Identification and characterization of cDNA clones specific for cholesterol side-chain cleavage cytochrome P-450.
Identification and characterization of cDNA clones specific for cholesterol side-chain cleavage cytochrome P-450.
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胆固醇侧链裂解细胞色素 P-450 特异性 cDNA 克隆的鉴定和表征。
DOI：
10.1073/pnas.81.18.5628
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发表时间：
1984
影响因子：
11.1
通讯作者：
Waterman,MR
中科院分区：
文献类型：
--
作者：
John,ME;John,MC;Ashley,P;MacDonald,RJ;Simpson,ER;Waterman,MR
两个重叠的cDNA克隆（pBSCC-1和pBSCC-2）轴承插入约等于425和约等于950碱基对长，分别是特异性的牛胆固醇侧链裂解细胞色素P-450（P-450 SCC），已确定通过使用两个差异杂交筛选程序，然后通过杂交选择RNA翻译。通过使用这些克隆的cDNA作为杂交探针，确定了一种RNA种类，其具有预期的P-450 SCC特异性mRNA的组织特异性、促肾上腺皮质激素（ACTH）介导的合成调节和体外合成的蛋白质产物的大小的特性。在从牛肾上腺皮质、牛黄体和培养的牛肾上腺皮质细胞获得的RNA样品中，发现P-450 SCC由大约等于2000个碱基长的mRNA种类编码，其中大部分是聚腺苷酸化的。从牛心脏、肝脏和肾脏制备的RNA样品中未检测到P-450 SCC mRNA。用ACTH处理培养的牛肾上腺皮质细胞，在8小时内出现P-450 SCC mRNA水平升高。因此，ACTH促进P-450 SCC基因转录的增强或起到稳定转录物的作用。当pBSCC-2 cDNA被用来探测高分子量牛DNA与限制性内切酶处理后，观察到一个简单的杂交模式，表明P-450 SCC可能是由一个单一的基因编码。
Two overlapping cDNA clones (pBSCC-1 and pBSCC-2) bearing inserts approximately equal to 425 and approximately equal to 950 base pairs long, respectively, which are specific for bovine cholesterol side-chain cleavage cytochrome P-450 (P-450scc), have been identified by using two differential hybridization screening procedures followed by hybrid-selected RNA translation. By using these cloned cDNAs as hybridization probes, an RNA species was identified that had the properties expected of mRNA specific for P-450scc with respect to tissue specificity, corticotropin (ACTH)-mediated regulation of synthesis, and size of the protein product synthesized in vitro. In RNA samples obtained from bovine adrenal cortex, from bovine corpus luteum, and from cultured bovine adrenocortical cells, it was found that P-450scc is encoded by mRNA species approximately equal to 2000 bases long, a majority of which are polyadenylylated. P-450scc mRNA was not detected in RNA samples prepared from bovine heart, liver, and kidney. Treatment of cultured bovine adrenocortical cells with ACTH resulted in the appearance of elevated levels of P-450scc mRNA within 8 hr. Thus, ACTH promotes the enhancement of P-450scc gene transcription or acts to stabilize the transcripts. When pBSCC-2 cDNA was used to probe high molecular weight bovine DNA following treatment with restriction endonucleases, a simple pattern of hybridization was observed indicating that P-450scc may be encoded by a single gene.