Proc. Nati. Acad. Sci. USA Vol. 77, No. 7, pp. 385-3854, July 1980 Biochemistry Amplification of integrated viral DNA sequences in polyoma virus-transformed cells (polyoma large tumor antigen/tandem integration/restriction enzyme mapping) VITTORIO COLANTUONI, LISA DAILEY, AND CLAUDIO BASILICO Department of Pathology, New York University School of Medicine, New York, New York 10016 Communicated by Severo Ochoa, April 3,1980 ABSTRACT Polyoma virus (Py) transformation of rat cells so far always contain the origin of viral DNA replication. The requires integration of viral genomes into the host DNA, which results also show that full tandem integration can be generated generally occurs in a partial or full head-to-tail tandem ar- rangement. The instability of this structure was previously at times subsequent to the initial transformation event. demonstrated by the high rate of loss of integrated Py genomes in the presence of viral large tumor (T) antigen. We now show MATERIALS AND METHODS that integrated Py DNA sequences can also undergo amplifi- cation. We studied two rat cell lines transformed by the ts-a Py Cells. The general properties of the F2408 Fisher rat cells mutant, which codes for a thermolabile large T antigen. In a transformed by Py used in these experiments have been de- derivative of the ts-a H6A cell line, we have observed loss of scribed (7, 8). The lines used, ts-a H6A and ts-a H3A, were both full-length Py DNA molecules from the integrated tandem transformed by the temperature-sensitive (ts)-a mutant (10) ("curing'), accompanied by the creation of new tandem repeats of Py and grown in Dulbecco's modified Eagle's medium of two segments of viral DNA corresponding to 38% and 10% of the viral genome, each containing the origin of DNA repli- containing 10% calf serum. cation. In the ts-a H3A cell line, which contains an integrated The ts-a H6 and H3 lines were produced by infection at 50 partial tandem of about 1.3 viral genomes with three distinct plaque-forming units per cell. Cells were plated in soft agar at deletions, propagation at 330C resulted in the generation of full 330C for 5 days, then shifted to 39.50C, and transformed tandem repeats of a 94% Py DNA "unit" (including two 3% colonies were isolated (2). These cell lines were recloned in agar deletions), an 85% "unit" (including a 3% and the 12% dele- at 39.50C to produce subclones designated H6A and H3A. tion), or both. Amplification of integrated viral DNA was not These cells were always propagated at 39.50C unless otherwise observed in cells propagated at 39.50C, the nonpermissive temperature for large T antigen function. Amplification of in- stated. tegrated Py DNA sequences thus requires an active large T an- DNA Extraction and Restriction Enzyme Analysis by Blot tigen and can generate a full tandem of integrated viral DNA Hybridization. Chromosomal DNA was prepared from cells molecules long after the initial integration event. grown at 39.50C and digested with restriction endonucleases (New England BioLabs) (Fig. 1) as described (2,6). The DNA Transformation of rat cells by polyoma virus (Py) is charac- samples were loaded on a 16 X 18 X 0.5 cm I% agarose hori- terized by integration of the viral genome into the host DNA zontal slab gel and run for 15-18 hr at 40 V. After alkali dena- and expression of early viral functions. Integration has been turation the DNA was transferred to nitrocellulose filter paper observed so far to occur exclusively in a partial or full head- as described (2, 6). Hybridization with denatured 32P-labeled to-tail tandem arrangement, and the viral DNA regions where nick-translated (11, 12) Py DNA (specific activity 5 X 107 to insertion into the host DNA occurs do not seem to be specific 1 X 109 cpm/,Lg) was carried out for 20-24 hr at 650C, in the (1-6). Therefore, tandem integration preserves the integrity presence of 40 ,g/ml of sonicated and denatured calf thymus of viral genes coding for transforming functions. DNA. After hybridization the filter was rinsed as described (2, Py DNA integration is not totally stable. Transformed rat cells 6) and exposed at -700C to XR-2 X-ray film (Kodak) with or have been shown to produce free viral DNA molecules, which without an intensifying screen (LightningPlus, Du Pont). originate from the integrated ones by induction occurring in a minority of the cell population (7, 8). Excision of integrated RESULTS viral DNA sequences can also occur (2, 5), and both production of free viral DNA (7) and excision (2) require an active viral The transformed cell lines used in this study had all been pro- large tumor (T) antigen, a protein known to be involved in the duced by infection with the ts-a mutant (10) of Py, which codes initiation of viral DNA replication (9). In general, excision leads for a temperature-sensitive large T antigen (13-15). This allows to the loss of an integral number of viral DNA molecules, the monitoring of the cultures under conditions permissive suggesting the involvement of an intramolecular recombination (330C) or nonpermissive (39.5"C) for this viral function. event (5). The analysis of integrated viral DNA sequences in these lines These and other findings led us to investigate whether am- was performed by digestion of high molecular weight cellular plification of integrated viral DNA sequences could also occur DNA with restriction enzymes of different specificities for Py in Py-transformed cells, and whether this process exhibited DNA (Fig. 1), followed by fractionation on agarose gels, transfer requirements similar to those observed for excision. The results to nitrocellulose paper, and hybridization with 32P-labeled Py presented in this paper show that, under conditions permissive DNA ("Southern" blot) (16). for large T antigen function, integrated viral DNA sequences Amplification of Viral DNA Sequences in a Derivative of can undergo amplification. The amplified sequences observed the ts-a H6A Cell Line. The ts-a H6A line contains a single insertion of integrated viral DNA consisting of full length Py As The publication costs of this article were defrayed in part by page molecules arranged in a head-to-tail tandem repeat (2, 5). charge payment. This article must therefore be hereby marked "ad- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate Abbreviations: Py, polyoma virus; T antigen, tumor antigen; kb, kilo- this fact. base(s); m.u., map unit(s); ts, temperature-sensitive. 3850 Downloaded by guest on October 1, 2021 Biochemistry: Colantuoni et al. Proc. Natl. Acad. Sci. USA 77 (1980) 3851 i. 11 Early I t |1]i |~~11 I,, At "P ,. 0 ,41-40 ,: *k -.0. A B FIG. 2. Analysis ofthe Py DNA sequences integrated in the DNA ofthe SS1A cell line, the parental line ts-a H6A-1, and one ofthe H6A "cured" derivatives. About 10 jg ofhigh molecular weight DNA per sample was digested with restriction endonucleases, fractionated through a 1% agarose slab gel, denatured, and transferred to nitro- Late cellulose membrane filters. Specific Py sequences were detected by FIG. 1. Physical map of Py DNA [5.3 kilobases (kb)], showing the hybridization with denatured 32P-labeled nick-translated Py DNA sites ofcleavage ofthe restriction enzymes used in these experiments. and visualized by autoradiography. The positions and the sizes (in Ori, origin of viral DNA replication. kb) of some linear DNA fragments containing Py sequences are in- dicated. They were calculated from the mobility of X phage DNA fragments produced by cleavage with Bgl II and Eco RI enzymes, as previously described (2), propagation of the H6A line at the well as from the mobility of marker Py DNA fragments. (A) Lanes temperature permissive for large T antigen function allows the 1-4, Bgl II digestion of purified Py DNA marker, ts-a H6A-1 DNA, isolation of a high frequency of "cured" cells which, through SS1A DNA, and "cured" H6A DNA. The two bands appearing in lane excision, have lost 1 correspond to supercoiled form I (the fastest), and relaxed form II the tandem arrangement of integrated viral Py DNA. (B) Lanes 1-4, Eco RI digestion of Py DNA marker, H6A-1 molecules, while the regions of the viral DNA adjacent to the DNA, SS1A DNA, and "cured" H6A DNA. Lanes 5-8, Bam I diges- host DNA remain unchanged. These cells have undergone re- tion of Py DNA marker, H6A-1 DNA, SS1A DNA, and "cured" H6A version to an "intermediate" transformed phenotype (2). The DNA. Lanes 9-12, double digestion with Bgl H and Hae I ofPy DNA ts-a H6A line was recloned in agar at 39.50C to produce a marker, H6A-1 DNA, SS1A DNA, and "cured" H6A DNA. subclone designated H6A-1, whose Py integration pattern was indistinguishable from that of H6A. The ts-a H6A-1 line was fragment of a higher molecular weight than that of the other propagated at 330C for 2 weeks and revertants were isolated. revertants, suggesting the presence of more Py sequences within While all of the other revertants were indistinguishable from the insertion. EcoRI cleavage (Fig. 2B, lane 3) showed that al- those previously studied (2, 5), one of these, designated SS1A, though the 5.3-kb monomer was absent, one flanking sequence had an integration pattern not previously seen. of SS1A migrated as did that of H6A-1 and the other "cured" The analysis of the integrated viral DNA sequences in these lines (Fig. 2) shows that EcoRI cleavage of H6A-1 DNA (Fig.