This request for an ADAMHA RSDA (Level II) describes a study of ethanol-induced changes in neuronal gene expression. Ethanol produces a well recognized spectrum of neurologic sequelae following acute and chronic exposure. The development of tolerance/dependence to this drug, together with the consequent neurodegenerative effects of chronic ethanol abuse pose a major public health concern. Recent studies have documented that ethanol can modulate the expression of specific gene products. This might be the ultimate mechanism in cellular adaptation to chronic ethanol exposure. This proposal describes a comprehensive biochemical/molecular genetic approach to the study of ethanol effects on gene expression in the NG108-15 glioma x neuroblastoma cell line. This system has been well characterized as a model system for studying the biochemistry of ethanol effects on neurons. The ultimate goal of this study is the identification, isolation and characterization of ethanol responsive genes (ERGs). Initial experiments will extend current studies with Northern blot analysis of specific marker genes that have been shown to respond (induction and inhibition) to chronic ethanol. The effects of ethanol on NG108-15 cell mRNA will also be studied by 2-dimensional gel analysis of in vitro translation products. These studies will then be extended to include a detailed analysis of protein synthesis in cells exposed to acute or chronic ethanol. Finally, molecular cloning will be used to isolate ERGs. Differential and/or subtractive hybridization screening will be used to select genes that are preferentially modulated by ethanol. Biochemical purification of ERGs will also be used as a method for cloning these genes. The identification and isolation of ERGs may provide powerful molecular tools for the study of ethanol tolerance/dependency both in experimental models and in humans. The cloning of specific ERGs may also enable a molecular study of the genetics of alcoholism. This award will allow considerable professional growth on part of the principal investigator through the research plan outlined above as well as by participation in a well established, multi-disciplinary center dedicated to the study of alcoholism and alcohol-related neurologic disease. The resources of UCSF and the Dept. of Neurology will also provide ready access to many excellent leaders of academic medicine and basic research in a variety of areas. The opportunity to spend nearly all of my time in basic research activities will allow important progress toward my development as a molecular neurobiologist interested in alcoholism and will be an invaluable aid toward my career in academic medicine.
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