Biology Reference
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above for ectopic
cis
-complementation (see Sect. 4.6). The
wt
gene is subcloned
in an FRT containing rescue plasmid. This construct is subjected to random Tn
mutagenesis, as described for small plasmids. Mutants are sequenced and a com-
prehensive set of mutants is selected. Recombination between the FRT transfer
plasmids carrying the mutants and the BAC lacking the gene of interest, mediated
by the FLP recombinase, provides genomes that are
cis
-complemented by the
mutant set. The complemented genomes are tested one by one for virus rescue. In
combination with standard biochemical or cell biological assays, this procedure
allowed genetic analysis of essential gene functions of MCMV at high resolution
(Bubeck et al. 2004; Lotzerich et al. 2006). The method easily maps functionally
important sites in essential viral proteins.
Identification and Analysis of Dominant Negative Mutants
The function of nonessential genes is studied by gene deletion and by loss-of-function
mutants. By
cis
-complementation assays functionally important sites of essential genes
can be mapped. Unfortunately, a major target of genetics, the null phenotype of an
essential gene is generally hard to come by. This requires the cumbersome establish-
ment and optimization of a
trans-
complementation system for each gene under study.
Here, we try to develop a systematic approach. Dominant negative (DN) mutants are
special null mutants that induce the null phenotype even in the presence of the
wt
allele.
DN mutants of cellular genes have been proven to be a valuable for genetic analysis of
complex pathways (Herskowitz 1987). Knowledge of protein structure, protein func-
tions or sequence motifs aid the design of DN mutants (Crowder and Kirkegaard 2005).
Unfortunately, the information on the majority of herpesvirus proteins is too limited for
knowledge based construction of DN mutants.
Therefore, we set up a random approach to isolate DN mutants of CMV genes.
The Tn7-based linker-scanning mutagenesis introduces 5-aa insertions into coding
sequences and provides a comprehensive set of subtle insertion mutants of the
ORF. Nonfunctional mutants are selected from a library by a
cis
-complementation
screen as described above. These mutants can then be introduced into the
wt
MCMV genome. This allows testing their inhibitory potential (Fig. 7a). If the
mutant interferes with the function of the essential
wt
allele, virus reconstitu-
tion is inhibited. As we showed for both M50 (Rupp et al. 2007) and the M53
(Z. Ruzsics and U.H. Koszinowski, unpublished data), such mutants represent
only a small proportion of the null mutants but can be isolated by a standardized
procedure.
Transfection of the viral nucleic acid is error-prone and not only a DN function,
but also unrelated effects may prevent virus reconstitution. Conditional expression of
the inhibitory mutants in the context of the
wt
genome should allow virus reconstitu-
tion in the off state and should induce the null phenotype when turned on (Fig. 7b).
We constructed a regulated expression system for MCMV (Rupp et al. 2005) in
which the constitutive viral expression of the TetR blocks the transcription of the
