水稻齿叶矮缩病毒双链RNA的体外翻译

水稻齿叶矮缩病毒(Rice Ragged Stunt Virus,以下简称RRSV)的基因组双链RNA经羟甲基汞变性处理后,能在兔网状红细胞体外翻译体系中有效地指导蛋白质生物合成。利用该病毒的抗血清免疫沉淀体外翻译产物,结合SDS-聚丙烯酰胺凝胶电泳分析,证明RRSV基因组RNA体外翻译的主要产物有十种左右,其中八种能被该病毒抗血清专一沉淀。用纯化的水稻齿叶矮缩病毒直接作蛋白质电泳分析,证明上述八种体外翻译产物与该病毒结构蛋白的分子量相当。Schiff试剂染色实验未能检测出糖蛋白。     

T4病毒研究进展

T4病毒科由一类单股正链RNA病毒组成,分为松天蛾β样病毒属和松天蛾ω样病毒属。这2个属的病毒具有不同的基因组结构,β样病毒含单组分基因组,其结构蛋白由一亚基因组RNA表达; 而ω样病毒含双组分基因组,2个RNA分子分别编码复制酶蛋白和结构蛋白。在T4病毒基因组RNA 3′端有类似tRNA的二级结构。ω样病毒壳蛋白的氨基酸序列一致性高达66%～86%, 而β样病毒壳蛋白的氨基酸同源性则要低得多。在昆虫细胞中表达壳蛋白基因时都能形成病毒类似粒子。该文还介绍了T4病毒复制机理以及T4病毒与其他病毒的进化关系。     

马铃薯Y病毒属病毒的分子生物学研究进展

马铃薯Y病毒属（Potyvirus）是种类最多的一类植物病毒,现在国际病毒分类委员会（ICTV）承认的成员有168种。它们的病毒粒子都是弯曲线状的,长度在680-900nm,在植物细胞内可以形成圆柱形（风轮状）内含体或卷旋状内含体。基因组为正义单链RNA,翻译时先翻译成多聚蛋白（polyprotein）。本属病毒可由蚜虫传播。Riechmann等曾在1992年介绍过其基因组结构、基因组表达等方面的进展,本文主要介绍1992年以后的研究进展。1基因组结构与功能马铃薯Y病毒属的基因组为正单链RNA,约10,000个核着酸,5’．端具有病毒基因组连接蛋白（VPg）…     

RNA分子的生物学功能

RNA是一些病毒的遗传物质,能复制和突变;RNA是DNA与蛋白质之间的桥梁,具有编码、翻译和拓展遗传信息的功能;RNA逆转录产生cDNA,在基因组形成中起关键作用。RNA具催化活性,可以催化mRNA的剪接,DNA和RNA的水解、连接,RNA聚合,RNA的磷酸化等多种生化过程;某些微小RNA的时序表达,在个体发育中有重要调节作用;由双链RNA介导的转录后基因沉默对生物体抵御外来遗传元件RNA有重要作用。     

生物防治因子

将一种双链DNA分子插入植物细胞基因组。双链DNA分子中有一个在植物细胞内有功能病毒RNA顺序的启动区,病毒RNA的cDNA,能把多腺昔酸化核普酸加到RNA3产端的非翻译区。(魏钧)882303产生杀菌素的内共生微生物及其制备与使用方法仁专     

RNA:诱导基因沉默

在生物体中,双链RNA(double-strand RNA,dsRNA)裂解后的小RNA可以诱导细胞质和基因组水平外源基因沉默。所谓基因沉默(gene silencing)是指生物体中特定基因由于种种原因不表达。小RNA能诱导互补信使RNA在转录后降解。RNA沉默是基因组水平的免疫现象,代表了进化过程中原始的基因组对抗外源基因序列表达的保护机制,在动植物进化中起着重要作用,RNA沉默具有抵抗病毒入侵、抑制转座子活动等作用,并调控蛋白编码基因的表达,具有十分诱人的应用前景。     

小RNA病毒3'非编码区结构与功能的研究进展

小RNA病毒基因组的两侧分别为5′非编码区(UTR)和3′UTR。研究表明:5′/3′UTR能形成复杂的RNA结构,如颈-环结构、三叶草结构和假结体结构等,在病毒的复制或翻译过程中发挥重要调节作用。本文即对近10年来有关小RNA病毒3′UTR的结构与功能研究方面的进展情况进行综述。     

小RNA病毒蛋白翻译调控元件研究进展

真核生物的起始复合物并不是在起始AUG处形成 ,而是在mRNA的 5′末端形成 ,其识别信号就是 5′末端的帽子结构。小RNA病毒科成员RNA 5′末端没有帽子结构 ,而有一个病毒编码的小蛋白质与基因组共价相连。小RNA病毒的蛋白翻译起始于 5′非翻译区中的内部顺式调控元件 ,称为内部核糖体进入位点 (IRES)。口蹄疫病毒 (foot and mouthdiseasevirus,FMDV)是该科病毒的典型代表 ,引起偶蹄动物的急性接触性传染病。完整FMDV含有单链正股RNA、衣壳蛋白及少量装配过程中夹带的非结构蛋白和宿主细胞肌动蛋白 ,其基因组RNA全长约 8 5kb ,可直接作为信使RNA。对IRES的一、二级结构进行了比较 ,对IRES与翻译起始因子的相互作用以及对病毒毒力的影响作了综述。     

登革病毒（DENV）基因组的5'和3'末端所蕴含的生物调控信息

登革病毒(Dengue virus,DENV)为正链RNA黄病毒,其基因组进入宿主细胞后经历了蛋白翻译和正链-负链-正链的RNA复制过程,病毒基因组的5'末端区域和3'末端区域的RNA序列和结构在上述过程中发挥不同的功能,病毒自身因此能更好地完成其生命活动。本文对DENV基因组末端区域各个序列和结构的基本功能,帽子结构依赖或非经典的非帽非IRES依赖的蛋白翻译,以环化为核心的5'–3'RNA长程相互作用的复制方式和3'UTR进化出的重复序列及串联结构在适应不同宿主所发挥的功能进行综述。     

昆虫小RNA病毒研究进展

昆虫小RNA病毒依据基因组结构特点分为家蚕软化病毒组、蟋蟀痹病毒组和豌豆蚜病毒组。昆虫小RNA病毒蛋白质翻译具有独特性：不依赖于帽子结构,不需要启始因子eIF1、eIF1A和帽子结合蛋白eIF4E;而且蟋蟀痹病毒组和豌豆蚜病毒组结构蛋白翻译从内部核糖体进入位点独立启始,启始密码子是CUU。该文还简介了昆虫小RNA病毒复制机理及与其它动、植物小RNA病毒或类小RNA病毒的亲缘关系等的研究进展。     

Sequence and translation of the murine coronavirus 5''-end genomic RNA reveals the N-terminal structure of the putative RNA polymerase.

A 28-kilodalton protein has been suggested to be the amino-terminal protein cleavage product of the putative coronavirus RNA polymerase (gene A) (M.R. Denison and S. Perlman, Virology 157:565-568, 1987). To elucidate the structure and mechanism of synthesis of this protein, the nucleotide sequence of the 5' 2.0 kilobases of the coronavirus mouse hepatitis virus strain JHM genome was determined. This sequence contains a single, long open reading frame and predicts a highly basic amino-terminal region. Cell-free translation of RNAs transcribed in vitro from DNAs containing gene A sequences in pT7 vectors yielded proteins initiated from the 5'-most optimal initiation codon at position 215 from the 5' end of the genome. The sequence preceding this initiation codon predicts the presence of a stable hairpin loop structure. The presence of an RNA secondary structure at the 5' end of the RNA genome is supported by the observation that gene A sequences were more efficiently translated in vitro when upstream noncoding sequences were removed. By comparing the translation products of virion genomic RNA and in vitro transcribed RNAs, we established that our clones encompassing the 5'-end mouse hepatitis virus genomic RNA encode the 28-kilodalton N-terminal cleavage product of the gene A protein. Possible cleavage sites for this protein are proposed.     

Identification of polypeptides encoded in open reading frame 1b of the putative polymerase gene of the murine coronavirus mouse hepatitis virus A59.

The polypeptides encoded in open reading frame (ORF) 1b of the mouse hepatitis virus A59 putative polymerase gene of RNA 1 were identified in the products of in vitro translation of genome RNA. Two antisera directed against fusion proteins containing sequences encoded in portions of the 3'-terminal 2.0 kb of ORF 1b were used to immunoprecipitate p90, p74, p53, p44, and p32 polypeptides. These polypeptides were clearly different in electrophoretic mobility, antiserum reactivity, and partial protease digestion pattern from viral structural proteins and from polypeptides encoded in the 5' end of ORF 1a, previously identified by in vitro translation. The largest of these polypeptides had partial protease digestion patterns similar to those of polypeptides generated by in vitro translation of a synthetic mRNA derived from the 3' end of ORF 1b. The polypeptides encoded in ORF 1b accumulated more slowly during in vitro translation than polypeptides encoded in ORF 1a. This is consistent with the hypothesis that translation of gene A initiates at the 5' end of ORF 1a and that translation of ORF 1b occurs following a frameshift at the ORF 1a-ORF 1b junction. The use of in vitro translation of genome RNA and immunoprecipitation with antisera directed against various regions of the polypeptides encoded in gene A should make it possible to study synthesis and processing of the putative coronavirus polymerase.     

Tissue-specific expression of the rat glutathione S-transferases

Tissue-specific patterns of rat glutathione S-transferase expression have been demonstrated by in vitro translation of purified poly(A) RNAs and by protein purification. Poly(A) RNAs from six rat tissues including heart, kidney, liver, lung, spleen, and testis were used to program in vitro translation with the rabbit reticulocyte lysate system and [35S]methionine. The glutathione S-transferase subunits synthesized in vitro were purified from the translation products by affinity chromatography on S-hexylglutathione-linked Sepharose 6B columns. The affinity bound fractions were analyzed by Na dodecyl SO4-polyacrylamide gel electrophoresis and fluorography. A subunit of Mr = 22,000 detected in the in vitro translation products of poly(A) RNAs from heart, kidney, lung, spleen, and testis is missing from the translation products of liver poly(A) RNAs. This Mr = 22,000 subunit is present only in the anionic glutathione S-transferase fraction purified from rat heart, kidney, lung, spleen, and testis. Purified anionic glutathione S-transferase from rat liver does not contain this subunit. The relative specific activities toward a dozen different substrates also demonstrate the nonidentity between liver and kidney anionic glutathione S-transferases. In addition, among the glutathione S-transferase subunits expressed in the liver, some of them could not be detected in the other tissues investigated. Our results indicate that tissue-specific expression of rat glutathione S-transferases may occur pretranslationally.     

Viral influences on aflatoxin formation by Aspergillus flavus

Summary A nontoxigenic isolate of Aspergillus flavus (NRRL 5565) contains a viral genome consisting of 3 double-stranded RNA (ds-RNA) components with molecular weights of approximately 3 kb each. It thus shares a characteristical feature with a virus occuring in strains of Penicillium chrysogenum.Application of known inhibitors of doublestranded RNA virus synthesis results in stable aflatoxin formation by this originally nontoxigenic strain and the simultaneous loss of its ds-RNA traits. Since the inhibitor induced toxicity can be completely reverted by incubation with a virus from Penicillium chrysogenum (PcV), it is presumed that PcV or a functional related virus possibly constitutes the aflatoxin repressing determinant in Aspergillus flavus.     

Protein synthesis directed by encephalomyocarditis virus RNA. II. The in vitro synthesis of high molecular weight proteins and elements of the viral capsid

RNA from the encephalomyocarditis virus directs the cell-free synthesis of several discrete, high molecular weight proteins. The largest of these have molecular weights of approximately 110,000, 82,000, 73,000, 61,000 and 44,000 Daltons. In addition, tryptic digestion of the in vitro products gives rise to a number of peptides corresponding to those derived from the viral capsid. The data suggest that approximately one-third of the information encoded by the EMC genome is translated in vitro as a single polypeptide chain, that this translation proceeds in an appropriate phase, and that portions of the genome corresponding to structural proteins of the virus are translated.     

Candidate product of the FBJ murine osteosarcoma virus oncogene: characterization of a 55,000-dalton phosphoprotein

Sera from rat bearing tumors induced by inoculation of FBJ murine osteogenic sarcoma virus (FBJ-MSV) nonproducer rat cells precipitate two proteins with molecular weights of 55,000 (p55) and 39,000 (p39) from FBJ-MSV-transformed cells. These proteins cannot be precipitated from uninfected cells or cells transformed by other strains of murine sarcoma virus, nor can they be precipitated by sera specific for the viral structural proteins. A methionine tryptic peptide mapping analysis showed that p55 and p39 have little or no homology and that they are not related to the helper virus gag and env gene products. p55 could also be detected among the in vitro translation products of 70S RNA from FBJ murine leukemia virus plus FBJ-MSV virions but not among those from FBJ murine leukemia virus alone. This suggests that p55 is encoded by the FBJ-MSV genome, whereas p39, which was not detected among the in vitro translation products, may not be virus encoded. Another difference between p55 and p39 is that p55 is phosphorylated, with most of the phosphate on a serine residue(s), whereas p39 is phosphorylated to a much lesser extent, if at all. No protein kinase activity was associated with p55 and p39 immune complexes under standard conditions. Our data suggest that p55 is a strong candidate for the FBJ-MSV oncogene product.     

Structure and function of rat liver polysome populations. II. Characterization of polyadenylate-containing mRNA associated with subpopulations of membrane-bound particles

Poly(A)+RNA fractions prepared from free and loosely and tightly membrane-bound polysome populations (poly(A)+RNAfree, poly(A)+RNAloose, and poly(A)+RNAtight) were used to drive cDNA in homologous and heterologous hybridization reactions. A large fraction by mass of sequences was shared among the three poly(A)+RNA populations, but shared sequences exhibited distinct frequency distributions within the different populations. 13-15 in vitro translation products of poly(A)+RNAfree and poly(A)+RNAloose detected by gel electrophoresis were shared. Most of these were produced in different relative quantities by the two RNA populations. Five or six higher mol wt polypeptides were produced by poly(A)+RNAloose that were not detected as products of either poly(A)+free or poly(A)+RNAtight. We suggest that loosely bound polysomes may not be artifactually derived as reflected in their quantitatively distinct poly(A)+RNA population. Two tightly membrane-bound RNP fractions were prepared from rat liver on the basis of their release from or retention on purified rough microsomes or a crude membrane fraction after in vitro disaggregation of polysomes with high-salt and puromycin. Homologous and heterologous hybridizations involving their poly(A)+RNA fractions revealed that a large portion by mass of sequences was shared but that these sequences exhibited distinct frequency distributions in the two fractions. The RNA fractions produced exhibited distinct frequency distributions in the two fractions. The RNA fractions produced an identical set of in vitro translation products but individual polypeptides were produced in different relative quantities. This indicates that the two RNP fractions do not arise by any random artifactual process and suggests that they may represent functionally distinct populations.     

In vitro translation of infectious flacherie virus RNA in a wheat germ and a rabbit reticulocyte system

Infectious flacherie virus is an insect picornavirus isolated from the silkworm, Bombyx mori. Its RNA was found to act as an efficient mRNA in a wheat germ extract and a rabbit reticulocyte lysate translation system. In either system the sum of molecular weights of translation products far exceeded the coding capacity of the virus genome, which suggests the occurrence of proteolytic cleavage of large primary products to smaller polypeptides as reported for other picornaviruses and/or premature termination of translation. The highest molecular weight product of 200 000 (polyprotein-like product) could be translated in both systems. One of the antigenic products common to both systems had a molecular weight of 130 000, which corresponds to the sum of molecular weights of the four major viral proteins. Another product, which comigrated with viral protein 0, the largest viral structural protein in SDS-polyacrylamide gel electrophoresis, also showed antigenicity. Peptide mapping of these polypeptides showed that the two in vitro systems translated the same cistron in the viral RNA and that the smaller polypeptide was a part of the 130 000 Da product.