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Published on: October 25, 2016
Generation of a large library of point mutations in polyoma middle T antigen
1Division of Cellular and Molecular Biology, Dana-Faber Cancer Institute, Boston, MA.
Abstract:
Polyoma middle T antigen (MTAg) transforms cells by associating with and activating a variety of intracellular proteins, including src family members and a phosphatidylinositol-3 kinase. In order to assist in the study of the relative importance of the various associated biochemical activities for transformation by polyomavirus MTAg, a library of MTAg mutants was constructed. Chemically mutagenized MTAg DNA was purified from wild-type DNA by separation on denaturing gradient gels and placed into a recombinant retrovirus vector. Utilizing the resultant library of MTAg-expressing retroviruses, fibroblast cell lines expressing retroviruses, fibroblast cell lines expressing individual MTAg mutants were generated and screened for a non-transformed morphology. Of the first seven non-transformed clones tested, all express the MTAg protein. We estimate that approximately 24% of the G418-resistant colonies contain a transformation-defective MTAg mutant. A more thorough evaluation of one such clone revealed four single base-pair changes as compared to wild-type. Further genetic dissection of this mutant reveals that substituting leucine for proline at amino acid 248 results in a completely transformation defective MTAg. The utility of this mutagenesis and screening procedure as well as the description of several new MTAg mutants is described. This library of mutations should be of general interest for studying the transforming ability of MTAg.
Insights
Researchers created polyoma middle T antigen (MTAg) mutants to study cell transformation. A specific mutation (proline to leucine at amino acid 248) completely abolished MTAg
Area of Science:
- Molecular Biology
- Virology
- Cell Biology
Background:
- Polyoma middle T antigen (MTAg) drives cell transformation by interacting with proteins like src family kinases and phosphatidylinositol-3 kinase.
- Understanding the specific biochemical activities of MTAg crucial for transformation is essential.
Purpose of the Study:
- To generate and characterize a library of MTAg mutants to investigate the relative importance of its associated biochemical activities in polyomavirus-mediated cell transformation.
Main Methods:
- Chemical mutagenesis of MTAg DNA followed by purification using denaturing gradient gel electrophoresis.
- Cloning mutagenized MTAg into a retroviral vector for expression in fibroblast cell lines.
- Screening for cell lines exhibiting non-transformed morphology to identify transformation-defective MTAg mutants.
Main Results:
- A library of MTAg mutants was successfully generated, with approximately 24% of screened colonies containing transformation-defective mutants.
- One mutant clone with four single base-pair changes was identified.
- A specific mutation, substituting proline with leucine at amino acid 248, rendered MTAg completely transformation-defective.
Conclusions:
- The developed mutagenesis and screening strategy is effective for identifying functionally impaired MTAg mutants.
- The identified MTAg mutants, particularly the proline-248-to-leucine substitution, provide valuable tools for dissecting the molecular mechanisms of MTAg-induced cell transformation.
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