REGULATION OF THE PGH SYNTHASE GENE

增加PGH合成酶的合成的因素似乎是这样做的 通过增加PGH合成酶基因的转录。我们已经隔离了 小鼠生长激素合成酶基因的基因组克隆及测序 在转录起始点的5‘侧约2200个碱基。我们 已经确定了与AP-1相关的共识增强子序列(它介导 佛波酯、血清和Fos诱导Jun/Fos转录增强 生长因子)和二恶英反应元件(DRE)(它介导 由芳香烃(AH)受体增加转录 对多环芳烃(二恶英/三氯二苯、多氯联苯、多溴联苯)的反应。我们 还发现了一种可能的糖皮质激素负性调节成分 (NGRE)可能下调基因转录以响应 糖皮质激素。我们建议测试AP-1、DRE和NGRE 调节PGH合成酶基因转录的序列并确定 存在与已知的增强子元件不同的其他序列，其 调节PGH合成酶基因的转录。 具体目标#1和#3是制备含有5‘-侧翼的质粒 与氯霉素相连的前列腺素H合成酶基因序列 乙酰转移酶(CAT)报告基因及其在 转染实验确定AP-1和DRE的共同序列或 基因5‘侧翼区的任何其他序列都会增强 PGH合酶基因转录对血清-、PDGF-或 佛波酯刺激物。 具体目标#2是使用为特定目的构建的pGHS-CAT载体 目标#1和#2确定负性糖皮质激素是否对 我们已经在PGH的5‘-侧翼序列中鉴定出的元素 合成酶基因或侧翼序列的任何其他区域都可以调节 糖皮质激素通过糖皮质激素受体抑制转录 对基础、PDGF、血清或TPA刺激的依赖抑制 PGH合成酶基因的转录。 具体目的#4是确定是否缺少PGH合成酶的剪接变体 氨基酸523-585(包括活性部位丝氨酸)显示过氧化物酶。 活动。含有该剪接的cDNA的pSVT7表达载体 将构建突变体，将其导入cos-1细胞，并进行检测 过氧化物酶活性。

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.