Amplification of polyomavirus DNA sequences stably integrated in rat cells.
To investigate the mechanism by which the polyomavirus large T antigen (T-Ag) promotes amplification of integrated viral sequences, we constructed a rat cell line, Hy2-ts5, carrying two different inserts of polyomavirus DNA. The first insert, designated the middle T (pmt) locus, was devised to analyze homologous recombination between two defective copies of pmt lying 3.3 kb apart on the same chromosome. Reconstitution of a functional pmt by spontaneous recombination occurred at a rate of about 2 x 10(-7) per cell generation. The second locus contained the polyomavirus large T (plt) gene carrying a temperature-sensitive mutation and producing a nonfunctional large T-Ag at 39 degrees C. A shift to the permissive temperature for as little as 24 h induced the production of a functional large T-Ag which, in turn, promoted homologous recombination in the pmt locus at a rate close to 1.0 per cell generation. The particularity of this system is that it allowed recombination products to be analyzed as early as a single cell doubling following the initial recombinational event. Amplification occurred by successive duplications of a discrete sequence in the viral insert. Unequal sister chromatid exchange was ruled out as the recombination mechanism promoted by large T-Ag. Instead, we proposed a model of nonconservative recombination involving mispairing between homologous sequences.
St-Onge
L
L
Department of Microbiology, Université de Sherbrooke, Quebec, Canada.
Bastin
M
M
D000818:Animals; D000957:Antigens, Viral, Tumor / Q000235:genetics*; D015139:Blotting, Southern; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics*; D005784:Gene Amplification; D008957:Models, Genetic; D009154:Mutation; D011120:Polyomavirus / Q000235:genetics*; D051381:Rats; D011995:Recombination, Genetic; D013696:Temperature; D014170:Transformation, Genetic
D000957:Antigens, Viral, Tumor; D004279:DNA, Viral