Purified Saccharomyces cerevisiae RNA polymerase II interacts homologously with two different promoters as revealed by P1 endonuclease analysis

Summary The intergenic region of the Saccharomyces cerevisiae GAL1-GAL10 divergent promoters has been circularized in vitro in different topological states. In defined conditions, purified homologous RNA polymerase II forms two stable complexes (half-life -5 h) with this DNA in the presence of the four ribonucleotides, as determined by measurement (Gamper and Hearst 1983) of the amount and stability of the resulting unwinding. Each stable complex induces in the closed DNA domain a region of hypersensitivity to P1 endonuclease. The two induced hypersensitive regions are very similar: each maps on one promoter, spans over the 100 bp DNA sequence that encompasses the RNA Initiation Sites (RIS) and the TATA box, is composed by three subregions (one on the RIS, one proximal or overlapping the TATA sequence, one intermediate). We show that this promoter-localized interaction is supercoil-dependent.     

RNA干扰在肿瘤基因治疗中的应用策略

运用RNA干扰（RNAi）技术可以通过以下策略进行肿瘤的靶向治疗：抑制癌基因、生长因子及其受体的过表达,从而抑制细胞生长;干扰细胞周期蛋白及其相关基因的表达,从而抑制细胞增殖;抵抗致癌病毒的入侵;抑制抗凋亡基因的表达;上调和恢复抑癌基因的功能;抑制肿瘤发生过程中的关键酶;抑制与肿瘤转移有关的血管生成;靶向端粒酶;靶向耐药基因。尽管目前RNAi已经较为广泛地应用于基因功能研究和肿瘤疾病的基因治疗研究中,但其在应用过程中还有许多亟待解决的问题。我们就RNAi及其在肿瘤疾病基因治疗中的应用策略和存在的问题做一综述。     

Spacing requirements for simultaneous recognition of the adenovirus major late promoter TATAAAAG box and initiator element

The distance between the TATAAAAG box and initiator element of the strong adenovirus major late promoter was systematically altered to determine the optimal spacing for simultaneous recognition of both elements. We find that the TATAAAAG element is strongly dominant over the initiator for specification of the start site. The wild type spacing of 23 base pairs between TATAAAAG and +1A is optimal for promoter strength and selective recognition of the A-start. Initiation is constrained to a window spaced 19-26 base pairs downstream of (-31)-TATAAAAG-(-24), and A-starts are favored over alternate starts only when spaced between 21 and 25 base pairs downstream of TATAAAAG. We report an expanded TATAAAAG and initiator promoter consensus for vertebrates and plants. Plant promoters of this class are (A-T)-rich and have an A-rich (non-template strand) core promoter sequence element downstream of +1A.     

人工 microRNA 技术研究进展

RNA干扰（RNAi）是由双链RNA触发的在mRNA水平进行的特异靶序列的基因沉默现象,广泛存在于动物、植物和病毒中,主要包括小干扰RNA（siRNA）及微小RNA（miRNA）两种作用途径。人工miRNA（amiRNA）是将天然miRNA的成熟序列替换成人工设计的靶向其他感兴趣基因的反义序列,通过天然miRNA的生成和作用途径达到RNAi的效果,具有干扰效果明显、作用迅速、毒性低等优点,拥有广阔的应用前景。我们对基于amiRNA的基因沉默技术进行了较为系统的介绍和总结,梳理了该技术的优缺点和适用范围,并展望了其进一步发展的方向和应用前景。     

基因枪在RNA干扰中的应用研究进展

RNA干扰（RNA interference,RNAi）是指双链RNA（double-strand RNA,dsRNA）特异性降解同源mRNA,从而引发基因转录后水平沉默的现象,是一种高效、高特异性抑制基因表达的途径。自1998年Fire等发现RNA干扰现象以来,其特异性降解目的基因的优势吸引了众多研究者的目光。本文在简要综述RNAi技术在基因功能研究、抗病毒治疗,肿瘤基因治疗等领域的应用后,重点归纳了基因枪技术在RNAi研究即siRNA导入细胞中的应用,并简单分析其优势与意义。     

RNAi的作用机制及其在抗病毒领域的应用

RNA干扰是外源性或内源性双链RNA诱发的mRNA水平上的基因沉默机制。RNA技术具有高效性、特异性。最近将RNA干扰应用于许多病毒性疾病的治疗研究均取得了显著的基因沉默效果,为病毒的预防和治疗开辟了一条新途径。就RNA干扰作用机制及抗病毒效应作一综述。     

Small interfering RNA–mediated gene suppression as a therapeutic intervention in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the lethal and difficult-to-cure cancers worldwide. Owing to the late diagnosis and drug resistance of malignant hepatocytes, treatment of this cancer by conventional chemotherapy agents is challenging, and researchers are seeking new alternative treatment options to overcome therapy resistance in this neoplasm. RNA interference (RNAi) is a potent and specific approach in targeting gene expression and has emerged as a novel therapeutic tool for many diseases, including cancers. Small interfering RNA (siRNA) is a type of RNAi that is produced intracellularly from exogenous synthetic oligonucleotides and can selectively knock down target gene expression in a sequence-specific manner. Various factors play roles in the initiation and progression of HCC and provide multiple candidate targets for siRNA intervention. In addition, due to the liver's unique architecture and availability of some hepatic siRNA delivery methods, this organ has received much more attention as a target tissue for such oligonucleotide action. Recent advances in designing nanoparticle systems for the in vivo delivery of siRNAs have markedly enhanced the potency of siRNA-mediated gene silencing under clinical development for HCC therapy. The utility of siRNAs as anti-HCC agents is the subject of the current review. siRNA-based gene therapies could be one of the main feasible approaches for HCC therapy in the future.     

碳纳米管作为siRNA转运载体的研究进展

随着人们对RNA干扰分子机理的研究愈加深入,siRNA作为一种新的基因治疗药物极有可能为人类攻克癌症等难以治愈的疾病带来希望。然而,目前在RNA干扰应用中遇到的最大挑战就是如何有效地将siRNA导入靶细胞且不致引起严重的细胞毒性。碳纳米管在药物传递和基因传递等生物医学领域的潜在应用受到广泛关注;但要实现碳纳米管在基因治疗领域的应用,碳纳米管的功能化是关键,也是近几年来研究的重点。综述近年来碳纳米管作为siRNA转运载体在基因治疗领域的研究进展。     

DNA-dependent RNA polymerase II from nuclei of suspension-cultured tobacco cells

From isolated nuclei of suspension cultured cells of Nicotiana tabacum. DNA-dependent RNA polymerase II (E.C. 2.7.76) has been purified to homogeneity as evidenced by polyacrylamidegel electrophoresis under non-denaturing conditions. The purified enzyme had a specific activity of more than 15 nmol min^-1·mg^-1 with denatured calf thymus DNA as template. Sodium-dodecyl-sulfate gel electrophoresis and protein highperformance liquid chromatography revealed a subunit composition of four proteins with molecular weights of 165 000, 135 000, 35 000 and 25 000 and with a stoichiometry of 1:1:2:2. The RNA polymerase did not exhibit any detectable proteinkinase activity. The 25 000 subunit binds ADP in a molar ratio of 1:1; it could not be decided whether this subunit has an ATPase activity or is merely an acceptor of ADP.Abbreviations HPLC high-performance liquid chromatography - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecyl sulfate This contribution is dedicated to Professor Fritz Cramer on the occasion of his 60th birthday     

RNA干扰技术治疗疾病

RNA干扰(RNA interference,RNAi)现象最早发现于秀丽隐杆线虫(Caenorhabditis elegans),随后发现该现象普遍存在于真菌、植物和哺乳动物等真核生物,并行使基因调控和抵御外源基因片段侵袭的作用。目前,RNAi分子机制和RNAi在基因功能方面的研究已经取得了突破性的进展。鉴于RNAi在基因沉默中的特异性、高效性和易操作,其在药物筛选和疾病治疗等方面有着广泛的应用前景。然而,RNAi技术用于治疗疾病的安全性尚待确定,分子传递途径也有待进一步的研究。