Biology Reference
In-Depth Information
Fig. 1
Different methods of herpesvirus mutagenesis.
a
Site-directed mutagenesis in eukaryotic
cells. A linear DNA fragment containing a marker along with the mutation (
M
) flanked by
homologies to the viral target sequence is transfected into virus infected cells. By homologous
recombination (
dashed lines
) the marker gene and the mutation insert into some of the virus
genomes deleting the wild type sequence (wt) Recombinant viruses and wild type viruses need
further separation.
b
Cosmid mutagenesis in eukaryotic cells. Overlapping viral fragments span-
ning the entire genome are cloned as cosmids. A mutation (
M
) is introduced into one fragment.
After transfection of the linearized cosmid clones into permissive cells, the virus genome can be
reassembled by several homologous recombination steps generating the mutant virus.
c
Principle
of the mutagenesis with bacterial artificial chromosomes (BACs) in
E. coli
. Recombinant viral
BACs can be generated using various site-directed and random mutagenesis approaches.
Recombinant viral BAC DNA with a mutation (
M
) is then transfected into permissive eukaryotic
cells and the mutant virus progeny is thereby reconstituted
virus reconstitution became an issue (Horsburgh et al. 1999). Since virus recon-
stitution relies on several recombination events in eukaryotic cells, changes in the
recombinant genomes may occur (Kemble et al. 1996). The cosmid-based muta-
genesis is based on recombination in cells and requires the regeneration of a rep-
lication-competent virus genome. Therefore, mutant genomes in which an
essential gene is affected are difficult, if not impossible, to construct. Unfortunately,
revertants cannot be constructed without unreasonable efforts, since the genera-
tion of a revertant in its strict sense would require cosmid cloning of the newly
generated recombinant genome. Therefore, the usage of this elegant method was
restricted to mutagenesis of genes with a known phenotype.
