Biology Reference
In-Depth Information
Mutagenesis of the Cytomegalovirus Genome
Z. Ruzsics , U. H. Koszinowski (
ΓΌ
)
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
42
CMV Genetics in Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
42
Forward (Classical) Genetics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
42
Reverse Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
43
CMV Genetics in Bacteria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
45
Bacterial Artificial Chromosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
45
Cloning and Maintenance of CMV Genomes as BACs . . . . . . . . . . . . . . . . . . . . . . . . . . . .
45
Allelic Exchange by Shuttle Plasmid Mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
46
Allelic Exchange Using Linear Fragment Mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . .
47
Transposon Mutagenesis for Reverse and Forward Genetics . . . . . . . . . . . . . . . . . . . . . . . .
49
Mutants, Revertants and the Mutation-Phenotype Connection . . . . . . . . . . . . . . . . . . . . . . .
51
Genetic Analysis of Essential Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
53
Comprehensive Mutational Analysis of Essential CMV Genes . . . . . . . . . . . . . . . . . . . . . .
53
Identification and Analysis of Dominant Negative Mutants . . . . . . . . . . . . . . . . . . . . . . . . .
55
Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
57
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
57
Abstract
Bacterial artificial chromosomes (BACs) are DNA molecules assembled
in vitro from defined constituents and are stably maintained as one large DNA
fragment in
Escherichia coli
. Artificial chromosomes are useful for genome
sequencing programs, for transduction of DNA segments into eukaryotic cells, and
for functional characterization of genomic regions and entire viral genomes such
as cytomegalovirus (CMV) genomes. CMV genomes in BACs are ready for the
advanced tools of
E. coli
genetics. Homologous and site-specific recombination,
or transposon-based approaches allow for the engineering of virtually any kind of
genetic change.
U.H. Koszinowski
Max von Pettenkofer Institute, Dept. of Virology, Gene Center,
Ludwig-Maximilians-University , 81377 Munich , Germany
koszinowski@mvp.uni-muenchen.de
