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Construction and analysis of viable deletion mutants of polyoma virus
Abstract:
Viable mutants of polyoma with small deletions ranging in size from 2 to 75 base pairs were obtained by infecting 3T3 cells with polyoma DNA that had been cleaved once with HaeII endonuclease or with DNase-Mn2+ digestion. The HaeII endonuclease-cleaved DNA yielded mutants with deletions at map position 72--73, whereas the mutants generated by DNase I-Mn2+ digestion had deletions either at map position 72--73 or within the map coordinates 92 and 99. Both groups of mutants appeared to grow as well as wild-type virus in 3T3 cells. The deletions at map position 72--73 did not alter the virus's ability to transform rat cells. Hence, the region just to the early side of the origin of DNA replication is not essential for vegetative growth or transformation. But the mutants with deletions in the region between map coordinates 92 and 99, a segment thought to code for polyoma large and middle T antigens (Hutchinson et al., Cell 15:65--77, 1978; Smart and Ito, Cell 15:1427--1437, 1978; Soeda et al., Cell 17:357--370, 1979), transformed rat cells at 0.2 to 0.05 the efficiency of wild-type virus.
Insights
Researchers created polyoma virus mutants with small deletions to study gene function. Deletions near the DNA replication origin did not affect growth or transformation, but those in a specific region reduced transformation efficiency.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Polyoma virus is a model system for studying eukaryotic DNA replication and gene expression.
- Understanding the function of viral genes is crucial for comprehending viral pathogenesis and developing antiviral strategies.
Purpose of the Study:
- To investigate the role of specific regions in the polyoma virus genome in viral replication and cell transformation.
- To identify non-essential regions for viral growth and transformation.
Main Methods:
- Generation of viable polyoma virus mutants with small deletions (2-75 base pairs) using HaeII endonuclease cleavage or DNase I-Mn2+ digestion.
- Infection of 3T3 cells with wild-type and mutant polyoma DNA.
- Assessment of viral growth in 3T3 cells.
- Evaluation of rat cell transformation efficiency by wild-type and mutant viruses.
Main Results:
- Mutants with deletions at map position 72-73 (near the origin of DNA replication) grew as well as wild-type virus and retained their ability to transform rat cells.
- Mutants with deletions between map coordinates 92 and 99 exhibited a 0.05 to 0.2 fold reduced efficiency in transforming rat cells compared to wild-type virus.
- The region near the origin of DNA replication is not essential for polyoma virus vegetative growth or transformation.
Conclusions:
- The region of the polyoma virus genome between map coordinates 92 and 99 is important for efficient cell transformation, likely due to its role in coding for large and middle T antigens.
- The region flanking the origin of DNA replication is dispensable for both viral replication and oncogenic transformation.