Localization of a transcription promoter within the second exon of the cytochrome P-450c27/25 gene for the expression of the major species of two-kilobase mRNA.
Localization of a transcription promoter within the second exon of the cytochrome P-450c27/25 gene for the expression of the major species of two-kilobase mRNA.
复制标题
转录启动子定位于细胞色素 P-450c27/25 基因的第二个外显子内，用于表达主要物种的两千碱基 mRNA。
DOI：
10.1021/bi00042a003
复制
发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Avadhani,NG
中科院分区：
文献类型：
--
作者：
Mullick,J;Addya,S;Sucharov,C;Avadhani,NG
7995年8月31日接收的修订版Mandarin pt ®摘要：大鼠P-450 c27/25（CYP 27）基因表达为2和2.3 kb的两种不同大小的mRNA（CYP酶）。2 kb的mRNA是肝脏中的主要形式，2.3 kb的种类可忽略不计。另一方面，大鼠肾脏和肝癌含有显著水平的2.3 kb物质。大鼠CYP 27基因由11个外显子组成，外显子长度为80-415个核苷酸，内含子长度为83个碱基，内含子长度约为10 kb。S1核酸酶保护和引物延伸分析显示，从外显子2开始的下游86个核苷酸处有一个突出的5 '末端。该位点被指定为+1，是2kb mRNA的起始位点。大鼠肾和肝癌RNA的5 'RACE分析显示存在一个5'延伸的mRNA，其序列与Spi 2 mRNA互补。一个隐蔽的TATA盒（TTTAAA）位于2kb mRNA转录起始位点上游24个核苷酸的+1。一个106 bp的DNA片段（序列-83至+23），其中含有假定的TATA基序形成三个不同的迁移复合物与核提取物从小鼠3 T3细胞。DNA酶I足迹法和与合成DNA的竞争表明，复合物A代表结合的Spl因子，复合物B和C是由于分别与-83至-71和-20至-12序列结合的未知因子。用克隆在CAT报告质粒中的-840/+23 DNA及其5 '缺失进行的体内转录分析表明，基本启动子元件位于基因的序列-45至+23内。最后，在HeLacell nuclearextract中的体外转录分析表明，来自该区域的完整TTTAAA基序和复杂的C形成序列对于启动子+1位置的转录起始是必需的。我们的研究结果表明，2 kb的mRNA转录作为一个独立的转录驱动的直接上游启动子位于外显子2。
Revised Manuscript Received August 31, 7995® abstract: The rat P-450c27/25 (CYP27) gene is expressed as two distinctly sized mRNAs of 2 and 2.3 kb (kilobase). The 2 kb mRNA is the predominant form in the liver with negligible 2.3 kb species. Rat kidney and hepatoma, on the other hand, contain significant levels of the 2.3 kb species. Rat CYP27 gene contains 11 exons of 80—415 nucleotides that are separated by 10 introns of 83 bases to~ 10 kb. S1 nuclease protection and primer extensionanalyses using liver RNA showed a prominent 5'terminus 86 nucleotides downstream from the start of exon 2. This site, designated as+ 1, is the start site for the 2 kb mRNA. 5'RACE analysis of rat kidney and hepatoma RNAs showedthe presence of a 5'extended mRNA with a sequence complementary to theSpi2 mRNA. A cryptic TATA box (TTTAAA) is located 24 nucleotides upstream of the 2 kb mRNA transcription initiation site at+ 1. A 106 bp DNA fragment (sequence-83 to+ 23) that houses the putative TATA motif forms three differently migrating complexes with nuclear extract from the murine 3T3 cells. DNAse I footprinting and competition with synthetic DNA showed that complex A represents the bound Spl factor and complexes B and C are due to unknown factors binding to the—83 to—71 and—20 to—12 sequences, respectively. In vivo transcription analysis using—840/+ 23 DNA and its 5'deletions cloned in a CAT reporter plasmid suggests that the basal promoter elements are located within sequence—45 to+ 23 of the gene. Finally, in vitro transcription analysis in HeLacell nuclearextract showed that intact TTTAAAmotif and complex C-forming sequence from this region are essential for transcription initiation at the+ 1 position of the promoter. Our results demonstrate that the 2 kb mRNA is transcribed as an independent transcript driven by an immediate upstream promoter located within exon 2.