Structural determination and promoter analysis of the chicken mitogen-inducible prostaglandin G/H synthase gene and genetic mapping of the murine homolog.
Structural determination and promoter analysis of the chicken mitogen-inducible prostaglandin G/H synthase gene and genetic mapping of the murine homolog.
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鸡促有丝分裂原诱导的前列腺素 G/H 合酶基因的结构测定和启动子分析以及小鼠同源物的遗传图谱。
DOI：
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发表时间：
1993
影响因子：
3.9
通讯作者：
Daniel L. Simmons
中科院分区：
文献类型：
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作者：
Weilin Xie;J. Merrill;William S. Bradshaw;Daniel L. Simmons
我们之前报道过从鸡胚成纤维细胞中分离和表征了一种新形式的前列腺素 G/H 合酶（PGHS，环氧合酶）（PGHS-2）。为了进一步研究该基因的调控和结构，我们从鸡基因组文库中克隆了整个鸡 PGHS-2（以前称为 miPGHSch）基因及其 5' 侧翼区域。基因组 Southern 印迹显示单个 PGHS-2 基因的存在。通过DNA测序和聚合酶链式反应分析，该基因的大小估计为8.9 kb。 PGHS-2 基因被发现含有 10 个外显子，使其结构与人类 PGHS-1 和鼠 PGHS-2 基因相似。通过引物延伸确定转录起始位点，确定转录起始位点紧上游的5'侧翼区的1.6kb的核苷酸序列。启动子序列包含 TATA 盒和多种增强子元件，包括血清反应元件、AP-1、NF-κ B 以及多个 SP-1 和 AP-2 位点。氯霉素乙酰转移酶 (CAT) 测定表明，启动子的前 158 个核苷酸在血清刺激的细胞中有效驱动 CAT 报告基因的转录。地塞米松是一种有效的前列腺素合成抑制剂，对 CAT 活性没有影响，尽管已知该药物可显着降低体内 PGHS-2 mRNA。这表明地塞米松可能在转录后水平抑制 PGHS-2 mRNA 表达。使用放射性标记的 cDNA 探针对仓鼠/小鼠体细胞杂交体进行的分析表明，PGHS-1 映射到小鼠基因组的 2 号染色体，PGHS-2 映射到小鼠基因组的 1 号染色体。
We have previously reported the isolation and characterization of a new form (PGHS-2) of prostaglandin G/H synthase (PGHS, cyclooxygenase) from chicken embryo fibroblasts. To further study the regulation and structure of the gene, we have cloned the entire chicken PGHS-2 (previously termed miPGHSch) gene with its 5'-flanking region from a chicken genomic library. A genomic Southern blot showed the existence of a single PGHS-2 gene. The size of the gene was estimated at 8.9 kb through DNA sequencing and polymerase chain reaction analysis. The PGHS-2 gene was found to contain 10 exons, giving it a structure similar to that of the human PGHS-1 and murine PGHS-2 genes. The transcription start site was determined by primer extension, and the nucleotide sequence of 1.6 kb of the 5'-flanking region immediately upstream of the transcription start site was determined. The promoter sequence contained a TATA box and a variety of enhancer elements, including a serum response element, an AP-1, an NF-kappa B, and several SP-1 and AP-2 sites. Chloramphenicol acetyltransferase (CAT) assays showed that the first 158 nucleotides of the promoter efficiently drove transcription of the CAT reporter gene in serum-stimulated cells. Dexamethasone, a potent inhibitor of prostaglandin synthesis, had no effect on CAT activity, although this drug is known to markedly decrease PGHS-2 mRNA in vivo. This suggests that dexamethasone may inhibit PGHS-2 mRNA expression at the post-transcriptional level. Analysis of hamster/mouse somatic cell hybrids with radiolabeled cDNA probes demonstrated that PGHS-1 mapped to chromosome 2 and PGHS-2 mapped to chromosome 1 of the mouse genome.