Biology Reference
In-Depth Information
CMV Genetics in Bacteria
Bacterial Artificial Chromosomes
The desire to clone large eukaryotic genomes in order to acquire contiguous physical
chromosome maps brought emphasis to cloning vectors of larger insert capacity.
Although yeast artificial chromosomes (YACs) can encompass DNA fragments
larger than 1000 kbp, YACs are marred by spontaneous rearrangements, insert
instability and yeast DNA contamination (Ramsay 1994; Schalkwyk et al. 1995).
Stable maintenance of foreign DNA larger than 300 kbp in size was reported using
either a fertility factor (F-factor) replicon based bacterial artificial chromosome
(BAC) (Shizuya et al. 1992) or a bacteriophage P1 replicon-based cloning system
called PAC (Ioannou et al. 1994). In contrast to YACs or cosmids, the BAC clones
show surprising sequence stability in appropriate strains of
E. coli
. As a rule, most
of the useful BAC hosts are derivatives of DH10B, pointing to the importance of
the genetic background of the
E. coli
host strain (Shizuya et al. 1992; Tao and
Zhang 1998). Human genome fragments as BACs were maintained over 100
generations in bacteria without detectable changes (Shizuya et al. 1992). The strict
control of the F-factor-based replicon keeps one copy of the BAC per cell and
reduces recombination events via repetitive DNA elements present in the eukaryotic
DNAs. BACs now play a central role in genome research.
Cloning and Maintenance of CMV Genomes as BACs
We pioneered BAC cloning and mutagenesis of MCMV (Messerle et al. 1997).
This concept was quickly taken up by several groups. By now, many genomes of
many herpesviruses, including different strains of CMVs from various species,
have been cloned (for review see Brune et al. 2000). The construction of a herpes-
virus BAC starts with conventional mutagenesis procedures. First, the BAC vector
sequences flanked with appropriate viral sequences are introduced into the genome
by homologous recombination in cells. The linear double-stranded DNA genome
of herpesviruses circularizes after infection and these replication intermediates of
the BAC-containing herpesvirus genome are transferred by transformation into
E. coli
. This transformation step is needed only once. In
E. coli
, virus functions do
not need to be expressed for either genome amplification or mutagenesis procedures.
Potential size constraints of the CMV genomes with regard to packaging limits due
to the oversize of the inserted BAC cassette can be solved by deletion of nonessen-
tial genomic sequences. (Messerle et al. 1997; Borst et al. 1999; Wagner et al.
1999). The deleted sequences can be reinserted after the cloning procedure (Wagner
et al. 1999). To regenerate infectious virus, the herpesvirus BACs are transfected
into permissive host cells (Fig. 1c). Herpesvirus genomes in BACs are ready for the
advanced tools of
E. coli
genetics, which include homologous and site-specific
