Trends in Genetics
Volume 16, Issue 6, 1 June 2000, Pages 254-259
Journal home page for Trends in Genetics

Review
Forward with BACs: new tools for herpesvirus genomics

https://doi.org/10.1016/S0168-9525(00)02015-1Get rights and content

Abstract

The large, complex genomes of herpesviruses document the high degree of adaptation of these viruses to their hosts. Not surprisingly, the methods developed over the past 30 years to analyse herpesvirus genomes have paralleled those used to investigate the genetics of eukaryotic cells. The recent use of bacterial artificial chromosome (BAC) technology in herpesvirus genetics has made their genomes accessible to the tools of bacterial genetics. This has opened up new avenues for reverse and forward genetics of this virus family in basic research, and also for vector and vaccine development.

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.

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