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Intracistronic complementation in the simian virus 40 A gene
Abstract:
A set of eight simian virus 40 mutants was constructed with lesions in the A gene, which encodes the large tumor (T) antigen. These mutants have small deletions (3-20 base pairs) at either 0.497, 0.288, or 0.243 map units. Mutants having both in-phase and frameshift mutations at each site were isolated. Neither plaque formation nor replication of the mutant DNAs could be detected after transfection of monkey kidney cells. Another nonviable mutant, dlA2459, had a 14-base-pair deletion at 0.193 map unit and was positive for viral DNA replication. Each of the eight mutants were tested for ability to form plaques after cotransfection with dlA2459 DNA. The four mutants that had in-phase deletions were able to complement dlA2459. The other four, which had frameshift deletions, did not. No plaques were formed after cotransfection of cells with any other pair of group A mutants. This suggests that the defect in dlA2459 defines a distinct functional domain of simian virus 40 T antigen.
Insights
Researchers created simian virus 40 (SV40) mutants with deletions in the large tumor antigen gene. In-phase deletions complemented a nonviable mutant, suggesting distinct functional domains within the SV40 T antigen.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Simian virus 40 (SV40) is a well-characterized DNA tumor virus.
- The SV40 large tumor (T) antigen is crucial for viral replication and oncogenesis.
- Understanding T antigen domains is key to deciphering its functions.
Purpose of the Study:
- To investigate the functional domains of the SV40 T antigen.
- To correlate specific mutations within the T antigen gene with functional complementation.
- To analyze the impact of in-phase versus frameshift deletions on T antigen activity.
Main Methods:
- Construction of eight SV40 mutants with small deletions (3-20 base pairs) in the A gene at specific map units.
- Isolation of mutants with both in-phase and frameshift deletions.
- Transfection of monkey kidney cells to assess plaque formation and DNA replication.
- Complementation assays involving cotransfection with a nonviable mutant (dlA2459).
Main Results:
- Mutants with frameshift deletions failed to replicate or form plaques.
- A mutant with an in-phase deletion (dlA2459) was replication-positive.
- Four mutants with in-phase deletions complemented dlA2459, enabling plaque formation.
- Mutants with frameshift deletions did not complement dlA2459.
Conclusions:
- The results suggest that in-phase deletions within the SV40 T antigen gene can restore function through complementation.
- Frameshift deletions lead to a loss of function, indicating their critical impact.
- The defect in dlA2459 likely represents a distinct functional domain of the SV40 T antigen, separable from other domains affected by frameshift mutations.