THE MOLECULAR EVOLUTION OF CUCUMBER MOSAIC VIRUS

黄瓜花叶病毒的分子进化

基本信息

  • 批准号:
    6215627
  • 负责人:
  • 金额:
    $ 11.07万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    1977
  • 资助国家:
    美国
  • 起止时间:
    1977-06-01 至 2004-06-30
  • 项目状态:
    已结题

项目摘要

We will characterize a number of cucumber mosaic virus (CMV) isolates found on the Island of Hawaii. We will also determine the nucleotide sequences of the RNA genomes of these viruses using reverse transcriptase/PCR technology. This first portion of the project will yield information regarding the existing diversity of CMV on the island. Further, by correlating sequence differences with host range and symptom differences, we will identify nucleotides important in determining these viral phenotypes. Our preliminary results show 91% sequence identify between two CMV strains found on different hosts, but less than 10 miles preliminary results show 91% sequence identify between two CMV strains found on different hosts, but less than 10miles part, and 99.1% sequence identify between two strains found on different host species separated by less than 20 feet. The cDNAs for one of the CMV strains will then be cloned for propagation in Escherichia coli. This will serve to provide a source of a homogenous viral population, which can also be targeted for site-directed mutagenesis experiments. The homogeneous viral population will be used to perform experiments in which we infect plants, and then analyze by sequencing the rate at which mutations become fixed during CMV infection. We will infect a single leaf, and after infection becomes systemic, analyze virus in the inoculated leaf as compared to other parts of the plant. We will then passage the virus to other plants of the same as well as different species and similar analyze the viral sequences. This study will reveal the rates of mutations and whether or not they can be accelerated in certain hosts. We will also target the CMV cDNA clones for site-directed mutagenesis. Our earlier analysis will have implicated certain nucleotide positions as candidates for determining certain viral phenotypes, and these mutagenesis studies will narrow the field and eventually identify specific important nucleotides. This analysis will in turn identify important amino acid residues on specific viral proteins. Undergraduate studies will be trained in the handling and manipulating of eucaryotic RNA viruses, without being at risk for infection. During viral characterization and sequence analysis, they will also gain experience in the enzyme linked immunosorbent assay (ELISA), reverse transcription, and the polymerase chain reaction (PCR). The will also become very familiar with nucleotide sequence analysis, cDNA synthesis and cloning, which in turn involves the restriction enzyme treatment and ligation of DNAs, and propagation and transformation of E. coli. Finally, the mutagenesis studies will provide the students with experience in site- directed mutagenesis of DNA and with in vitro mRNA transcription.
我们将描述一些黄瓜花叶病毒(CMV)分离株发现的夏威夷岛。我们还将使用逆转录酶/PCR技术确定这些病毒的RNA基因组的核苷酸序列。该项目的第一部分将产生有关岛上CMV现有多样性的信息。此外,通过将序列差异与宿主范围和症状差异相关联,我们将确定在确定这些病毒表型中重要的核苷酸。初步结果表明,不同寄主上的两个CMV株系之间的序列同源性为91%,但在10英里以内;不同寄主上的两个CMV株系之间的序列同源性为99.1%,但在20英尺以内。然后将CMV菌株之一的cDNA克隆用于在大肠杆菌中繁殖。这将用于提供同源病毒群体的来源,其也可以被靶向用于定点诱变实验。 同质病毒群体将用于进行实验,在实验中我们感染植物,然后通过测序分析CMV感染期间突变固定的速率。我们将感染一片叶子,在感染成为系统性的之后,分析接种叶子中的病毒与植物的其他部分相比。然后,我们将通过病毒到其他植物的相同以及不同的物种和类似的分析病毒序列。这项研究将揭示突变的速率,以及它们是否可以在某些宿主中加速。我们还将针对CMV cDNA克隆进行定点突变。我们早期的分析将涉及某些核苷酸位置作为确定某些病毒表型的候选者,这些诱变研究将缩小该领域并最终确定特定的重要核苷酸。这种分析将反过来识别特定病毒蛋白上的重要氨基酸残基。本科研究将在处理和操纵真核RNA病毒方面进行培训,而不会有感染的风险。在病毒表征和序列分析过程中,他们还将获得酶联免疫吸附试验(ELISA),逆转录和聚合酶链反应(PCR)的经验。本课程还将非常熟悉核苷酸序列分析、cDNA合成和克隆,这又涉及到DNA的限制性内切酶处理和连接,以及E.杆菌最后,诱变研究将为学生提供DNA定点诱变和体外mRNA转录的经验。

项目成果

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MICHAEL H SHINTAKU其他文献

MICHAEL H SHINTAKU的其他文献

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{{ truncateString('MICHAEL H SHINTAKU', 18)}}的其他基金

THE MOLECULAR EVOLUTION OF CUCUMBER MOSAIC VIRUS
黄瓜花叶病毒的分子进化
  • 批准号:
    6630594
  • 财政年份:
    2002
  • 资助金额:
    $ 11.07万
  • 项目类别:
THE MOLECULAR EVOLUTION OF CUCUMBER MOSAIC VIRUS
黄瓜花叶病毒的分子进化
  • 批准号:
    6471799
  • 财政年份:
    2001
  • 资助金额:
    $ 11.07万
  • 项目类别:
THE MOLECULAR EVOLUTION OF CUCUMBER MOSAIC VIRUS
黄瓜花叶病毒的分子进化
  • 批准号:
    6495391
  • 财政年份:
    2001
  • 资助金额:
    $ 11.07万
  • 项目类别:

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