REGULATION OF THE PGG/H SYNTHASE GENE

PGG/H 合酶基因的调控

• 批准号: 3298549
• 负责人: David Lee DEWITT
• 金额: $ 11.71万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 1991-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3298549
• 关键词: acyltransferase; cell cycle; complementary DNA; cycloheximide; gene expression; gene induction /repression; genetic enhancer element; genetic library; genetic manipulation; genetic promoter element; genetic regulation; genetic transcription; messenger RNA; molecular cloning; molecular genetics; nuclear runoff assay; nucleic acid probes; nucleic acid sequence; oncogenes; platelet derived growth factor; prostaglandin F; prostaglandin endoperoxide synthase; regulatory gene; reporter genes; tissue /cell culture; transposon /insertion element

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.

前列腺素 G/H 合酶催化转化 花生四烯酸转化为前列腺素内过氧化物 G2 和 H2， 合成具有生物活性的前列腺素的第一步。 这 拟议研究的主要目标是检验以下假设： PGG/H合酶基因是一种立即早期的“能力” 基因，如 c-fos 和 c-myc，其激活是细胞的激活所必需的 复制。 该假设基于两个观察：(a) 刺激后的 3T3 细胞中 PGG/h 合酶蛋白水平升高 血小板衍生生长因子（PDGF）大约在同一时间 c-myc、c-fos和其他“立即早期”基因的表达； (b) PDGF 刺激的复制需要 PGE2 3T3细胞。 我们最近分离并测序了全长 编码绵羊 PGG/H 合酶的 cDNA。 我们将使用 绵羊 cDNA 作为探针，用于分离近全长 cDNA 小鼠 PGG/H 合酶（具体目标#1）。 然后我们将使用 小鼠 cDNA 与小鼠 3T3 细胞以确定 PGG/H 是否 合成酶基因具有立即早期基因的特征。 具体来说，使用小鼠 cDNA 和抗 PGG/H 合酶 IgG 已经可用，我们建议： 具体目标 #2： 通过蛋白质印迹法确定时间进程 处理的 3T3 细胞中免疫反应性 PGG/H 合酶的变化 与PDGF。 具体目标#3：使用 32p 标记来确定 cDNA 探针，PGG/H 合酶变化的时间过程，c- 经处理的 3T3 细胞中的 fos、c-myc 和 beta-actin mRNA 血小板衍生生长因子。 具体目标#4：确定是否像其他直接目标一样 早期能力基因，PGG/H合酶基因是 在环己酰亚胺存在下超诱导。 具体目标#5： 为了确定，使用核径流分析，是否存在 3T3 中 PGG/H 合酶基因的转录率增加 用PDGF处理的细胞。 具体目标#6：确定 PGG/H 合酶基因 5'-侧翼区域的序列 来自小鼠基因组文库制备的克隆，并且 确定是否存在可识别的调控序列 侧翼区域与增强子序列同源 即早基因。 具体目标#7：准备构建体 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域对于 PDGF诱导的基因表达。