Analysis of genetic variability and mapping of point mutations in influenza virus by the RNase A mismatch cleavage method.
Analysis of genetic variability and mapping of point mutations in influenza virus by the RNase A mismatch cleavage method.
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通过 RNase A 错配切割方法分析流感病毒的遗传变异性和点突变图谱。
DOI：
10.1073/pnas.85.10.3522
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发表时间：
1988
影响因子：
11.1
通讯作者：
Perucho,M
中科院分区：
文献类型：
--
作者：
Lopez-Galindez,C;Lopez,JA;Melero,JA;delaFuente,L;Martinez,C;Ortin,J;Perucho,M
我们以流感病毒为模型系统，应用RNase A错配切割方法分析了RNA病毒的遗传变异。用克隆的某一病毒株的血凝素基因合成的统一标记的RNA探针与从其他具有特征或非特征遗传组成的病毒株分离的RNA杂交。RNA。用RNase a酶切含有可变碱基错配的RNA异双链，并通过变性聚丙烯酰胺凝胶电泳分析抗性产物。我们发现许多这些单碱基错配被RNase A切割，产生针对每种不同病毒株的独特和特征的抗性RNA片段模式。对裂解模式的比较分析可以对田间菌株的遗传亲缘性和进化进行定性估计。我们还发现，RNase A在单碱基错配处的切割可以很容易地检测和定位单克隆抗体抗性变异中存在的点突变。该方法在RNA病毒的研究中具有广泛的应用前景，不仅可用于流行病学分析，还可用于一些诊断问题，如表型突变的表征。
We have applied the RNase A mismatch cleavage method to analyze genetic variability in RNA viruses by using influenza virus as a model system. Uniformly labeled RNA probes synthesized from a cloned hemagglutinin gene of a given viral strain were hybridized to RNA isolated from other strains of characterized or uncharacterized genetic composition. The RNA.RNA heteroduplexes containing a variable number of base mismatches were digested with RNase A, and the resistant products were analyzed by denaturing polyacrylamide gel electrophoresis. We show that many of these single base mismatches are cleaved by RNase A, generating unique and characteristic patterns of resistant RNA fragments specific for each of the different viral strains. Comparative analysis of the cleavage patterns allows a qualitative estimation of the genetic relatedness and evolution of field strains. We also show that cleavage by RNase A at single base mismatches can readily detect and localize point mutations present in monoclonal antibody-resistant variants. This method should have wide applications in the study of RNA viruses, not only for epidemiological analysis but also in some diagnostic problems, such as characterization of phenotypic mutants.