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Molecular cloning and physical mapping of murine cytomegalovirus DNA
Abstract:
Murine cytomegalovirus (MCMV) Smith strain DNA is cleaved by restriction endonuclease HindIII into 16 fragments, ranging in size from 0.64 to 22.25 megadaltons. Of the 16 HindIII fragments, 15 were cloned in plasmid pACYC177 in Escherichia coli HB101 (recA). The recombinant plasmid clones were characterized by cleavage with the enzymes XbaI and EcoRI. In addition, fragments generated by double digestion of cloned fragments with HindIII and XbaI were inserted into the plasmid vector pACYC184. The results obtained after hybridization of 32P-labeled cloned fragments to Southern blots of MCMV DNA cleaved with HindIII, XbaI, EcoRI, BamHI, ApaI, ClaI, EcoRV, or KpnI allowed us to construct complete physical maps of the viral DNA for the restriction endonucleases HindIII, XbaI, and EcoRI. On the basis of the cloning and mapping experiments, it was calculated that the MCMV genome spans about 235 kilobase pairs, corresponding to a molecular weight of 155,000,000. All fragments were found to be present in equimolar concentrations, and no cross-hybridization between any of the fragments was seen. We conclude that the MCMV DNA molecule consists of a long unique sequence without large terminal or internal repeat regions. Thus, the structural organization of the MCMV genome is fundamentally different from that of the human cytomegalovirus or herpes simplex virus genome.
Insights
Murine cytomegalovirus (MCMV) DNA was mapped using restriction endonuclease analysis and molecular cloning. MCMV possesses a unique genome structure, differing significantly from human cytomegalovirus and herpes simplex virus.
Area of Science:
- Molecular Biology
- Virology
- Genomics
Background:
- Murine cytomegalovirus (MCMV) is a significant model organism in herpesvirus research.
- Understanding the MCMV genome structure is crucial for comparative genomics and viral pathogenesis studies.
Purpose of the Study:
- To construct a detailed physical map of the MCMV Smith strain genome.
- To elucidate the structural organization of the MCMV genome and compare it to other herpesviruses.
Main Methods:
- Restriction endonuclease digestion (HindIII, XbaI, EcoRI, BamHI, ApaI, ClaI, EcoRV, KpnI).
- Molecular cloning of MCMV DNA fragments into Escherichia coli.
- Southern blot hybridization with radiolabeled cloned fragments.
Main Results:
- A complete physical map for HindIII, XbaI, and EcoRI restriction enzymes was established.
- The MCMV genome size was estimated at approximately 235 kilobase pairs (155,000,000 molecular weight).
- The MCMV genome consists of a unique sequence lacking significant terminal or internal repeats.
Conclusions:
- The MCMV genome exhibits a unique structural organization.
- This structure fundamentally differs from the genomes of human cytomegalovirus and herpes simplex virus.
- The findings provide a foundational map for future MCMV genetic and functional studies.