Читать книгу Principles of Virology - Jane Flint, S. Jane Flint - Страница 144

Before the advent of recombinant DNA technology, it was extremely difficult for investigators to determine the locations of mutations in viral genomes. The marker rescue technique (described in “Introducing Mutations into the Viral Genome” below) was a solution to this problem, but before it was developed, other, less satisfactory approaches were exploited.

Recombination mapping can be applied to both DNA and RNA viruses. Recombination results in genetic exchange between genomes within the infected cell. The frequency of recombination between two mutations in a linear genome increases with the physical distance separating them. In practice, cells are coinfected with two mutants, and the frequency of recombination is calculated by dividing the titer of phenotypically wild-type virus (Box 3.7) obtained under restrictive conditions (e.g., high temperature) by the titer measured under permissive conditions (e.g., low temperature). The recombination frequency between pairs of mutants is determined, allowing the mutations to be placed on a contiguous map. Although a location can be assigned for each mutation relative to others, this approach does not result in a physical map of the actual location of the base change in the genome.

In the case of RNA viruses with segmented genomes, the technique of reassortment allows the assignment of mutations to specific genome segments. When cells are coinfected with both mutant and wild-type viruses, the progeny includes reassortants that inherit RNA segments from either parent. The origins of the RNA segments can be deduced from their migration patterns during gel electrophoresis (Fig. 3.11) or by nucleic acid hybridization. By analyzing a panel of such reassortants, the segment responsible for the phenotype can be identified.