Amplification of polyomavirus DNA sequences stably integrated in rat cells

Abstract

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.

High-frequency recombination mediated by polyomavirus large T antigen defective in replication

“…The structure or configuration of the insert also may be critical in determining by which pathway homologous sequences will recombine in a given cell line. For example, Hy2, which essentially differs from Hy5 by the position of the viral replication origin, undergoes amplification by successive duplications of a discrete sequence within the insert (31). We interpret the lack of amplification as arguing against a role for an onionskin structure in the recombination pathway mediated by large T-Ag in Hy5.…”

“…The reasons for this are not completely understood. A possibility may be the absence of selection, although amplification of the pmt insert does not seem to confer any selective advantage in Hy2 (31). The structure or configuration of the insert also may be critical in determining by which pathway homologous sequences will recombine in a given cell line.…”

“…We previously described amplification of a polyomavirus insert in a rat cell line, designated Hy2, that did not occur via the resolution of an onionskin structure. Instead, we proposed that large T-Ag promoted successive duplications of a discrete sequence within the insert by a mechanism involving mispairing between homologous sequences (31).…”

Amplification mediated by polyomavirus large T antigen defective in replication

“…This is illustrated in Fig. 2 with 7a2, a cell line that was shown previously to carry a single duplication of the insert (24). On the basis of band intensities, we determined that some transformants underwent only one duplication (e.g., 13val21, 16valll, d113-11, dll3-24, 13vallO, 13va1l5, and 16val16), while others contained more than one pmt copy (e.g., 13vaI13, 13vaI25, 13vaI29, d113-15, and d113-32).…”

Elements of the polyomavirus replication origin required for homologous recombination mediated by large T antigen

“…One particularly attractive model invokes local polytenization at the integrated viral origin to generate an intermediate structure resembling an onion skin (4). Other studies have proposed that large T-ag promotes successive duplications of a discrete sequence within the insert by a mechanism of slippedstrand mispairing (27,31). In addition, while some replicationdefective mutants are defective in recombination, others can promote a variety of recombination events, such as gene conversion, amplification, chromosomal inversion, and unequal sister chromatid exchange (28)(29)(30).…”