TRANSCRIPTION ANALYSIS OF THE SV40 EARLY AND LATE PROMOTER

SV40早期和晚期启动子的转录分析

• 批准号: 3963502
• 负责人: J BRADY
• 金额: --
• 依托单位: DIVISION OF CANCER EPIDEMIOLOGY AND GENETICS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3963502
• 关键词: DNA directed RNA polymerase; gene expression; genetic manipulation; genetic mapping; genetic transcription; mutant; nucleic acid sequence; simian virus 40; tissue /cell culture; virus DNA; virus RNA; virus genetics

We have analyzed the effect of insertion mutants between the SV40 21-base pair (bp) repeats and the early-early (EE) TATA sequence. Insertion of 4-, 42- or 90-bp of DNA at the SV40 NcoI site (m.p. 37) have been analyzed for their effect on SV40 early expression and positioning of the RNA 5' ends. Insertion of 4-bp reduced SV40 early promoter dependent CAT expression by 6- to 8-fold. Increasing the size of the insertion to 42- or 90-bp resulted in a further drop in early gene expression to basal levels. At the RNA level, the 4-bp insertion reduced EE RNA synthesis approximately 10-fold. No concomitant increase in late-early (LE) RNA synthesis was observed. Insertion of 42- or 90-bp of DNA resulted in a decrease in EE RNA synthesis and a stimulation of LE RNA synthesis. Deletion of the SV40 72-bp repeats from the insertion mutants demonstrated that some, but not all, of the LE RNA depends upon the presence of these sequences. These studies suggest that the ability of RNA polymerase II to utilize the EE (TATA-directed) transcriptional control sequence requires an interaction with the upstream 21-bp repeats and/or the 72-bp repeats. The fact that LE RNA levels in pJI1-in42 CAT and pJI1-in90 CAT were equivalent to the level of EE RNA in pJI1-CAT, yet the level of CAT gene expression was decreased greater than 10-fold, suggests that LE mRNA is under translational control.

我们分析了SV 40 21-碱基之间插入突变体的影响， 对（bp）重复序列和早期-早期（EE）TATA序列。 插入4-， 42-在SV 40 NcoI位点（m.p.37）的90-bp的DNA中， 它们对SV 40早期表达和RNA 5'端定位的影响。 插入4-bp通过以下途径降低SV 40早期启动子依赖性CAT表达： 6-8倍。 将插入片段的大小增加到42或90 bp 导致早期基因表达进一步下降到基础水平。 在 在RNA水平上，4-bp的插入使EE RNA合成降低了约10%， 十倍 晚期-早期（LE）RNA合成没有伴随增加， 观察 插入42-或90-bp的DNA导致EE降低 RNA合成和LE RNA合成的刺激。 删除SV 40 插入突变体的72-bp重复序列表明，一些，但不是 所有的LE RNA依赖于这些序列的存在。 这些研究表明，RNA聚合酶II的能力，利用 EE（TATA定向）转录控制序列需要相互作用 具有上游21-bp重复序列和/或72-bp重复序列。 事实上，LE pJI 1-in 42 CAT和pJI 1-in 90 CAT中的RNA水平相当于 在pJI 1-CAT中，EE RNA的表达量显著降低，而CAT基因的表达量显著降低 大于10倍，表明LE mRNA受翻译控制。