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A temperature-sensitive mutant of the myeloproliferative sarcoma virus, altered by a point mutation in the mos
Abstract:
The myeloproliferative sarcoma virus (MPSV) is a mos-oncogenic retrovirus which induces an acute myeloproliferative disease in adult mice. The isolation and molecular cloning of two mutants of MPSV temperature sensitive (ts) for mos transformation (Kollek et al., J. Virol. 50:717-724, 1984) have been described previously. In this report, we describe the biological activity of these clones, the molecular basis of the ts lesion of one clone, and the construction of a selectable vector based on the MPSV ts genome. Both molecular clones, ts159 and ts124, proved to have retained the ts phenotype, the former being tighter for the induction and maintenance of the transformed phenotype. A single transition (G----A) at position 1888 in the mos coding region, resulting in the change of Gly to Arg at position 307, was responsible for the ts phenotype of clone ts159. Substitution of sequences carrying this mutation with the corresponding sequences of the wild-type virus generated a virus that was ts for transformation. Insertion of the dominant selectable marker gene for geneticin resistance (neor) into ts159 did not disrupt mos expression or its ts phenotype. neor-ts159 facilitates the study of mos action by allowing the selection of infected cells at the nonpermissive temperature before mos transformation has been induced. Furthermore, infected cells which show no obvious phenotype alteration due to mos expression can be identified by their Neor phenotype.
Insights
Researchers identified a specific mutation in the mos oncogene responsible for temperature-sensitive transformation by the myeloproliferative sarcoma virus (MPSV). They also developed a selectable vector for studying mos oncogene function in infected cells.
Area of Science:
- Molecular Virology
- Oncology
- Retroviral Research
Background:
- Myeloproliferative sarcoma virus (MPSV) is a mos-oncogenic retrovirus causing acute myeloproliferative disease in mice.
- Previous isolation and cloning of temperature-sensitive (ts) mutants for mos transformation were reported.
- Understanding the molecular basis of ts mutations is crucial for studying oncogene function.
Purpose of the Study:
- To characterize the biological activity of MPSV ts mutants.
- To elucidate the molecular basis of the ts lesion in a specific MPSV clone.
- To construct a selectable vector for studying mos oncogene action.
Main Methods:
- Biological activity assays of MPSV ts clones (ts159 and ts124).
- Molecular analysis to identify mutations in the mos coding region.
- Site-directed mutagenesis to confirm the role of the identified mutation.
- Construction of a selectable vector by inserting the geneticin resistance (neor) gene into ts159.
Main Results:
- Both ts159 and ts124 retained the ts phenotype; ts159 showed a tighter phenotype.
- A single G-to-A transition at position 1888 in the mos gene (Gly307Arg) caused the ts phenotype of ts159.
- Reversion to wild-type sequences abolished the ts phenotype, confirming the mutation's role.
- Insertion of the neor gene into ts159 did not affect mos expression or the ts phenotype, creating neor-ts159.
Conclusions:
- The study precisely mapped the molecular determinant of temperature sensitivity for mos-mediated transformation.
- The developed neor-ts159 vector enables selection of infected cells at non-permissive temperatures, facilitating mos oncogene studies.
- This tool allows for the identification of cells with altered phenotypes due to mos expression.