| Budget Amount *help |
¥7,600,000 (Direct Cost: ¥7,600,000)
Fiscal Year 1997: ¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1996: ¥4,600,000 (Direct Cost: ¥4,600,000)
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| Research Abstract |
The gene encoding the rat V1a arginine vasopressin (AVP) receptor was isolated, and its structural organization and 5' -flanking region were characterized. In addition, the complete cDNA sequence of the major transcript of the rat V1a receptor gene was determined. Southern blots demonstrated a single copy of the V1a receptor gene in the rat genome, spanning a region of 3.8 kilobases (kb) and consisting of two exons and one intron (1.8kb). The location of the intron was unique among G protein-coupled receptor genes in that the first exon encodes six of the seven transmembrane regions, the seventh region being encoded by the second exon. Primer extension, RNase protection, and rapid amplification of the 5' -end of the cDNA identified three transcriptional initiation sites (-405, -243, and -237), the major transcription initiation sites being mapped to prositions -243 and -237 base pairs (bp) upstream of the ATG initiation codon (+1 bp). This portion of the 5'-flanking region has neither
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a TATA nor a CCAAT box, is GC-rich but has no GC box motif, and has features of promoters seen in housekeeping genes. Chimeras containing 2.2 kb of the 5'-flanking region and deletion analyses using the chloramphenicol acetyltransferase gene indicated that a "minimal" regiion, exhibiting promoter activity and tissue specificity, is located between nucleotides -296 and -221, when transfected into vascular smooth muscle cells. Gel mobility shift assay and Southwestern blotting suggested that -3O-and -28-kDa nuclear proteins specifically bind to this region. Rapid amplification of the 3' -end of the cDNA showed that the major transcript terminates 442 bp downstream of the stop codon, in agreement with the mRNA size (2.1 kb). This study demonstrated a distinctive feature in the structural organization of the AVP-oxytocin receptor family genes, and characterization of the 5'-flanking re-gion reported here will lead to a better understanding of the mechanism of transcriptional regulation of the rat V1a AVP receptor gene. Less
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