Insertion and deletion mutagenesis of the human cytomegalovirus genome
Studies on human cytomegalovirus (CMV) have been limited by a paucity of molecular genetic techniques available for manipulating the viral genome. The authors have developed methods for site-specific insertion and deletion mutagenesis of CMV utilizing a modified Escherichia coli lacZ gene as a genetic marker. The lacZ gene was placed under the control of the major ..beta.. gene regulatory signals and inserted into the viral genome by homologous recombination, disrupting one of two copies of this ..beta.. gene within the L-component repeats of CMV DNA. They observed high-level expression of ..beta..-galactosidase by the recombinant in a temporally authentic manner, with levels of this enzyme approaching 1% of total protein in infected cells. Thus, CMV is an efficient vector for high-level expression of foreign gene products in human cells. Using back selection of lacZ-deficient virus in the presence of the chromogenic substrate 5-bromo-4-chloro-3-indolyl ..beta..-D-galactoside, they generated random endpoint deletion mutants. Analysis of these mutant revealed that CMV DNA sequences flanking the insert had been removed, thereby establishing this approach as a means of determining whether sequences flanking a lacZ insertion are dispensable for viral growth. In an initial test of the methods, they have shown that 7800 base pairs of one copy of L-component repeat sequences can be deleted without affecting viral growth in human fibroblasts.
- Research Organization:
- Stanford Univ. School of Medicine, CA (USA)
- OSTI ID:
- 6994579
- Journal Information:
- Proc. Natl. Acad. Sci. U.S.A.; (United States), Vol. 84:20, Issue 20
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
62 RADIOLOGY AND NUCLEAR MEDICINE
DNA
AUTORADIOGRAPHY
VIRUSES
GENE REGULATION
MUTAGENESIS
ELECTROPHORESIS
ESCHERICHIA COLI
FIBROBLASTS
HYBRIDIZATION
MAN
METHIONINE
PHOSPHORUS 32
SULFUR 35
AMINO ACIDS
ANIMAL CELLS
ANIMALS
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CARBOXYLIC ACIDS
CONNECTIVE TISSUE CELLS
DAYS LIVING RADIOISOTOPES
DRUGS
EVEN-ODD NUCLEI
ISOTOPES
LIGHT NUCLEI
LIPOTROPIC FACTORS
MAMMALS
MICROORGANISMS
NUCLEI
NUCLEIC ACIDS
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PARASITES
PHOSPHORUS ISOTOPES
PRIMATES
RADIOISOTOPES
SOMATIC CELLS
SULFUR ISOTOPES
VERTEBRATES
550401* - Genetics- Tracer Techniques
550601 - Medicine- Unsealed Radionuclides in Diagnostics