Generation of a large library of point mutations in polymamiddle T antigen

Brian J. Druker, Thomas M. Roberts

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

Polyoma middle T antigen (RflTAg) 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 RflTAg mutants was constructed. Chemically mutagenized MTAg DNA was purified from wild-type DNA by separation on denaturing gradient gels and placed into a recombinant retrovlrus vector. Utilizing the resultant library of RflTAgexpressing retroviruses, fibroblast cell lines expressing individual RflTAg mutants were generated and screened for a non-transformed morphology. Of the first seven non-transformed clones tested, all express the RflTAg protein. We estimate that approximately 24% of the G418-resistant colonies contain a transformationdefective 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 RflTAg. 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.

Original languageEnglish (US)
Pages (from-to)6855-6861
Number of pages7
JournalNucleic acids research
Volume19
Issue number24
DOIs
StatePublished - Dec 25 1991

ASJC Scopus subject areas

  • Genetics

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