Structural determination and promoter analysis of the chicken mitogen-inducible prostaglandin G/H synthase gene and genetic mapping of the murine homolog.
We have previously reported the isolation and characterization of a new form (PGHS-2) of prostaglandin G/H synthase (PGHS, cyclooxygenase) from chicken embryo fibroblasts. To further study the regulation and structure of the gene, we have cloned the entire chicken PGHS-2 (previously termed miPGHSch) gene with its 5'-flanking region from a chicken genomic library. A genomic Southern blot showed the existence of a single PGHS-2 gene. The size of the gene was estimated at 8.9 kb through DNA sequencing and polymerase chain reaction analysis. The PGHS-2 gene was found to contain 10 exons, giving it a structure similar to that of the human PGHS-1 and murine PGHS-2 genes. The transcription start site was determined by primer extension, and the nucleotide sequence of 1.6 kb of the 5'-flanking region immediately upstream of the transcription start site was determined. The promoter sequence contained a TATA box and a variety of enhancer elements, including a serum response element, an AP-1, an NF-kappa B, and several SP-1 and AP-2 sites. Chloramphenicol acetyltransferase (CAT) assays showed that the first 158 nucleotides of the promoter efficiently drove transcription of the CAT reporter gene in serum-stimulated cells. Dexamethasone, a potent inhibitor of prostaglandin synthesis, had no effect on CAT activity, although this drug is known to markedly decrease PGHS-2 mRNA in vivo. This suggests that dexamethasone may inhibit PGHS-2 mRNA expression at the post-transcriptional level. Analysis of hamster/mouse somatic cell hybrids with radiolabeled cDNA probes demonstrated that PGHS-1 mapped to chromosome 2 and PGHS-2 mapped to chromosome 1 of the mouse genome.
Xie
W
W
Department of Chemistry, Brigham Young University, Provo, Utah 84602.
Merrill
J R
JR
Bradshaw
W S
WS
Simmons
D L
DL
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002642:Chick Embryo; D002645:Chickens; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D002874:Chromosome Mapping; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D003907:Dexamethasone / Q000494:pharmacology; D004742:Enhancer Elements, Genetic; D005091:Exons; D005347:Fibroblasts / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D005822:Genetic Vectors; D015698:Genomic Library; D007438:Introns; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis / Q000235:genetics*; D012326:RNA Splicing; D011994:Recombinant Proteins / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D016385:TATA Box; D014157:Transcription Factors / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014157:Transcription Factors; D003907:Dexamethasone; D004247:DNA; D011451:Prostaglandin-Endoperoxide Synthases; D015500:Chloramphenicol O-Acetyltransferase