REGULATION OF THE PGG/H SYNTHASE GENE

PGG/H 合酶基因的调控

• 批准号: 3298545
• 负责人: David Lee DEWITT
• 金额: $ 12.28万
• 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/3298545
• 关键词: acyltransferase; cell cycle; complementary DNA; cycloheximide; gene expression; genetic enhancer element; genetic library; genetic manipulation; genetic promoter element; genetic regulation; genetic transcription; messenger RNA; molecular cloning; molecular genetics; 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 花生四烯酸转化为前列腺素内过氧化物G2和H2， 生物活性前列腺素合成的第一步。 的 拟议研究的主要目标是检验假设， PGG/H合酶基因是一种立即早期“感受态”， 基因，如c-fos和c-myc，其激活是细胞增殖所必需的。 复制的 这一假设基于两个观察：（a） PGG/h合酶蛋白水平在用以下刺激的3 T3细胞中升高： 血小板衍生生长因子（PDGF）在大约相同的时间， c-myc、c-fos等“即刻早期”基因的表达; 和（B）PGE 2是PDGF刺激的 3 T3细胞。 我们最近分离并测序了 编码绵羊PGG/H合酶的cDNA。 我们将使用 以绵羊cDNA为探针， 小鼠PGG/H合酶（特异性目标#1）。 然后我们将使用 小鼠cDNA与小鼠3 T3细胞进行杂交，以确定PGG/H 合酶基因具有立即早期基因的特征。 具体地说，用小鼠cDNA和抗PGG/H合酶， IgG，这是已经可用，我们建议：具体目标#2： 通过蛋白质转移印迹法确定 3 T3细胞中PGG/H合成酶的变化 PDGF。 具体目标#3：使用32 p标记的 cDNA探针，PGG/H合酶，c- fos、c-myc和β-肌动蛋白mRNA在3 T3细胞中的表达 PDGF。 具体目标#4：确定是否像其他直接- 早期感受态基因，PGG/H合酶基因， 在环己酰亚胺的存在下超诱导。 具体目标#5： 为了确定，使用核径流分析，如果有一个 3 T3中PGG/H合酶基因的转录速率增加 用PDGF处理的细胞。 具体目标#6：确定 PGG/H合酶基因的5 ′-侧翼区的序列 从小鼠基因组文库制备的克隆，和 确定是否有可识别的调控序列中， 侧翼区同源的增强子序列 立即早期基因。 具体目标#7：制备结构 PGG/H合酶基因的基因组调控区， 报告基因氯霉素乙酰转移酶， 确定基因的哪些调控区域对 PDGF诱导的基因表达。