豇豆单孢锈菌中病毒样颗粒中双链RNA的存在

从北京西郊清华园附近田间豇豆上采集的豇豆单孢锈菌夏孢子。萌发后提取双链ＲＮＡ，电泳分析可侧出３００－８０００碱基对的三组双链ＲＮＡ。从萌发的孢子中通过差速离心提取病毒样颗粒，可获得二种类型的病毒样颗粒，一种直径为３５－４０ｎｍ的等轴颗粒，加一种为长短不等的棒状颗粒，用提纯物提取核酸电泳分析与直接从孢子中提取的双链ＲＮＡ有相同的核酸带，从而证明这些双链ＲＮＡ存在于病毒样颗粒中。     

a-鹅膏蕈碱对绒毛烟斑驳病毒及其拟病毒复制的影响

在接种了绒毛烟斑驳病毒(VTMoV)的原生质体培养物中加入50μg／ml的a-鹅膏蕈碱和10μCi／ml的3H-尿嘧啶核苷,培养72小时后,从感病的原生质体内提取核酸,然后对核酸进行凝腔电泳分析并测定’H一放射活性,以此探讨a鹅膏蕈碱对绒毛烟斑驳病毒壳内拟病毒RN^复制的作用。结果表明,拟病毒RNA的复制不受该抑制剂的影响。用酶联免疫吸附分析法(E LJsA)测定从感病的原生质体内提取的病毒颗粒,表明a一鹅膏蕈碱对绒毛烟斑驳病毒的复制无影响。本文还对拟病毒RNA的复制机制进行了探讨。     

黑曲霉病毒的形态和特性及其在细胞内的表现

从产生糖化酶的黑曲霉(Aspergillus niger)菌株中分离到一种等轴对称、衣壳表面有突起、内含双链RNA的病毒颗粒。病毒颗柱在电镜下直径为28—33nm,呈六面体晶格排列。病毒在蔗糖密度梯度离心中有三个分部,分析超离心所得沉降系数为161S,118S和94S。病毒在凝胶电泳中为一条带,在SDS-聚丙烯酰胺凝胶电泳中外壳蛋白分子量为90,000、82,000、76,000、52,0000、42,000 道尔顿。提取的病毒与其抗血清在免疫双扩散实验中出现一条沉淀线。病毒核酸有5个组分,与聚肌苷：聚胞苷[poly(1)：poly?]抗血清反应中出现一条沉淀线。在早期菌丝细胞超薄切片中,病毒颗粒多近似球状紧密聚集,外被以膜结构,聚生或散生于胞质中;后期菌丝细胞中病毒颗粒则多散生于胞质中。     

基于多重置换扩增技术的RNA病毒基因组扩增方法研究

为了便于新发或罕见病毒性传染病的筛查检测,本研究利用多重置换扩增技术,以负链RNA病毒—发热伴血小板减少综合征病毒和正链RNA病毒—登革病毒为模拟样本探索临床样本中RNA病毒基因组非特异性扩增方法。研究中通过梯度稀释的RNA病毒模拟样本中可能存在的不同丰度的病原体,样本核酸依次加工成单链cDNA、双链cDNA、T4DNA连接酶处理后的双链cDNA以及添加外源辅助RNA后合成并连接的双链cDNA形式,然后进行Phi29DNA聚合酶等温扩增,使用荧光定量PCR方法比较各种方法对RNA病毒核酸扩增的影响。结果显示,对于不同类型的RNA病毒模拟标本,多重置换扩增对于单链及双链cDNA的扩增效果有限,而双链cDNA经DNA连接酶处理后的扩增能达到6×103倍;在cDNA合成过程中加入外源辅助RNA,模拟样本中病毒基因组的扩增可达2×105倍,尤其是对含有低丰度病原体的模拟样本扩增效果的改善更为明显。本研究摸索建立了基于多重置换扩增技术的RNA病毒基因组扩增方法,能够对样本中低丰度RNA病毒基因组实现有效扩增,可满足开展多种病原体筛查检测的需求。     

两种核酸提取方法对小鼠诺如病毒RNA提取效能的比较

目的比较两种核酸提取方法对小鼠诺如病毒RNA的提取效能。方法用Trizol提取法和QIAamp Vira lRNA Min iKit提取法分别提取感染小鼠诺如病毒（Murine Norovirus,MNV）的小鼠小肠组织样品RNA和细胞培养物RNA,测定RNA浓度;用MNV特异的引物对分离的核酸样品进行一步法RT—PCR扩增。结果Trizol提取法提取小肠组织的RNA浓度高于QIAamp Viral RNA Mini Kit提取法;QIAamp Viral RNA Mini Kit提取得到的细胞培养物RNA浓度高于Trizol提取法。经QIAamp Viral RNA Mini Kit提取的两种核酸样品均能扩增出特异条带,而Trizol提取的核酸样品未见特异条带。结论在MNV的检测中,QIAampViralRNAKit更适合组织样品中MNV病毒核酸的提取。     

芜菁花叶病毒（TUMV）核酸cDNA的合成与克隆

从接种芜菁花叶病毒后发病芥菜(Brassica Juneaen)中提取病毒,然后提取其核酸(TuMV-RNA)。以纯化的TuMV-RNA为模板,以Oligo(dT)_(12-18)及小牛胸腺DNA水解物为引物,合成双链cDNA,双链cDNA长度约500—4300bp。将双链cDNA补齐后,钝端连接到pUC19质粒的Smal位点,转化E.coli DH,获得500多个白色克隆。菌落原位杂交及酶切分析表明,重组质粒中的插入片段大多数为芜菁花叶病毒RNA的互补DNA,长度为500—4000bp。     

一种含有单链RNA的香菇球状病毒

从生长不正常的香菇(Lentinus edodes(Berk.)Sing)菌株中分离到一种等轴对称含单链RNA的病毒颗粒。病毒颗粒在电镜下直径为33～34nm,在SDS-聚丙烯酰胺凝胶电泳中病毒外壳蛋白分子量为22000道尔顿。病毒核酸径DNase1和SI酶解试验及热变性紫外吸收曲线试验证明为单链RNA,在1.5％的琼脂糖凝胶电泳中,病毒核酸呈现一条带,分子量为2.38×10~6道尔顿。     

我国水稻齿叶矮缩病毒的分离提纯及病毒形态和核酸的研究

应用比较简单的一次四氯化碳澄清,二次聚乙二酵浓缩沉淀,差速离心和20％—50％的蔗糖密度梯度离心,可以获得分离提纯效果较好的水稻齿叶病病毒(RRSV)制剂,在260nm处有最大吸收值,在A_(260)/A_(280)的比值为1.6。用醋酸铀负染方法,可以观察到RRSV为直径54nm—65nm的球状颗粒,具有底部较宽的突起,突起的高度为8—11nm,宽度约为22nm。病毒的核酸为双链RNA,共有十组分散的基因组,这一结果和四方报道的一致,在这一提纯病毒的制剂中,尚发现有其他三种大小不同的球状和线状颗粒,对它们的可能来源进行了讨论。     

在离体转录中产生的一些比TMV-RNA短的片段

体内合成的TMV特异的双链型RNA(RF和RI)在98℃加热45秒的热变性条件下可拆成完整的TMV—RNA,而用感染rMV的烟叶无细胞提取物(zo,000×g沉淀)在放线菌素D存在下离体合成的病毒特异的双链型RNA(具有与RF和RI相同的表观分子置)在同一条件下只产生8—10个比病毒粒子RNA短的片断,其中有几个在大小上与Begchy和Zaitlin(1977)[1]在TMV制品的短颗粒中发现的RNA颇为一致。讨论了这些部分转录产生的RNA片段作为TMV特异的mRNA的可能性。     

核酸分子中碱基含量的计算

关于核酸分子中碱基含量的计算,在遗传学和高中生物教学中相当重要,但在教科书中通常没有专门讲述。我们根据碱基互补配对规律及中心法则进行归纳总结,从DNA结构、DNA复制、转录、翻译等方面探讨了DNA、RNA、蛋白质3者之间的关系,分析了核酸分子中碱基的含量。互核酸分子中碱基含量的计算1．且已知双链DNA分子中一种碱基的含量,推断其他碱基的含量：例1：一双链‘DNA分子中,（A－C）占碱基总量的Zo％。求A、T、G、C各占多少？解：在双链DNA分子中,据规律知,1．2由碱基含量推断核酸分子的结构——单链或双链、DNA或RNA…     

Analysis of double-stranded RNA and virus-like particles in trichothecene-producing strains of<Emphasis Type="Italic">Fusarium graminearum</Emphasis>

Double-stranded RNAs (dsRNAs) have been found in two isolates of the plant pathogenic fungus Fusarium graminearum which produce trichothecene mycotoxins. The isolates 8.2 and 19.2 had dsRNAs in the size of about 2.0 kb and 6.0 kb, respectively, which were associated with capsid proteins and persisted within the cytoplasm of the infected host cells as encapsidated virus-like particles (VLPs). The dsRNAs contained in the VLP pellets were the same size as the dsRNA isolated in total nucleic acid preparations. In the VLP pellets the isolate 19.2 had a second dsRNA with the size of about 1.6 kb. After mycovirus purification one icosahedral particle of about 28 nm in diameter from the isolate 8.2 and two icosahedral particles of about 28 nm and 38 to 40 nm in diameter from the isolate 19.2 could be identified with electron microscopy. SDS-PAGE analysis of the VLPs from the isolate 8.2 revealed one major protein component of approximately 65 kDa, while the isolate 19.2 had two major protein bands at about 94 kDa and 105 kDa. Both isolates were studied for potential trichothecene production. Tox5 PCR showed a 658 bp fragment in each isolate. In addition, both strains were able to produce the trichothecenes deoxynivalenol (DON), the derivatives acetyl-DON (3-A-DON, 15-A-DON) and nivalenol (NIV) in vitro.     

Three different M1 RNA-containing viruslike particle types in Saccharomyces cerevisiae: in vitro M1 double-stranded RNA synthesis.

Killer strains of Saccharomyces cerevisiae bear at least two different double-stranded RNAs (dsRNAs) encapsidated in 39-nm viruslike particles (VLPs) of which the major coat protein is coded by the larger RNA (L-A dsRNA). The smaller dsRNA (M1 or M2) encodes an extracellular protein toxin (K1 or K2 toxin). Based on their densities on CsCl gradients, L-A- and M1-containing particles can be separated. Using this method, we detected a new type of M1 dsRNA-containing VLP (M1-H VLP, for heavy) that has a higher density than those previously reported (M1-L VLP, for light). M1-H and M1-L VLPs are present together in the same strains and in all those we tested. M1-H, M1-L, and L-A VLPs all have the same types of proteins in the same approximate proportions, but whereas L-A VLPs and M1-L VLPs have one dsRNA molecule per particle, M1-H VLPs contain two M1 dsRNA molecules per particle. Their RNA polymerase produces mainly plus single strands that are all extruded in the case of M1-H particles but are partially retained inside the M1-L particles to be used later for dsRNA synthesis. We show that M1-H VLPs are formed in vitro from the M1-L VLPs. We also show that the peak of M1 dsRNA synthesis is in fractions lighter than M1-L VLPs, presumably those carrying only a single plus M1 strand. We suggest that VLPs carrying two M1 dsRNAs (each 1.8 kilobases) can exist because the particle is designed to carry one L-A dsRNA (4.5 kilobases).     

High-frequency occurrence of virus-like particles with double-stranded RNA genome in Fusarium poae

Abstract Fifty-five geographically different strains of Fusarium poae were assayed for the presence of extrachromosomal nucleic acid elements. All strains were found to harbour double-stranded RNA (dsRNA) elements and encapsidated virus-like particles (VLP). There were great individual differences in dsRNA patterns of the various strains, but numbers and sizes characteristic for a given isolate remained unchanged after repeated subculturing of the fungi. Morphological alterations or signs of degeneration were not observed in dsRNA-containing isolates. This is the first report on the ubiquitous occurrence of dsRNAs in a hyphomycete fungus species.     

Presence of double-stranded RNA and virus-like particles in Rhizopus isolates

Fungal isolates belonging to four Rhizopus species were screened for the presence of double-stranded RNA (dsRNA) molecules. Five (two R. stolonifer, two R. microsporus, and one R. oryzae) of the 27 isolates examined harboured such genetic elements. Electrophoresis of the nucleic acids revealed five RNA patterns, with 1-5 discrete dsRNA bands. The molecular sizes corresponding to these bands were 2.2-14.8 kb. Gel electrophoresis of purified virus-like particles (VLPs) indicated only one capsid of similar size in all virus-harbouring strains; when investigated by electron microscopy, they were found to be polyhedral VLPs 40 nm in diameter. In one of the R. microsporus isolates an uncapsidated large dsRNA segment (14.8 kb) was observed. No phenotypic differences were observed between uninfected and virus-harbouring Rhizopus isolates.     

A double-stranded RNA mycovirus from the plant pathogenic fungus, Fusarium solani f. sp. robiniae

Abstract Two kinds of double-stranded RNA (dsRNA), estimated to be 1.9 and 1.7 kb in size, were detected in the plant pathogenic fungus, Fusarium solani f. sp. robiniae . Isometric virus-like particles (VLPs), 30 nm in diameter, were recovered from cell extracts as a discrete band when centrifuged through a CsCl density gradient. The dsRNA molecules extracted from VLP preparations were identical in electrophoretic mobility to the dsRNAs obtained directly from cells. SDS-PAGE analysis of the VLPs revealed a single polypeptide of 38 kDa. The dsRNAs obtained directly from cells. SDS-PAGE analysis of the asexual cycle).     

Presence of non-suppressive, M2-related dsRNAs molecules in Saccharomyces cerevisiae strains isolated from spontaneous fermentations

Total dsRNA extractions in five killer K2 strains of Saccharomyces cerevisiae isolated from spontaneous fermentations revealed the presence of a novel dsRNA fragment (which we named NS dsRNA) of approximately 1.30 kb, together with L and M2 dsRNAs. NS dsRNA appeared to be encapsidated in the same kind of viral particles as L and M2 dsRNA. Northern blot hybridization experiments indicated that NS dsRNA was derived from M2 dsRNA, likely by deletion of the internal A+U-rich region. However, unlike S dsRNAs (suppressive forms derived from M1 dsRNA in K1 killers), NS dsRNA did not induce exclusion of the parental M2 dsRNA when the host strain was maintained for up to 180 generations of growth.     

Virus-like particle capsid proteins encoded by different L double-stranded RNAs of Saccharomyces cerevisiae: their roles in maintenance of M double-stranded killer plasmids.

The plasmid determinants of killer phenotypes in type K1 and K2 killer yeast cells are the 1.9-kilobase (kb) M1 and 1.7-kb M2 double-stranded RNAs (dsRNAs), respectively. These are dependent for their maintenance and encapsidation, in Saccharomyces cerevisiae virus ScV-M1 or ScV-M2 virus-like particles, on the capsid provided by one of a group of moderately related 4.7-kb dsRNAs called LA. The L1A and L2A dsRNAs found in naturally isolated K1 and K2 killers encode 88-kilodalton VL1A-P1 and 86-kilodalton VL2A-P1 capsids, respectively. These are competent for encapsidating homologous LA dsRNAs as well as M dsRNAs. Most strains of S. cerevisiae, including killers, contain one of a second group of closely related 4.7-kb dsRNAs called LBC. These encode their own 82-kilodalton capsid protein, VLBC-P1, which, at least in strains containing only LBC, encapsidates homologous dsRNA in ScV-LBC virus-like particles. In a K1 killer strain containing both L1A and LBC, ScV-M1 particles contain only VL1A-P1. In such strains it is probable that each virus-like particle contains a single capsid type and that each L dsRNA is encapsidated by a homologous capsid.     

Nonhost resistance of rice to rust pathogens

Rice is atypical in that it is an agricultural cereal that is immune to fungal rust diseases. This report demonstrates that several cereal rust species (Puccinia graminis f. sp tritici, P. triticina, P. striiformis, and P. hordei) can infect rice and produce all the infection structures necessary for plant colonization, including specialized feeding cells (haustoria). Some rust infection sites are remarkably large and many plant cells are colonized, suggesting that nutrient uptake occurs to support this growth. Rice responds with an active, nonhost resistance (NHR) response that prevents fungal sporulation and that involves callose deposition, production of reactive oxygen species, and, occasionally, cell death. Genetic variation for the efficacy of NHR to wheat stem rust and wheat leaf rust was observed. Unlike cereal rusts, the rust pathogen (Melampsora lini) of the dicotyledenous plant flax (Linum usitatissimum) rarely successfully infects rice due to an apparent inability to recognize host-derived signals. Morphologically abnormal infection structures are produced and appressorial-like structures often don't coincide with stomata. These data suggest that basic compatibility is an important determinate of nonhost infection outcomes of rust diseases on cereals, with cereal rusts being more capable of infecting a cereal nonhost species compared with rust species that are adapted for dicot hosts.     

Double-stranded RNA and virus-like particles in the edible basidiomycete Flammulina velutipes (Enokitake)

One of the commercial strains of Flammulina velutipes was analyzed for the presence of double-stranded RNA (dsRNA) elements to examine the underlying mechanism of strain degeneration. As a result, two dsRNA elements sized 1.9 and 1.8 kb were detected in mycelium derived from spontaneously brown-colored fruit body. They were not detected in the normal strains or in fruiting-impaired degenerative isolates. The dsRNAs were not in the nuclear or mitochondrial fractions, but were located in the cytoplasmic fraction. The presence of virus-like particles of ca. 50 nm diameter associated with the dsRNAs was confirmed by electron microscopic observation.     

Dynamics of double-stranded RNA segments in a Helicobasidium mompa clone from a tulip tree plantation

Eighty-three isolates of the violet root rot fungus, Helicobasidium mompa, were collected in a tulip tree plantation and analyzed for the dynamics of double-stranded (ds) RNA for five years. They were divided into eight mycelial compatibility groups (MCGs). Prevalent MCGs 60 and 68 included 61 and 11 isolates, respectively. Electrophoretic profiles of dsRNA in the first year collection of MCG 60 contained no or a single large dsRNA (more than 10 kb) with or without small dsRNAs (ca. 2.0-2.5 kb). Additional dsRNA fragments, i.e., a middle dsRNA (ca. 8.0 kb) or another type of small dsRNAs, became evident within MCG 60 isolates with time. Northern hybridization revealed the relatedness of all large and middle dsRNA fragments within MCG 60 but small fragments of dsRNA were variable. Large dsRNA fragment differed from that in other MCGs even in the same field. Correlation between specific dsRNA fragments and hypovirulence was not observed. Possible explanations for the accumulation of dsRNA fragments during the growth of disease patch by MCG 60 are discussed in terms of their internal changes such as evolution of novel dsRNA fragments from pre-existing viruses or fungal genomic DNA and horizontal transmissions.