Three-dimensional structures of the TAFII-containing complexes TFIID and TFTC

TBP (TATA-binding protein)-associated factors (TAF(II)s) are components of large multiprotein complexes such as TFIID, TFTC, STAGA, PCAF/GCN5, and SAGA, which play a key role in the regulation of gene expression by RNA polymerase II. The structures of TFIID and TFTC have been determined at 3.5-nanometer resolution by electron microscopy and digital image analysis of single particles. Human TFIID resembles a macromolecular clamp that contains four globular domains organized around a solvent-accessible groove of a size suitable to bind DNA. TFTC is larger and contains five domains, four of which are similar to TFIID.     

Two domains of yeast U6 small nuclear RNA required for both steps of nuclear precursor messenger RNA splicing

U6 is one of the five small nuclear RNA's (snRNA's) that are required for splicing of nuclear precursor messenger RNA (pre-mRNA). The size and sequence of U6 RNA are conserved among organisms as diverse as yeast and man, and so it has been proposed that U6 RNA functions as a catalytic element in splicing. A procedure for in vitro reconstitution of functional yeast U6 small nuclear ribonucleoproteins (snRNP's) with synthetic U6 RNA was applied in an attempt to elucidate the function of yeast U6 RNA. Two domains in U6 RNA were identified, each of which is required for in vitro splicing. Single nucleotide substitutions in these two domains block splicing either at the first or the second step. Invariably, U6 RNA mutants that block the first step of splicing do not enter the spliceosome. On the other hand, those that block the second step of splicing form a spliceosome but block cleavage at the 3' splice site of the intron. In both domains, the positions of base changes that block the second step of splicing correspond exactly to the site of insertion of pre-mRNA-type introns into the U6 gene of two yeast species, providing a possible explanation for the mechanism of how these introns originated and adding further evidence for the proposed catalytic role of U6 RNA.     

Cathepsin B基因的沉默对香蕉穿孔线虫繁殖力的影响

【目的】通过研究cathepsin B基因对香蕉穿孔线虫(Radopholus similis)繁殖力的影响,探索cathepsin B基因的功能,为利用该基因防治香蕉穿孔线虫和植物寄生线虫组织蛋白酶的进一步研究提供科学依据。【方法】根据已知香蕉穿孔线虫cathepsin B基因(Rs-cb-1)的序列,从香蕉穿孔线虫克隆cathepsin B基因,以含有目的基因的质粒DNA为模板合成特异的双链RNA(dsRNA),采用dsRNA浸泡的方法对香蕉穿孔线虫进行RNA干扰(RNAi)试验,通过室内接种胡萝卜愈伤组织繁殖线虫的方法,研究cathepsin B基因的沉默效应对香蕉穿孔线虫繁殖力的影响。【结果】用Rs-cb-1dsRNA浸泡12、24、48、72h后香蕉穿孔线虫的平均繁殖倍数分别为165、93、54、53,而未经dsRNA处理的该线虫繁殖倍数均大于420;并且Rs-cb-1dsRNA浸泡的时间不同,对应各处理之间的繁殖倍数差异显著。RT-PCR检测,经Rs-cb-1dsRNA浸泡12h后,目的基因在香蕉穿孔线虫的表达量明显减弱,浸泡24h后其表达量进一步减弱,但还有微量表达,经dsRNA浸泡48、72h后目的基因基本不表达。【结论】Rs-cb-1与香蕉穿孔线虫的繁殖力相关;Rs-cb-1dsRNA的浸泡可以明显抑制cathepsin B基因的表达量,从而影响香蕉穿孔线虫的繁殖力,但浸泡时间不同Rs-cb-1的沉默效率也不同,沉默效率最好的干涉时间是48h。     

靶向ORFV-DNA ploymerase基因shRNA表达载体的构建

羊口疮（Orf）是由羊口疮病毒（ORFV）引起的人畜共患的一种急性、接触性和具有高度嗜上皮性传染病,主要感染绵羊、山羊和人。由于该病缺乏全身性感染症状,因此在防治上也缺乏有效的疫苗或抗体。RNA干扰技术是目前较为成熟的抑制目的基因表达生物技术。ORFV的DNA ploymerase是病毒复制的关键酶。研究运用RNA干扰技术针对ORFV的DNAploymerase基因进行体外基因沉默研究,通过网络自动筛选平台设计并合成3个short-hairpin RNAs（shRNAs）片段,并与含U6启动子的pLL3.7质粒构建重组载体,结果表明pLL3.7-D596为重组阳性,进一步测序结果正确。研究可以为靶向ORFV-DNAploymerase基因的体内基因沉默提供参考数据。     

拟南芥miRNA及其靶序列配对特征分析

microRNAs(miRNAs)是真核生物中一类长度约为22 nt的非编码小分子RNA,miRNA与AGO等蛋白形成RISC沉默复合体,通过剪切或翻译抑制对靶基因起负调控作用。对拟南芥miRNA序列及其配对的靶序列间的特征进行了统计分析,结果表明miRNA序列5′端富含A、U,第1、第19碱基位对U、C具有较强的倾向性;miRNA与靶序列间常有1~4个碱基错配,错配碱基常出现在第1,第2和第21位,而第3~第6,第9~第10,第16~第17碱基配对较为保守,为人工合成miRNA的设计及miRNA靶基因的预测以提供了依据。     

A subset of yeast snRNA's contains functional binding sites for the highly conserved Sm antigen

Autoimmune sera of the Sm specificity react with the major class of small nuclear RNA (snRNA)-containing ribonucleoprotein particles (snRNP's) from organisms as evolutionarily divergent as insects and dinoflagellates but have been reported not to recognize snRNP's from yeast. The Sm antigen is thought to bind to a conserved snRNA motif that includes the sequence A(U3-6)G. The hypothesis was tested that yeast also contains functional analogues of Sm snRNA's, but that the Sm binding site in the RNA is more strictly conserved than the Sm antigenic determinant. After microinjection of labeled yeast snRNA's into Xenopus eggs or oocytes, two snRNA's from Saccharomyces cerevisiae become strongly immunoprecipitable with human auto-antibodies known as anti-Sm. These each contain the sequence A(U5-6)G, are essential for viability, and are constituents of the spliceosome. At least six other yeast snRNA's do not become immunoprecipitable and lack this sequence; these non-Sm snRNA's are all dispensable.