Insertion and deletion mutagenesis of the human cytomegalovirus genome

Journal Article · Thu Oct 01 00:00:00 EDT 1987 · Proc. Natl. Acad. Sci. U.S.A.; (United States)

DOI:https://doi.org/10.1073/pnas.84.20.7213· OSTI ID:6994579

Spaete, R R; Mocarski, E S

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), Journal Name: Proc. Natl. Acad. Sci. U.S.A.; (United States) Journal Issue: 20 Vol. 84:20; ISSN PNASA
• Country of Publication: United States
• Language: English

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59 BASIC BIOLOGICAL SCIENCES
62 RADIOLOGY AND NUCLEAR MEDICINE
AMINO ACIDS
ANIMAL CELLS
ANIMALS
AUTORADIOGRAPHY
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CARBOXYLIC ACIDS
CONNECTIVE TISSUE CELLS
DAYS LIVING RADIOISOTOPES
DNA
DRUGS
ELECTROPHORESIS
ESCHERICHIA COLI
EVEN-ODD NUCLEI
FIBROBLASTS
GENE REGULATION
HYBRIDIZATION
ISOTOPES
LIGHT NUCLEI
LIPOTROPIC FACTORS
MAMMALS
MAN
METHIONINE
MICROORGANISMS
MUTAGENESIS
NUCLEI
NUCLEIC ACIDS
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PARASITES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PRIMATES
RADIOISOTOPES
SOMATIC CELLS
SULFUR 35
SULFUR ISOTOPES
VERTEBRATES
VIRUSES