Studies on the promoter region of the c-Ha-ras gene in FRTL5 rat thyroid cells

The functional activity of the promoter region of the rat c-Ha-ras gene was examined in FRTL5 rat thyroid cells, the cell type from which this promoter was cloned. A plasmid (p035-ras-CAT) was constructed containing the untranslated-1 exon as well as 172 base pairs (bp)5' to this exon inserted upstream of the chloramphenicol acetyl transferase (CAT) reporter gene. These 172 bp of 5'-flanking region contain two 10 bp GC box consensus sites and two CAAT boxes. Very weak promoter activity was observed in experiments involving transient transfection of FRTL5 cells with this plasmid, as well as with another plasmid (p5kb-ras-CAT) containing a much more extensive (3.5 kb) 5'-flanking region of the gene. In contrast, strong promoter activity was observed when the same plasmids were transfected into mouse 3T3 fibroblasts. When other promoters (pfos, RSV, and MMTV) were used to drive CAT activity, CAT activity in FRTL5 cells was about 10-fold less than in NIH-3T3 cells and rat embryo fibroblasts. However c-Ha-ras promoter activity was reduced out of proportion in FRTL5 thyroid cells relative to the other cell types (approximately 50-fold less). DNA gel-shift assays performed using crude extracts of FRTL5 and 3T3 nuclear proteins revealed quantitatively similar binding to the same promoter region in the c-Ha-ras 5'-flanking sequence. These data demonstrate that promoter activity of the rat c-Ha-ras gene is contained within the 172 bp 5'-flanking region of the gene. This promoter activity is expressed at a much lower level in slow-growing FRTL5 cells relative to other more rapidly growing cell types.     

Genomic organization and chromosomal location of the mouse vasoactive intestinal polypeptide 1 (VPAC1) receptor.

The gene encoding the mouse vasoactive intestinal polypeptide type 1 (VPAC1) receptor was cloned, and its structural organization was determined. The gene (Vipr1) is more than 16 kb in length and is divided into 13 exons. The 5'-flanking region is highly GC-rich and lacks an apparent TATA box, but contains a CCAAT box, three potential Sp1-binding sites, and two potential AP-2-binding sites. Promoter analysis of the 5'-flanking region of Vipr1 using a luciferase gene reporter system revealed that the isolated 5'-flanking region has functional promoter activity. The mouse Vipr1 gene is encoded by a single gene, which was mapped to the distal region of mouse chromosome 9. This region is syntenic with human chromosome 3p, where the human VPAC1 receptor gene has been mapped.