Mutagenesis

Spontaneous mutations occur because of chance errors during replication. Mutation frequency can be enhanced by treatment of virions or isolated viral nucleic acid with physical agents such as UV light or X-irradiation or with chemicals such as nitrous acid or nitrosoguanidine. Base analogs, such as 5'-fIuorouracil (for RNA viruses) or 5' bromodeoxyuridine (for DNA viruses), are mutagenic only when virus is grown in their presence because they are incorporated into the viral nucleic acid and produce mutations by miscoding during replication.

S i te-Specific Mu tngeucs is

Instead of relying on chance mutations anywhere in the genome, genetic engineering makes it possible to introduce mutations at any site of interest. Site-specific (or site-directed) mutagenesis enables the experimenter to substitute any selected nucleotide for that in a prescribed position in a DNA molecule (a DNA genome or complementary DNA (cDNA) transcribed from an RNA genome). Several techniques to achieve this are available. For example, the viral DNA is denatured and ssDNA is purified then transferred into an appropriate vector such as bacteriophage M13 A short synthetic oligonucleotide that is homologous to (he relevant region except that it contains the desired nucleotide is then annealed to the ssDNA, and the synthesis of this complementary strand primer is completed by DNA polymerase to yield a dsDNA molecule. The hybrid phage DNA is now propagated by transfection of permissive bacteria. Progeny phages containing the mutated genome are selected by hybridization with the oligonucleotide probe, and marker rescue is used to recover the mutated gene in infectious virus. The polymerase chain reaction can be used to verify the location of the genetic lesions.

Until recently, site-directed mutagenesis and other types of genetic engineering were restricted to DNA viruses and plus strand RNA viruses, from which cDNA could be produced by reverse transcription However, it has now become possible to apply these methods of reverse genetics to minus strand viruses, including those with segmented genomes. Site-directed mutagenesis has opened up new research areas, such as dissection of the function of individual genes and the proteins for which they code, or of particular regions of these genes and proteins. At a practical level, mutations can be introduced into particular genes, for example, those contributing to viral virulence, to produce mutants suitable for use as attenuated live-virus vaccines.

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