In FY 92, the Genetic Pharmacology Unit focused on the transcriptional regulation of genes encoding dopamine receptors, BDNF and POMC: 1. Transcriptional regulation of the D-1A dopamine receptor gene. A human genomic clone having the 5' flanking region of the D-1A gene was isolated and sequenced during FY 91. In FY 92, this gene was found to have a small intron in its 5' untranslated region; the true exon 1 was identified and transcription found to start at multiple points. The transcriptional activity of several deletion mutants of the D-1A promoter region was determined by subcloning them in a CAT expression vector. Strong enhancer and silencer regions were identified. Footprinting and gel shift assays suggested that AP2 in NS20Y nuclear extract binds to an AP2 consensus sequence in the promoter of this gene. No Sp1 binding was seen although the D-1A promoter has multiple consensus sequences for Sp1. We concluded that the D-1A gene, like """"""""housekeeping"""""""" genes, lacks a TATA box but is tissue-specific and highly regulated. 2. Promoter analysis of the D2 dopamine receptor gene. The rat genomic clone encoding the D2 receptor, which was isolated in FY 91, was further characterized in FY 92. Although transcription starts at multiple points, a clear preference is noted for three consecutive nucleotides located in an """"""""initiator""""""""-like sequence. Promoter analysis revealed an enhancer region harboring a functional Sp1 binding site and a silencer region having an Sp1 consensus sequence where Sp1 in NB41A3 nuclear extract binds only very weakly. In addition, sequences in exon 1 are found to contribute to the transcriptional regulation of this gene and to its cell-specific expression. We concluded that the D2 gene lacks a TATA box but appears to have a functional """"""""initiator"""""""" element. 3. Analysis of the 5' flanking region of the D3 receptor gene. This project was initiated during the last quarter of FY92. A D3 specific cDNA has been amplified from rat olfactory tubercle and several candidate clones harboring its 5' extent isolated. 4. Genetic regulation of the rat BDNF gene. Experiments to clone the gene for brain derived neurotrophic factor were begun during the last quarter of FY 92. To date, 18 candidate clones harboring the most upstream portion of this transcript have been isolated and are being sequenced. 5. Regulation of POMC gene transcription. The previously identified -137 to -106 region of this promoter was found to have significant enhancer activity when fused upstream of the SV40 promoter.
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