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Structural Determination and Promoter Analysis of the Chicken Mitogen-Inducible Prostaglandin G/H Synthase Gene and Genetic Mapping of the Murine Homolog
Ist Teil von
Archives of biochemistry and biophysics, 1993-01, Vol.300 (1), p.247-252
Ort / Verlag
San Diego, CA: Elsevier Inc
Erscheinungsjahr
1993
Link zum Volltext
Quelle
Elsevier Journal Backfiles on ScienceDirect (DFG Nationallizenzen)
Beschreibungen/Notizen
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-l and murine PGHS-2 genes. The transcription start site was determined by primer extension, and the nucleotide sequence of 1.6kb 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-κ13, and several SP- 1 and AP-2 sites. Chloramphenicol acetyltransferase AT) 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.