本氏烟Ⅰ型启动子的克隆及其转录起始位点分析

启动子是转录水平上一个重要的调控元件,其决定着基因的表达模式和表达强度。Ⅰ型启动子具有高转录活性和种属间特异性等特点。如将其应用于植物RNA病毒载体表达系统,有利于提高表达系统的表达效率和生物安全性。本氏烟(Nicotiana benthaminana)是一种被广泛地应用于植物生物反应器和植物病理学的模式生物,但是现有核酸数据库中尚没有其Ⅰ型启动子的相关信息。因此,克隆本氏烟Ⅰ型启动子并分析其转录起始位点就具有重要的应用价值。通过半巢式PCR获得了514 bp的本氏烟Ⅰ型启动子序列(KC352713);生物信息学分析初步预测其转录起始位点位于其核心序列TATA(G)TA(N)GGGGG中的第3位A处;通过植物RNA病毒表达载体和5'RACE技术在体内验证本氏烟Ⅰ型启动子转录起始位点与生物信息学预测结果一致。研究结果为深入研究Ⅰ型启动子和构建Ⅰ型启动子介导转录的植物RNA病毒载体表达系统奠定了基础。     

DNA Instability Maintains the Repeat Length of the Yeast RNA Polymerase II C-terminal Domain

The C-terminal domain (CTD) of RNA polymerase II in eukaryotes is comprised of tandemly repeating units of a conserved seven-amino acid sequence. The number of repeats is, however, quite variable across different organisms. Furthermore, previous studies have identified evidence of rearrangements within the CTD coding region, suggesting that DNA instability may play a role in regulating or maintaining CTD repeat number. The work described here establishes a clear connection between DNA instability and CTD repeat number in Saccharomyces cerevisiae. First, analysis of 36 diverse S. cerevisiae isolates revealed evidence of numerous past rearrangements within the DNA sequence that encodes the CTD. Interestingly, the total number of CTD repeats was relatively static (24–26 repeats in all strains), suggesting a balancing act between repeat expansion and contraction. In an effort to explore the genetic plasticity within this region, we measured the rates of repeat expansion and contraction using novel reporters and a doxycycline-regulated expression system for RPB1. In efforts to determine the mechanisms leading to CTD repeat variability, we identified the presence of DNA secondary structures, specifically G-quadruplex-like DNA, within the CTD coding region. Furthermore, we demonstrated that mutating PIF1, a G-quadruplex-specific helicase, results in increased CTD repeat length polymorphisms. We also determined that RAD52 is necessary for CTD repeat expansion but not contraction, identifying a role for recombination in repeat expansion. Results from these DNA rearrangements may help explain the CTD copy number variation seen across eukaryotes, as well as support a model of CTD expansion and contraction to maintain CTD integrity and overall length.     

Structure of RNA polymerase II pre-initiation complex at 2.9 Å defines initial DNA opening

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shRNA Transcribed by RNA Pol II Promoter Induce RNA Interference in Mammalian Cell

RNA interference is a powerful tool for gene functional analysis in mammals. Permanent gene suppression can be achieved by siRNAs as stem-loop precursors transcribed from RNA Pol III promoter such as H1 and U6 based on vector. This approach, however, has a major limitation: inhibition can not be controlled in a time or tissue specific manner because the RNA Pol III promoter is not time or tissue specific. To overcome these limitations, we designed a strategy that allows synthesis of small hairpin RNAs in a GFP-fused form mediated by RNA Pol II promoter CMV to efficiently and specifically knock down expression of both exogenous and endogenous genes in mammalian cells. As assayed by both fluorescence observing and quantitative RT-PCR, the protein and mRNA products of exogenous gene RFP were efficiently and specifically inhibited; quantitative RT-PCR and western blotting results respectively demonstrated that endogenous lamin B2 mRNA and protein was suppressed without global down-regulation of protein synthesis. Furthermore, GFP-fused shRNA efficacy for RNAi is dependent on target position based on this vector system. This method may provide a novel approach for the application of RNAi technology in suppressing gene expression in mammalian system. Jing Yuan, Xiaobo Wang and Ning Li - These authors contributed equally to this work.     

磷酸烯醇式丙酮酸羧化酶cDNA在T7 RNA多聚酶/启动子系统中专一性表达的研究

用PT7和pGP1—2质粒偶联表达系统,通过温度诱导(30～42℃),使温度敏感基因CI875失活;加入利福霉素选择性抑制E.coliRNA多聚酶的表达,从而使外源PEP羧化酶cDNA得到专一性的表达。产物经SDS—PAGE和Wesern杂交分析,表明该系统表达出两条PEP羧化酶带,分子量分别是78kD和80kD。     

The C-Terminal Domain of RNA Polymerase II Is a Multivalent Targeting Sequence that Supports Drosophila Development with Only Consensus Heptads

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Arginine Citrullination at the C-Terminal Domain Controls RNA Polymerase II Transcription

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Effect of Ischemia on the Activity of DNA-Dependent RNA Polymerase and DNA Polymerase

Abstract: Ischemia, anoxia, and hypoxia of the brain have been shown to inhibit protein synthesis in the central nervous system. To obtain data on the changes in DNA-dependent RNA and DNA polymerases as they pertain specifically to neurons and glia, nuclear enriched neuronal and glial fractions were prepared, by sucrose-gradient centrifugation, from spinal cords of adult dogs that had been subjected to prolonged ischemia. The isolated fractions were assayed for enzyme activity by a radiochemical technique. RNA polymerase was affected more than DNA polymerase, activity being reduced considerably in both neurons and glia. Possible causes of the difference in sensitivity to ischemia are discussed.     

RNA Polymerase I Activators Count and Adjust Ribosomal RNA Gene Copy Number

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