Generation of a large library of point mutations in polyoma middle T antigen

Brian Druker, Thomas M. Roberts

Research output: Contribution to journalArticle

10 Citations (Scopus)

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 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.

Original languageEnglish (US)
Pages (from-to)6855-6861
Number of pages7
JournalNucleic Acids Research
Volume19
Issue number24
StatePublished - Dec 25 1991
Externally publishedYes

Fingerprint

Viral Tumor Antigens
Antigens
Point Mutation
Libraries
Mutation
Mutant
Retroviridae
Clone
Clone Cells
DNA
Phosphatidylinositol 3-Kinase
Polyomavirus Transforming Antigens
Dissection
Mutagenesis
Proteins
Protein
Aptitude
Fibroblasts
Cell
Proline

ASJC Scopus subject areas

  • Genetics
  • Statistics, Probability and Uncertainty
  • Applied Mathematics
  • Health, Toxicology and Mutagenesis
  • Toxicology
  • Genetics(clinical)

Cite this

Generation of a large library of point mutations in polyoma middle T antigen. / Druker, Brian; Roberts, Thomas M.

In: Nucleic Acids Research, Vol. 19, No. 24, 25.12.1991, p. 6855-6861.

Research output: Contribution to journalArticle

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