Prostaglandin G/H synthase catalyzes the conversion of arachidonic acid to prostaglandin endoperoxides G2 and H2, the first step in the synthesis of biologically active prostanoids. The major goal of the proposed research is to test the hypothesis that the PGG/H synthase gene is an immediate-early """"""""competence"""""""" gene, like c-fos and c-myc, whose activation is required for cell replication. This hypothesis is based on two observations: (a) that PGG/h synthase protein levels rise in 3T3 cells stimulated with platelet-derived growth factor (PDGF) at about the same time as the expression of c-myc, c-fos and other """"""""immediate-early"""""""" genes; and (b) that PGE2 is required for replication of PDGF-stimulated 3T3 cells. We have recently isolated and sequenced a full-length cDNA coding for the ovine PGG/H synthase. We will use the sheep cDNA as a probe for isolating a near full-length cDNA for the mouse PGG/H synthase (Specific Aim #1). We will then use the mouse cDNA with mouse 3T3 cells to determine if the PGG/H synthase gene has characteristics of an immediate-early gene. Specifically, with the mouse cDNA and an anti-PGG/H synthase IgG, which is already available, we propose:
Specific Aim #2 : To determine, by western transfer blotting, the time course for changes in immunoreactive PGG/H synthase in 3T3 cells treated with PDGF.
Specific Aim #3 : To determine, using 32p-labeled cDNA probes, the time course for changes in PGG/H synthase, c- fos, c-myc and beta-actin mRNAs in 3T3 cells treated with PDGF.
Specific Aim #4 : To determine if, like other immediate- early competence genes, the PGG/H synthase gene is superinduced in the presence of cycolheximide.
Specific Aim #5 : To determine, using nuclear run-off assays, if there is an increased rate of transcription of the PGG/H synthase gene in 3T3 cells treated with PDGF.
Specific Aim #6 : To determine the sequences of the 5'-flanking region of the PGG/H synthase gene from clones prepared from a mouse genomic library, and to determine if there are identifiable regulatory sequences in the flanking region homologous to enhancer sequences from immediate-early genes.
Specific Aim #7 : To prepare constructs of genomic regulatory regions of the PGG/H synthase gene and the reporter gene chloramphenicol acetyltransferase and determine what regulatory regions of the gene are important for PDGF-induced gene expression.
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