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.     

Activation of a heterologous promoter in response to dexamethasone and cadmium by metallothionein gene 5'-flanking DNA

Human metallothionein-IIA (hMT-IIA) gene expression is regulated by heavy metals and glucocorticoids. When the cloned hMT-IIA gene or its 5'-flanking DNA structure fused to herpes simplex virus thymidine kinase (HSV-TK) structural gene sequences were transferred into TK- Rat 2 fibroblasts, both genes were inducible by Cd++ and/or dexamethasone. Placement of the hMT-IIA gene 5'-flanking region, either intact of deleted in its TATA box and cap site, upstream of the HSV-TK gene promoter rendered the latter both glucocorticoid- and heavy metal-inducible. Thus the structure that mediates both Cd++ and glucocorticoid responsiveness is present in the hMT-IIA gene 5'-flanking DNA, does not require its TATA box or cap site, and can activate a heterologous promoter.     

The human colipase gene: isolation, chromosomal location, and tissue-specific expression.

The digestion of dietary triglycerides occurs in the duodenum through the action of triglyceride lipase, a pancreatic exocrine protein. The activity of pancreatic lipase is inhibited by the bile salts normally found in the gut lumen. Another pancreatic exocrine protein, colipase, restores the lipolytic activity of triglyceride lipase. The synthesis and secretion of both triglyceride lipase and colipase is increased by dietary fats and secretin. An increase in mRNA accompanies the increased activity, suggesting that the genes for triglyceride lipase and colipase contain nucleotide elements responsive to dietary fats or secretin or both. To study the regulation of colipase expression, we have first isolated the gene for human colipase from a cosmid library with a cDNA probe. The gene was localized to chromosome 6 and is organized into three exons contained in a single 3.3-kb BamHI fragment. The 5'-flanking region of the gene contains a TATA box, a GC box, and a 28-bp region with homology to the rat pancreatic-specific enhancer. This region directs the tissue-specific expression of the chloramphenicol acetyltransferase gene in a transfected rat pancreatic acinar cell line, AR42-J. The same construct is inactive in HEPG2, C2C12, and COS-1 cells. These results demonstrate that the isolated gene for human colipase contains tissue-specific promoter activity in the 5'-flanking DNA. The 28-bp region specifically binds to a factor in nuclear extracts.     

Structure and Characterization of a Gene for Light-Harvesting Chi a/b Binding Protein from Rice

The gene for the light-harvesting chlorophyll a/b binding proteinfrom rice was cloned and se-quenced. The clone contains a 798-bpcoding sequence, which is identical to that of a cDNA for typeI LHCPII (Matsuoka 1990), and its 5'- and 3'-flanking regions.The coding region of this gene is not interrupted by interveningsequences, as reported for type I genes from other plants. Inthe 5'-flanking region, typical TATA and CAAT boxes are located30 and 92 bp upstream from the capping site (positions –30and –92), respectively. A putative phytochrome-responsiveelement (AAGATAAGG) is located at position –65 betweenthe TATA and CAAT boxes. Comparison of sequences in the 5'-flankingregions between this gene and genes for LHCPII from other gramineousplants indicates that the rice sequence has no apparent homologyto that of wheat. However, the rice sequence is highly homologousto the maize sequence, not only around the TATA and CAAT boxesbut also in regions further upstream. To investigate the promoter activity of the 5'-flanking regionof the gene, a chimeric gene was constructed by fusing the 5'-flankingregion to the coding sequence for ß-glucuronidase(GUS), and this chimeric gene was introduced into tobacco. Thehighest activity of GUS was observed in leaf tissue, indicatingthat the 5'-flanking region of the gene can act as a promoterin an organ-specific manner in tobacco. Histochemical analysisin situ was also performed to determine where GUS activity wasexpressed. The highest activity was found in leaf mesophyllcells. High activity was also observed in the vascular systemof stems and petioles, and low activity was found in root tissue. (Received August 20, 1990; Accepted January 21, 1991)