REGULATION OF THE PGH SYNTHASE GENE

PGH 合酶基因的调控

• 批准号: 3298550
• 负责人: David Lee DEWITT
• 金额: $ 13.6万
• 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 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3298550
• 关键词: DNA footprinting; animal tissue; beta galactosidase; chloramphenicol acetyltransferase; corticosteroid receptors; dexamethasone; dioxins; enzyme biosynthesis; fusion gene; gene deletion mutation; genetic enhancer element; genetic mapping; genetic terminator element; genetic transcription; glucocorticoids; laboratory mouse; molecular cloning; oncoproteins; peroxidases; phorbols; plasmids; platelet derived growth factor; prostaglandin endoperoxide synthase; receptor; tissue /cell culture; transcription factor; transfection; western blottings

Factors that increase synthesis of the PGH synthase enzyme appear to do so by increasing transcription of the PGH synthase gene. We have isolated and mapped genomic clones of the mouse PGH synthase gene and have sequenced approximately 2200 bp on the 5'-side of the transcriptional start site. We have identified consensus enhancer sequences related to AP-1 (which mediate increased transcription by Jun/Fos in response to phorbol esters, serum and growth factors) and to the dioxin responsive element (DRE) (which mediates increased transcription by the Aromatic hydrocarbon (Ah) receptor in response to polycyclic aromatic hydrocarbons (dioxin/TCDD, PCB, PBB). We have also identified a putative negative glucocorticoid regulatory element (nGRE) that may downregulate transcription of the gene in response to glucocorticoids. We propose to test whether the AP-1, DRE and nGRE sequences regulate PGH synthase gene transcription and to determine whether other sequences, different from known enhancer elements, are present which regulate transcription of the PGH synthase gene. Specific Aims #1 and #3 are to prepare plasmids containing 5'-flanking sequences of the PGH synthase gene adjoined to a chloramphenicol acetyltransferase (CAT) reporter gene and to use these plasmids in transfection assays to determine if the AP-1 and DRE consensus sequences or any other sequences in the 5'-flanking region of the gene enhance transcription of the PGH synthase gene in response to serum-, PDGF-, or phorbol ester-stimulation. Specific Aim #2 is to use the PGHS-CAT vectors constructed for Specific aims #1 and #2 to determine if the negative-glucocorticoid responsive element that we have identified in the 5'-flanking sequence of the PGH synthase gene, or any other region of the flanking sequence, can mediate transcriptional inhibition by glucocorticoids via glucocorticoid-receptor dependent inhibition of basal, PDGF-, serum-, or TPA-stimulated transcription of the PGH synthase gene. Specific Aim #4 is to determine if a Splice Variant of PGH synthase lacking amino acids 523-585 (including the active site serine) exhibits peroxidase activity. A pSVT7 expression vector containing the cDNA for this splice variant will be constructed, transfected into cos-1 cells, and tested for peroxidase activity.

增加 PGH 合酶合成的因素似乎确实如此 通过增加 PGH 合酶基因的转录。 我们已经隔离并 绘制了小鼠 PGH 合酶基因的基因组克隆图谱并进行了测序 转录起始位点 5' 侧约 2200 bp。 我们 已鉴定出与 AP-1 相关的共有增强子序列（介导 Jun/Fos 响应佛波酯、血清和 生长因子）和二恶英反应元件（DRE）（介导 芳香烃 (Ah) 受体的转录增加 对多环芳烃（二恶英/TCDD、PCB、PBB）的响应。 我们 还确定了假定的负糖皮质激素调节元件 （nGRE）可能会下调基因的转录以响应 糖皮质激素。 我们建议测试 AP-1、DRE 和 nGRE 是否 序列调节 PGH 合酶基因转录并确定是否 存在与已知增强子元件不同的其他序列 调节 PGH 合酶基因的转录。 具体目标 #1 和 #3 是制备含有 5'-侧翼的质粒 与氯霉素邻接的 PGH 合酶基因的序列 乙酰转移酶（CAT）报告基因并使用这些质粒 转染测定以确定 AP-1 和 DRE 共有序列或 基因 5' 侧翼区域中的任何其他序列都会增强 PGH 合酶基因的转录响应于血清、PDGF 或 佛波酯刺激。 具体目标 #2 是使用为具体目标构建的 PGHS-CAT 载体 目标#1和#2确定负糖皮质激素是否有反应 我们在 PGH 5' 侧翼序列中鉴定出的元素 合酶基因或侧翼序列的任何其他区域可以介导 糖皮质激素通过糖皮质激素受体进行转录抑制 基础、PDGF、血清或 TPA 刺激的依赖性抑制 PGH 合酶基因的转录。 具体目标 #4 是确定是否缺少 PGH 合酶的剪接变体 氨基酸 523-585（包括活性位点丝氨酸）表现出过氧化物酶 活动。 包含该剪接 cDNA 的 pSVT7 表达载体 将构建变体，转染到 cos-1 细胞中，并进行测试 过氧化物酶活性。