Analysis of genetic variability and mapping of point mutations in influenza virus by the RNase A mismatch cleavage method. 1988

C Lopez-Galindez, and J A Lopez, and J A Melero, and L de la Fuente, and C Martinez, and J Ortin, and M Perucho
Department of Biochemistry, State University of New York, Stony Brook 11794.

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.

UI MeSH Term Description Entries
D009154 Mutation Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations. Mutations
D009693 Nucleic Acid Hybridization Widely used technique which exploits the ability of complementary sequences in single-stranded DNAs or RNAs to pair with each other to form a double helix. Hybridization can take place between two complimentary DNA sequences, between a single-stranded DNA and a complementary RNA, or between two RNA sequences. The technique is used to detect and isolate specific sequences, measure homology, or define other characteristics of one or both strands. (Kendrew, Encyclopedia of Molecular Biology, 1994, p503) Genomic Hybridization,Acid Hybridization, Nucleic,Acid Hybridizations, Nucleic,Genomic Hybridizations,Hybridization, Genomic,Hybridization, Nucleic Acid,Hybridizations, Genomic,Hybridizations, Nucleic Acid,Nucleic Acid Hybridizations
D009980 Influenza A virus The type species of the genus ALPHAINFLUENZAVIRUS that causes influenza and other diseases in humans and animals. Antigenic variation occurs frequently between strains, allowing classification into subtypes and variants. Transmission is usually by aerosol (human and most non-aquatic hosts) or waterborne (ducks). Infected birds shed the virus in their saliva, nasal secretions, and feces. Alphainfluenzavirus influenzae,Avian Orthomyxovirus Type A,FLUAV,Fowl Plague Virus,Human Influenza A Virus,Influenza Virus Type A,Influenza Viruses Type A,Myxovirus influenzae-A hominis,Myxovirus influenzae-A suis,Myxovirus pestis galli,Orthomyxovirus Type A,Orthomyxovirus Type A, Avian,Orthomyxovirus Type A, Human,Orthomyxovirus Type A, Porcine,Pestis galli Myxovirus,Fowl Plague Viruses,Influenza A viruses,Myxovirus influenzae A hominis,Myxovirus influenzae A suis,Myxovirus, Pestis galli,Myxoviruses, Pestis galli,Pestis galli Myxoviruses,Plague Virus, Fowl,Virus, Fowl Plague
D010957 Plasmids Extrachromosomal, usually CIRCULAR DNA molecules that are self-replicating and transferable from one organism to another. They are found in a variety of bacterial, archaeal, fungal, algal, and plant species. They are used in GENETIC ENGINEERING as CLONING VECTORS. Episomes,Episome,Plasmid
D002460 Cell Line Established cell cultures that have the potential to propagate indefinitely. Cell Lines,Line, Cell,Lines, Cell
D002642 Chick Embryo The developmental entity of a fertilized chicken egg (ZYGOTE). The developmental process begins about 24 h before the egg is laid at the BLASTODISC, a small whitish spot on the surface of the EGG YOLK. After 21 days of incubation, the embryo is fully developed before hatching. Embryo, Chick,Chick Embryos,Embryos, Chick
D005814 Genes, Viral The functional hereditary units of VIRUSES. Viral Genes,Gene, Viral,Viral Gene
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D012259 Ribonuclease, Pancreatic An enzyme that catalyzes the endonucleolytic cleavage of pancreatic ribonucleic acids to 3'-phosphomono- and oligonucleotides ending in cytidylic or uridylic acids with 2',3'-cyclic phosphate intermediates. EC 3.1.27.5. RNase A,Ribonuclease A,Pancreatic RNase,RNase I,Ribonuclease (Pancreatic),Ribonuclease I,Pancreatic Ribonuclease,RNase, Pancreatic
D014644 Genetic Variation Genotypic differences observed among individuals in a population. Genetic Diversity,Variation, Genetic,Diversity, Genetic,Diversities, Genetic,Genetic Diversities,Genetic Variations,Variations, Genetic

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