REGULATION OF THE PGG/H SYNTHASE GENE

前列腺素G/H合成酶催化 花生四烯酸到前列腺素内氧化物G2和H2， 生物活性前列腺素合成的第一步。这个 这项拟议研究的主要目标是检验以下假设 PGG/H合酶基因是一种即刻早期的“能力” 基因，如c-fos和c-myc，其激活是细胞所必需的 复制。这一假设基于两个观察结果：(A) 3T3细胞中PGG/h合酶蛋白水平升高 血小板衍生生长因子(PDGF)几乎与 C-myc、c-fos等即刻早期基因的表达； 和(B)PDGF刺激的复制需要PGE2 3T3细胞。我们最近分离并测序了一个全长的 绵羊PGG/H合成酶的编码基因。我们将使用 以绵羊cDNAs为探针分离近全长基因 小鼠PGG/H合酶(特异目的1)。然后我们将使用 用小鼠3T3细胞测定小鼠的c DNA和pGG/H 合酶基因具有即早基因的特征。 具体地说，与小鼠的cDNA和抗PGG/H合成酶 已有的免疫球蛋白，我们建议：具体目标2： 用Western Transfer blotting法测定 3T3细胞处理后免疫活性PGG/H合酶的变化 使用PDGF。具体目标#3：使用32P标记的 C-DNA探针，PGG/H合酶的时间进程，c- 3T3细胞中fos、c-myc和β-肌动蛋白mRNAs的表达 PDGF。具体目标4：确定是否像其他直接的- 早期能力基因，PGG/H合酶基因是 在放线菌亚胺存在的情况下进行过诱导。具体目标5： 使用核径流分析来确定是否存在 3T3中PGG/H合酶基因转录速率的提高 用PDGF处理的细胞。具体目标#6：确定 PGG/H合酶基因5‘侧翼区的序列分析 从小鼠基因组文库中制备的克隆，并 确定是否存在可识别的调控序列 与以下增强子序列同源的侧翼区域 即刻早期基因。具体目标#7：准备构造 PGG/H合酶基因的基因组调控区和 报告基因氯霉素乙酰转移酶和 确定基因的哪些调控区对 PDGF诱导的基因表达。

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.