The research program described consists of two independent lines of investigation into the control of gene expression in nervous tissue. First, I propose to study the organization, structure, and control of gene activity for key enzymes in the catecholamine biosynthetic pathway. DNA complementary to mRNAs for tyrosine hydroxylase, dopamine-B-hydroxylase, and phenylethanolamine N-methyltransferase will be purified, and cloned in E. coli using the plasmid pBR322 as vector. Cloned cDNA fragments will be used as probes in molecular hybridization stucies designed to identify control mechanisms involved in the expression of the differentiated neuronal phenotype. Cloned cDNA will also be employed to screen a """"""""shotgun"""""""" cloned gene library for genomic DNA fragments containing TH, DBH, and PNMT coding sequences. Identificaton of the appropriate genomic fragments will permit detailed investigation into the organization and structure of catecholaminergic genes in question. The second project involves the identificaton of genes expressed specifically in cerebellum. These gene coding sequences will be identified by screening a """"""""brain-specific"""""""" cDNA library using radiolabeled mRNA from several major brain regions. Cloned cDNA fragments will be used as probes to study cerebellum-specific gene expression. Select developmentally regulated, region-specific cDNAs will be sequenced and peptides synthesized in vitro. Antibodies to these synthetic peptides will be raised in rabbits and used to locate the gene products in vivo using immunocytochemistry. For this aim, the applicant envision neuroanatomical study to be an area of scientific growth and development, which will be nurtured by the rapidly expanding neuroscience community at the applicant's new institutional affiliation. Successful completion of the proposed studies will (1) provide information on the organization and regulation of neurotransmitter enzyme gene expression, (2) yield much needed insight into the control of differentiated nerve cell function at the genetic level, and (3) begin to identify gene subsets involved in the development and maintenance of brain region-specific structure and function using cerebellum as a model system.
