candidate genes of importance, without bias about potential roles, using the technique of differential display. Inbred WSP-2 mice will be made physically dependent by inhalation, and mRNA from the brains of exposed and control mice will be analyzed by differential display. Dependent and control RNAs will be hybridized to modified oligo-dT primers and reverse transcribed. The reverse transcribed cDNA sequences will be amplified by PCR with subsets of random primers, and the products labeled and separated on a DNA sequencing gel. By comparing the banding patterns for the different groups, bands with similar intensities in both the control and treated samples will be ignored, leaving a number of gene products differentially displayed (i.e., either up- or down-regulated) presumably as a result of an effect of ethanol on gene expression.