Trends in Genetics
ReviewForward with BACs: new tools for herpesvirus genomics
Section snippets
Chemical mutagenesis
Any way to gain insight into the functional role of genes profits from genetic alterations. The introduction of random point mutations using mutagenic chemicals is a well-established means to produce a large number of mutants that can be screened for interesting phenotypes. This classical genetic approach was introduced into herpesvirus research 30 years ago for the production of temperature-sensitive (ts) virus mutants2. Generating such mutants is quite straightforward but identifying the
BAC technology
In mammalian genetics, the small size of plasmids and cosmids has limited their use for mapping and analysing large genomes. For this reason, new cloning vehicles were developed that could accommodate larger segments of genomic DNA. Yeast artificial chromosomes (YACs) are undoubtedly the largest, with a coding capacity of more than 2 Mbp (Ref. 8). However, YACs are not easy to handle and the recombination machinery of yeast renders YACs prone to unwanted rearrangements and deletions.
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Allelic exchange
Cloning a herpesvirus genome as an infectious BAC allows shuttling of the genome between eukaryotic and prokaryotic cells, but how does this facilitate site-directed mutagenesis? The large size of BACs precludes simple enzymatic cleavage and ligation approaches so mutations need to be introduced by homologous recombination, as in eukaryotic cells. In E. coli, homologous recombination usually requires the bacterial RecA and RecBCD enzymes19, 20. In many laboratory strains of E. coli, the recA
Transposon mutagenesis
Site-directed mutagenesis helps in the functional analysis of a gene of interest (reverse genetics). By contrast, the classical forward-genetic approach of identifying the gene responsible for an observed phenotype is inherently difficult. Reverse genetics merely allows hypotheses (‘candidate genes’) to be tested. In the absence of a candidate gene for a functional property of the genome, unbiased approaches are needed (i.e. rapid and efficient methods of random mutagenesis and suitable
Conclusions and perspectives
The intricate interaction of herpesviruses with their host cells has led to the use of similar techniques for their mutational analysis. The recent advances in this field through the introduction of BAC technology are especially welcome as complete sequences of large genomes become available. To translate this sequence data into meaningful functional information, genetic experiments are required. Importantly, the BACward approach facilitates both forward and reverse genetics of herpesviruses (
Acknowledgements
The authors’ work was supported by grants from the Deutsche Forschungsgemeinschaft, the Bundesministerium für Bildung und Forschung, the Fonds der chemischen Industrie and the European Union.
References (39)
Molecular engineering of the herpes simplex virus genomeinsertion of a second L-S junction into the genome causes additional genome inversions
Cell
(1980)- et al.
A generalized technique for deletion of specific genes in large genomesalpha gene 22 of herpes simplex virus 1 is not essential for growth
Cell
(1981) - et al.
Exchanging partnersrecombination in E. coli
Trends Genet.
(1996) A new logic for DNA engineering using recombination in Escherichia coli
Nat. Genet.
(1998)- et al.
Epstein–Barr virus vectors for gene expression and transfer
Curr. Opin. Biotechnol.
(1998) - et al.
Herpesvirus evasion of the immune system
Curr. Top. Microbiol. Immunol.
(1998) Temperature-sensitive mutants of herpesviruses
Curr. Top. Microbiol. Immunol.
(1975)Isolation and characterization of deletion mutants of herpes simplex virus type 1 in the gene encoding immediate-early regulatory protein ICP4
J. Virol.
(1985)Regeneration of herpesviruses from molecularly cloned subgenomic fragments
J. Virol.
(1988)Allele replacementan application that permits rapid manipulation of herpes simplex virus type 1 genomes
Gene Ther.
(1999)