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Characterization of proviruses cloned from mink cell focus-forming virus-infected cellular DNA
Abstract:
Two proviruses were cloned from EcoRI-digested DNA extracted from mink cells chronically infected with AKR mink cell focus-forming (MCF) 247 murine leukemia virus (MuLV), using a lambda phage host vector system. One cloned MuLV DNA fragment (designated MCF 1) contained sequences extending 6.8 kilobases from an EcoRI restriction site in the 5' long terminal repeat (LTR) to an EcoRI site located in the envelope (env) region and was indistinguishable by restriction endonuclease mapping for 5.1 kilobases (except for the EcoRI site in the LTR) from the 5' end of AKR ecotropic proviral DNA. The DNA segment extending from 5.1 to 6.8 kilobases contained several restriction sites that were not present in the AKR ecotropic provirus. A 0.5-kilobase DNA segment located at the 3' end of MCF 1 DNA contained sequences which hybridized to a xenotropic env-specific DNA probe but not to labeled ecotropic env-specific DNA. This dual character of MCF 1 proviral DNA was also confirmed by analyzing heteroduplex molecules by electron microscopy. The second cloned proviral DNA (designated MCF 2) was a 6.9-kilobase EcoRI DNA fragment which contained LTR sequences at each end and a 2.0-kilobase deletion encompassing most of the env region. The MCF 2 proviral DNA proved to be a useful reagent for detecting LTRs electron microscopically due to the presence of nonoverlapping, terminally located LTR sequences which effected its circularization with DNAs containing homologous LTR sequences. Nucleotide sequence analysis demonstrated the presence of a 104-base-pair direct repeat in the LTR of MCF 2 DNA. In contrast, only a single copy of the reiterated component of the direct repeat was present in MCF 1 DNA.
Insights
Researchers cloned two murine leukemia virus (MuLV) proviruses from infected mink cells. One clone, MCF 1, shows dual ecotropic and xenotropic characteristics, while MCF 2 aids in detecting long terminal repeats (LTRs).
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Murine leukemia viruses (MuLV) are retroviruses implicated in various cancers.
- Mink cell focus-forming (MCF) MuLV strains are of particular interest due to their unique biological properties.
- Understanding the genetic structure of MCF MuLV is crucial for deciphering its oncogenic potential.
Purpose of the Study:
- To clone and characterize novel MuLV proviruses from infected mink cells.
- To investigate the genetic makeup and structural features of two distinct MCF MuLV clones (MCF 1 and MCF 2).
- To explore the utility of cloned MCF MuLV DNA fragments as research tools.
Main Methods:
- Cloning of proviral DNA using a lambda phage host vector system.
- Restriction endonuclease mapping to analyze DNA fragment structure.
- Hybridization studies using specific DNA probes to identify viral gene sequences.
- Heteroduplex molecule analysis via electron microscopy.
- Nucleotide sequence analysis of long terminal repeat (LTR) regions.
Main Results:
- Two distinct MuLV proviral DNA fragments, MCF 1 and MCF 2, were successfully cloned.
- MCF 1 exhibited a hybrid character, containing sequences homologous to both ecotropic and xenotropic MuLV, confirmed by restriction mapping and hybridization.
- MCF 2, a 6.9-kilobase fragment with LTRs at both ends and a deletion in the envelope (env) region, proved useful for electron microscopic detection of LTRs.
- Nucleotide sequencing revealed a 104-base-pair direct repeat in the MCF 2 LTR, with only a single copy in MCF 1.
Conclusions:
- The cloned MCF MuLV proviruses represent distinct genetic entities with unique structural features.
- MCF 1 demonstrates a mosaic structure, potentially contributing to its biological activity.
- MCF 2 serves as a valuable molecular tool for studying MuLV LTRs and their organization.
- The findings provide insights into the genetic diversity and evolution of MCF MuLV strains.