bating 5 days at 37°C. NIH 3T3 cells were obtained from two different sources-. Geoffrey M. Cooper (Sidney Farber Cancer Institute, Boston,. MA) and Edward M.
Proc. Nati. Acad. Sci. USA Vol. 81, pp. 400-404, January 1984
Biochemistry
Microinjected pBR322 stimulates cellular DNA synthesis in Swiss 3T3 cells (plasmid/cloning vector/microinjection/cell cycle)
JULIA K. HYLAND*, RICKY R. HIRSCHHORN*, CARLO AVIGNOLO*, W. EDWARD MERCER*, MICHIO OHTA*, NORBEL GALANTI*, GERALD J. JONAKt, AND RENATO BASERGA* *Department of Pathology and Fels Research Institute, Temple University School of Medicine, Philadelphia, PA 19140; and tDupont Experimental Station, Wilmington, DE 19898
Communicated by Sidney Weinhouse, September 30, 1983
ABSTRACT When pBR322 is manually microinjected into the nuclei of quiescent Swiss 3T3 cells it stimulates the incorporation of [3H]thymidine into DNA. The evidence clearly shows that this increased incorporation that is detected by in situ autoradiography in microinjected cells represents cellular DNA synthesis and not DNA repair or plasmid replication. The effect is due to pBR322 and not due to impurities, mechanical perturbances due to the microinjection technique, or aspecific effects. This stimulation is striking in Swiss 3T3 cells. Some NIH 3T3 cells show a slight stimulation, but hamster cells, derived from baby hamster kidney (BHK) cells, are not stimulated when microinjected with pBR322. The preliminary evidence seems to indicate that the integrity of the pBR322 genome is important for the stimulation of cellular DNA synthesis in quiescent Swiss 3T3 cells. These results, although of a preliminary nature, are of interest because they indicate that a prokaryotic genome may alter the cell cycle of mammalian cells. From a practical point of view the stimulatory effect of microinjected pBR322 on cellular DNA synthesis has a more immediate interest, because pBR322 is the vector most commonly used for molecular cloning and 3T3 cells are very frequently used for gene transfer experiments.
pBR322 is the most commonly used vector in recombinant DNA technology. In previous experiments it appeared to be a suitable control for cell cycle studies with microinjected viral genes, because it failed to stimulate cell DNA synthesis when microinjected into quiescent Syrian hamster cells (1, 2). The present communication, though, shows that microinjected pBR322 stimulates cell DNA synthesis in Swiss 3T3 cells and that the stimulation is not due to impurities, mechanical perturbances caused by the microinjection technique, or aspecific effects. While confirming that Syrian hamster cells are totally refractory to stimulation by pBR322, our experiments also show that the presence of inserts in the pBR322 genome affects its ability to stimulate cellular DNA synthesis in Swiss 3T3 cells.
MATERIALS AND METHODS Cell Lines and Quiescence. Swiss 3T3 cells, originally obtained from Andrea Mastro (Pennsylvania State University), have been passaged in our laboratory for the past 2 years in Dulbecco's modified Eagle medium containing 10% fetal calf serum and antibiotics. Quiescent cultures were obtained by plating 1 X 105 cells in 60-mm Petri dishes containing a 22mm2 glass coverslip in medium with 1% calf serum and incubating 5 days at 37°C. NIH 3T3 cells were obtained from two different sourcesGeoffrey M. Cooper (Sidney Farber Cancer Institute, Boston, MA) and Edward M. Scolnick (Merck, Sharp & Dohme). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
tsAF8 and ts13 are G1 temperature-sensitive mutants of the cell cycle, originally derived by Meiss and Basilico (3) from baby hamster kidney (BHK) cells and passaged in our laboratory for >7 years. NIH 3T3, tsAF8, and ts13 cells were made quiescent and prepared for microinjection as described by Galanti et al. (2). Plasmids. pBR322 (4) was obtained from three different sources-our own laboratory, where it is grown in HB101, the laboratory of Kenneth J. Soprano (Dept. of Microbiology, Temple University Medical School), and a commercial source (New England Nuclear). pSV2G was constructed by Galanti et al. (2). It contains a 4,300-base-pair (bp) fragment of simian virus 40 (SV40), with an intact early region coding for the T antigens, cloned into the EcoRI and BamHI restriction sites of pBR322. When microinjected into cells it induces the appearance of T antigen and stimulates cell DNA synthesis (1, 2). dl 1046 is a deletion mutant of SV40, cloned in the BamHI restriction site of pBR322. It has been described by Pipas et al. (5). It does not express T antigen, and it does not stimulate DNA synthesis (6). All of the cloned fragments of Epstein-Barr virus (EBV) were obtained from Lars Rymo (University of Gothenburg, Gothenburg, Sweden). All of the clones used in these experiments were EBV fragments inserted into the BamHI or the EcoRI restriction sites of pBR322. The EcoRI fragment used (7) was the J fragment of -3.0 kilobases (kb). The BamHI fragments (B, K, R, and Z) were all derived from the EcoRI B fragment and their sizes range from 1.5 to 9 kb. The plasmid 1-6 was the gift of Edward M. Scolnick (Merck, Sharp & Dohme) and is described in the paper by Ellis et al. (8). 1-6 is a 3,060-bp fragment of the Harvey murine sarcoma virus cloned into the HindIII site of pBR322. It has a long terminal repeat but does not code for a p21 protein and does not transform. pUR222 has been described by Ruther et al. (9). It has the gene for ampicillin resistance but not the gene for tetracycline resistance. pDR42 (10) has a trp promoter insert at the Cla I site of pBR322. This plasmid is resistant to higher concentrations of tetracycline than pBR322. pSVPST was constructed in our laboratory. It contains a 1,200-bp fragment of SV40 cloned in the Pst I site of pBR322. The SV40 fragment includes the terminal portions of both the early and late regions and does not express T antigen. Preparation of Plasmid DNA. Two different methods were used to prepare plasmid DNA. In the first method, plasmid DNA was isolated and purified by phenol extraction and Sepharose 2B chromatography, as described by Meagher et al. (11) and Tilghman et al. (12). In the second method, the plasmids were purified from a Triton X-100-lysozyme cleared lysate on CsCl buoyant density gradients (twice), as described by Clewell and Helinski (13). Abbreviations: SV40, simian virus 40; bp, base pair(s); kb, kilobase(s); EBV, Epstein-Barr virus.
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Time after microinjection, days FIG. 1. Percentage of Swiss 3T3 cells labeled by [3H]thymidine after microinjection of pBR322. Quiescent Swiss 3T3 cells were microinjected with pBR322 at 0 time. Separate cultures were labeled with [3H]thymidine either from 0 to 24 hr. from 24 to 48 hr, or from 48 to 72 hr. Open symbols, microinjected cells; closed symbols, control cells (nonmicroinjected), on the same coverslips as the microinjected cells. Circles, experiment 1; squares, experiment 2; triangles, experiment 3; x, serum-stimulated Swiss 3T3 cells.
The purity of the preparations was also monitored on gels. Samples contaminated with cellular DNA or RNA were not used. Restriction endonuclease analysis was used to confirm the structure of the plasmids. These latter two procedures were carried out by standard methods (14). Microinjection. The standard buffer for microinjection is 10 mM Tris HCl (pH 7.3 at 250C). All DNAs and proteins were dissolved in this buffer. Unless otherwise stated, DNA was used at a final concentration of 0.1 mg/ml. Protein concentrations varied from 2 to 7.5 mg/ml. The basic technique is the one of Graessmann et al. (15). In our laboratory a small circle is etched on the coverslip before the cells are plated. All cells (or nearly all cells) in the circle are microinjected, and the cells outside the circle, on the same coverslip, serve as controls. Measurement of DNA Synthesis. For autoradiography we used standard techniques. The cells were labeled with [3H]thymidine (0.1-0.2 uCi/ml; 1 Ci = 37 gBq) for the desired period of time. For the determination of cellular DNA synthesis a total of 560 quiescent Swiss 3T3 cells was microinjected with pBR322. After microinjection, a cloning ring (encompassing =2,500 cells) was placed around the etched circle of microinjection, and the cells were left in their own medium. Cloning rings were also placed on separate cultures: some cultures were left undisturbed (quiescent nonmicroinjected controls) while in other cultures the cells within the ring were stimulat-
ed with 10% fetal calf serum (stimulated controls). About 20 hr after microinjection, the cells in the cloning rings were labeled for 6 hr with 32PO4 (72 pCi/ml) in Dulbecco's lowphosphate medium. After extensive washings the cells were trypsinized and collected and the DNA was extracted with phenol/chloroform/isoamyl alcohol and treatment with Pronase and RNase. Undigested DNAs and DNAs digested to completion with EcoRI were then subjected to electrophoresis overnight on a 1% agarose gel. The gel was dried and then autoradiographed in a cassette with two intensifying screens (Dupont Quanta III) using Kodak SV film at -70'C for 1 week. Measurements of DNA and RNA Amounts. These were carried out on fixed cells stained with acridine orange (16, 17). Under the conditions used, green fluorescence is a good measure of DNA amount, whereas red fluorescence is 90% due to RNA. Green and red fluorescences were quantitated by a computer-operated Zonax microspectrofluorimeter (Zeiss, West Germany). The mitotic index of cells was determined by exposing the cells to Nocodazole from 16 to 38 hr after microinjection. This drug causes metaphase-arrest (18).
RESULTS Microinjected pBR322 Stimulates DNA Synthesis in Quiescent Swiss 3T3 Cells. Swiss 3T3 cells made quiescent in low serum (1% calf serum) have a very low labeling index. When labeled with [3H]thymidine for periods of 24 hr, the percentage of labeled cells varies between 0.5% and 5%, depending on how long the cells have been kept quiescent. When quiescent Swiss 3T3 cells are microinjected with pBR322 there is a marked increase in the percentage of labeled cells, with a peak of labeling between 24 and 48 hr. Fig. 1 shows three separate experiments in which the microinjections were done by two different operators. The cells outside the circle of microinjected cells served as a control. The percentage of labeled cells increases modestly between 0 and 24 hr, reaches a peak between 24 and 48 hr, and after 48 hr returns
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Proc. NatL Acad Sci. USA 81
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FIG. 3. Determination of DNA and RNA content of Swiss 3T3 cells microinjected with pBR322. Nucleic acid content of individual cells was determined by measuring the intensity of acridine orange-stained cells with a computer-operated microspectrofluorimeter. Staining intensity is expressed in arbitrary units. Cellular DNA content is shown in A-C, whereas RNA content appears in D-F. (A and D) Serum-stimulated cells; (B and E) pBR322-microinjected cells; (C and F) quiescent nonmicroinjected cells.
to the level of control, nonmicroinjected cells. Serum-stimu-
lated cells reach a maximum of 90-95% labeled cells within 24 hr. pBR322 Induces Cellular DNA Synthesis in Swiss 3T3 Cells. The DNA synthesis detected by autoradiography (Fig. 1) could be due to cellular DNA synthesis or plasmid replication. Fig. 2 shows that microinjected pBR322 induces cellular DNA synthesis. In this experiment, 32P-labeled DNA from quiescent, pBR322-microinjected, and serum-stimulated cells was isolated. In Fig. 2, lane 3, the labeled DNA of microinjected cells is high molecular weight. No trace of a plasmid band (4.3 kb) is visible. After digestion with EcoRI, the labeled DNA of microinjected cells gives a smear like that of serum-stimulated cells. Again, no band is detectable at 4.3 kb. The presence of low molecular weight bands of satellite DNA indicates that the DNA was digested to completion. There are two reasons for the difference in intensity between microinjected and serum-stimulated cells. Serum stimulates a higher fraction (>90%) of cells than microinjection of pBR322 and, although the number of cells in the cloning ring is roughly the same, only about 20% were microinjected. Notice that the labeling intensity, though, is considerably higher in the microinjected group than in the quiescent cells. This experiment has been repeated. These findings show that the incorporation of [3H]thymidine stimulated by microinjection of pBR322 is due to cellular DNA synthesis and not plasmid replication. This is in agreement with the findings of Lusky and Botchan (19) and Peden et al. (20) that pBR322, and derivative plasmids, replicate poorly in mammalian cells. It is still possible that the labeling of cellular DNA may be due to DNA repair rather than DNA synthesis. This appeared to be unlikely because pBR322-microinjected cells incorporating [3H]thymidine showed an intense labeling, >100 grains per cell, like serum-stimulated cells. Furthermore, measurement of DNA amount in acridine orange-stained cells using a computer-operated microspectrofluorimeter showed that cells microinjected with pBR322 had an increased percentage of cells with a >2 n amount of DNA, in respect to quiescent cells (Fig. 3 B and However, there was no increase in the amount of cellular RNA in microinjected cells (Fig. 3 E and F). The measurable
amount of red fluorescence (in arbitrary units) averaged 14 (units per cell) in quiescent cells, 28 in serum-stimulated cells, and 12 in cells microinjected with pBR322. All of these measurements were carried out in Swiss 3T3 cells 38 hr after microinjection or serum-stimulation. In several experiments we looked for mitoses in cells microinjected with pBR322 and treated with Nocodazole (18) to arrest cells in metaphase. We never observed mitoses in cells microinjected with pBR322, whereas mitoses were abundant in serum-stimulated cells. Stimulation of DNA Synthesis Is Not Due to Mechanical Stimulation by the Microinjection Technique. Table 1 shows that microinjection per se does not stimulate DNA synthesis in quiescent Swiss 3T3 cells. Cells microinjected with Tris buffer or with protein solutions (albumin and IgG) did not show any increase in labeling index. Variations in the periods of labeling and in the length of quiescence account for the slight differences in the labeling indices of the control cells. Different buffers were used in case DNA slightly altered the pH of the solution. Other microinjected proteins from various sources, not shown in Table 1, were all incapable of stimulating Swiss 3T3 cells. Table 1. Stimulation of DNA synthesis in Swiss 3T3 cells microinjected with pBR322
Microinjection pBR322 pBR322* 10 mM Tris HCl pH 7.3t pH 7.5t
Cells in DNA synthesis, % Control Microinjected 44 56
1.5 6 5 2.3 1.6
1.1 2.2
1.4 6
8 pH 7.6t 5.8 Albumin 0.3 IgG All cells were labeled with l3H]thymidine from 0 to 24 hr after microinjection, unless indicated otherwise. pBR322 concentration varied from 0.1 to 0.5 mg/ml, and protein concentration varied from 2 to 7.5 mg/ml. The protein microinjections have been repeated with labeling from 24 to 48 hr, with exactly the same results. *Labeled from 24 to 48 hr. tLabeled from 0 to 48 hr.
Biochemistry: Hyland et aL Table 2. Stimulation of DNA synthesis by different plasmids microinjected into Swiss 3T3 cells Cells in DNA synthesis, % Microinjection Control Microinjected 44 pBR322 1.1 3.5 5.6 I-6 (insert at HindIll) dl 1046 (insert at BamHI) 7 6 EBV-BamHI K fragment 5.7 1.2 3.1 B fragment 1.2 7.6 R fragment 1.2 4.0 Z fragment 1.2 27.0 8.0 EBV-EcoRI J fragment 1.3 pUR222 (no TetR gene) 0.5 24.3 7.7 pDR42 (insert at Cla I) 19.0 5.0 pSVPST (insert at Pst I) All cells were labeled with [3H]thymidine from 0 to 24 hr. All plasmids, except pUR222 and pDR42, were pBR322 with inserts at the BamHI, HindIII, EcoRI, or Pst I restriction sites. Derivation of inserts are explained in the text. The same DNA concentration (0.1 mg/ml) was used for all plasmids.
Different preparations of pBR322 were used, including those made in our laboratory and one from a commercial source, and all gave similar results. In all cases, the purity of our preparations was monitored by gel electrophoresis and no contamination, by cellular DNA or RNA, was observed. Plasmid DNA in our laboratory was also prepared by two different methods (see Materials and Methods), and both gave the same results. This and some of the results with recombinant plasmids rule out a contaminant as the cause of stimulation of cell DNA synthesis (see below). Effect of Other Plasmids Microinjected into Swiss 3T3 Cells. A number of recombinant plasmids and plasmid vectors were tested for their ability to stimulate DNA synthesis in quiescent Swiss 3T3 cells (Table 2). Five different recombinant plasmids containing an insert at the BamHI restriction site of pBR322 (dl 1046 and the EBV BamHI fragments B, K, R, and Z) were microinjected. None of them stimulated DNA synthesis. Neither did 1-6, which contains a retroviral insert at the HindIII site of pBR322. pUR222 (a vector that lacks the TetR gene) also fails to stimulate cell DNA synthesis. The fact that several recombinant plasmids fail to stimulate DNA synthesis rules out an aspecific polyanion effect. Other recombinants stimulated DNA synthesis in microinjected Swiss 3T3 cells: pSVPST, with an insert at the Pst I restriction site of pBR322; pDR42, with an insert at the Cla I site; and the EBV-EcoRI J fragment. However, plasmids carrying inserts did not cause as large an increase in labeling index as did intact pBR322. Host Range of pBR322 Stimulation of DNA Synthesis. We have tested the ability of microinjected pBR322 to stimulate DNA synthesis in cells of mouse and hamster (Table 3). We Table 3. Host range of pBR322 stimulation of cell DNA synthesis Cells in DNA synthesis, % Microinjected with Cell type pBR322 pSV2G Control Swiss 3T3 39 32 4 NIH 3T3 (Cooper) 6 64 7 NIH 3T3 (Scolnick) 12 ND 2 tsAF8 3 69 8 27 90 ts13 25 All cells were labeled with [3H]thymidine from 0 to 24 hr after microinjection. ND, not done.
Proc. NatL. Acad. Sci. USA 81 (1984)
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have never been able to stimulate DNA synthesis by pBR322 in ts13 and tsAF8, both derived from BHK 21 cells (3) and therefore of Syrian hamster origin. This has now been true over a period of 5 years, with at least 10 different operators. Among mouse cell lines, Swiss 3T3 cells are the most sensitive to stimulation. NIH 3T3 cells vary in their response. One clone of NIH 3T3 did not respond to microinjection of pBR322, whereas another clone gave a very weak stimulation. For comparison, we report in Table 3 how these same cell lines respond to microinjection of pSV2G (2), our standard plasmid for stimulation of DNA synthesis. pSV2G contains the early region of SV40 inserted into the EcoRI and BamHI restriction sites of pBR322. The early region of SV40-i.e., the T-antigen coding gene-is known to stimulate cell DNA synthesis in microinjected cells (1, 2, 6, 15).
DISCUSSION Our results show that pBR322, when microinjected into quiescent Swiss 3T3 cells, stimulates cellular DNA synthesis. It is known that pBR322, and its recombinants, replicate very poorly in mammalian cells (19, 20). Although we cannot rule out a modest amount of plasmid replication or DNA repair, the cellular nature of the DNA that is synthesized in cells microinjected with pBR322 is supported by (i) the absence of any plasmid band in labeled DNA digested to completion with EcoRI; (ii) the increase in the amount of DNA per cell, demonstrated by computer-operated microspectrofluorimetry; and (iii) the labeling intensity of the microinjected cells. Although gene amplification of integrated pBR322 sequences cannot be ruled out, it seems unlikely that an integration event would occur in such a large percentage of microinjected cells in the time frame of these experiments. Incidentally, the increase in the amount of DNA per cell cannot be attributed to the microinjected DNA. We microinject -1,000 gene copies, or about 4 x 106 bp of pBR322, which is 1/1,000th of the diploid cellular genome, estimated at 3 x 109 bp. Control experiments with microinjection of buffer and proteins show that the increase in DNA synthesis is not due to mechanical stimulation by the microinjection technique. An unknown contaminant can be ruled out on the basis of four observations: (i) no contaminant could be detected by agarose gel electrophoresis; (ii) stimulation was achieved with three different preparations of pBR322 from three different sources; (iii) stimulation was obtained with pBR322 prepared in our laboratory by two different methods; and (iv) several recombinant plasmids, prepared by the same methods, failed to stimulate cell DNA synthesis. The integrity of the pBR322 genome appears to be necessary for maximal stimulation of DNA synthesis. Inserts into pBR322, especially at the BamHI site, decrease or abolish the effect. The failure of cells microinjected with pBR322 to increase their RNA amount and to enter mitosis indicates that pBR322-induced stimulation of DNA synthesis is probably similar to that induced by certain viruses, such as adenoviruses (21, 22), or by SV40 in cells blocked by butyrate (23). Stimulation of DNA synthesis without a concomitant increase in cellular RNA has also been reported after microinjection of certain deletion mutants of SV40 (6) and after alkaline shock of quiescent Swiss 3T3 cells (24). Incidentally, we could not test the ability of transfected pBR322 to induce DNA synthesis because, in our hands, the transfection procedure per se stimulates DNA synthesis in the cell lines tested (unpublished data). The limited host range of pBR322-induced cell DNA synthesis should not be too surprising. Both viruses (25) and ribosomal DNA promoters (26-29) require species-specific factors for expression. There are several reports in the literature indicating that pBR322 can be transcribed in eukaryotic cells and that some
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transcripts can be translated. Transcripts hybridizing to pBR322 DNA were detected by Wickens et al. (30) in microinjected Xenopus oocytes and by Kaufman and Sharp (31) in transfected Chinese hamster ovary (CHO) cells. Sassone-Corsi et al. (32) reported that, in vitro, RNA polymerase II transcribed pBR322. ,-Lactamase activity was detected in cultured cells incubated with pBR322-containing liposomes (33) and also in yeast cells transfected with pBR322 (34). However, to the best of our knowledge, it has not been previously reported that pBR322 stimulates DNA synthesis in mammalian cells. Moreover, pBR322 is the vector most commonly used for molecular cloning and it is frequently introduced into cells by microinjection and transfection techniques. Its capability to alter the cell cycle of some mammalian cells should be kept in mind, especially because 3T3 cells are very often used both for gene transfer and for cell proliferation studies. This research was supported by Grant CA 25898 from the National Cancer Institute. 1. Floros, J., Jonak, G., Galanti, N. & Baserga, R. (1981) Exp. Cell Res. 132, 215-223. 2. Galanti, N., Jonak, G. J., Soprano, K. J., Floros, J., Kaczmarek, L., Weissman, S., Reddy, V. B., Tilghman, S. M. & Baserga, R. (1981) J. Biol. Chem. 256, 6469-6474. 3. Meiss, H. K. & Basilico, C. (1972) Nature (London) New Biol. 239, 66-68. 4. Bolivar, F., Rodriguez, R. L., Greene, P. J., Betlach, M. C., Heyneker, H. L., Boyer, H. W., Crosa, J. H. & Falkow, S. (1977) Gene 2, 95-113. 5. Pipas, J. M., Peden, K. W. C. & Nathans, D. (1983) Mol. Cell. Biol. 3, 203-213. 6. Soprano, K. J., Galanti, N., Jonak, G. J., McKercher, S., Pipas, J. M., Peden, K. W. C. & Baserga, R. (1983) Mol. Cell. Biol. 3, 214-219. 7. Arrand, J. R., Rymo, L., Walsh, J. E., Bjorck, E., Lindahl, T. & Griffin, B. E. (1981) Nucleic Acids Res. 9, 2999-3014. 8. Ellis, R. W., DeFeo, D., Maryak, J. M., Young, H. A., Shih, T. Y., Chang, E. H., Lowy, D. R. & Scolnick, E. M. (1980) J. Virol. 36, 408-420. 9. Ruther, V., Koenen, M., Otto, K. & Muller-Hill, B. (1981) Nucleic Acids Res. 9, 4087-4098. 10. Herrin, G. L., Jr., Russell, D. R. & Bennett, G. N. (1982) Plasmid 7, 290-293.
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11. Meagher, R. B., Tait, R. C., Betlach, M. & Boyer, H. W. (1977) Cell 10, 521-536. 12. Tilghman, S. M., Kioussis, D., Gorin, M. B., Ruiz, J. P. B. & Ingram, R. S. (1979) J. Biol. Chem. 254, 7393-7399. 13. Clewell, D. B. & Helinski, D. R. (1970) Biochemistry 9, 44284440. 14. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Cloning (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). 15. Graessmann, A., Graessmann, M. & Mueller, C. (1980) Methods Enzymol. 65, 816-825. 16. Ashihara, T., Traganos, F., Baserga, R. & Darzynkiewicz, Z. (1978) Cancer Res. 38, 2514-2518. 17. Mercer, W. E., Avignolo, C., Galanti, N., Rose, K. M., Hyland, J. K., Jacob, S. T. & Baserga, R. Exp. Cell Res. (in press). 18. Zieve, G. W., Turnbull, D., Mullins, J. M. & McIntosh, J. M. (1980) Exp. Cell Res. 126, 397-405. 19. Lusky, M. & Botchan, M. (1981) Nature (London) 293, 79-81. 20. Peden, K. W. C., Pipas, J. M., Pearson-White, S. & Nathans, D. (1980) Science 209, 1392-13%. 21. Braithwaite, A. W., Murray, J. D. & Bellett, A. J. D. (1981) J. Virol. 39, 331-340. 22. Pochron, S., Rossini, M., Darzynkiewicz, Z., Traganos, F. & Baserga, R. (1980) J. Biol. Chem. 255, 4411-4413. 23. Daniell, E., Burg, J. L. & Fedor, M. J. (1982) Virology 116, 196-206. 24. Zetterberg, A., Engstrom, W. & Larsson, 0. (1982) Ann. N. Y. Acad. Sci. 397, 130-147. 25. Tooze, J. (1980) DNA Tumor Viruses (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). 26. Grummt, I. (1981) Nucleic Acids Res. 9, 6093-6102. 27. Learned, R. M. & Tjian, R. (1982) J. Mol. Appl. Genet. 1, 575584. 28. Miesfeld, R. & Arnheim, N. (1982) Nucleic Acids Res. 10, 3933-3949. 29. Wilkinson, J. K. & Sollner-Webb, B. (1982) J. Biol. Chem. 257, 14375-14383. 30. Wickens, M. P., Woo, S., O'Malley, B. W. & Gurdon, J. B. (1980) Nature (London) 285, 628-634. 31. Kaufman, R. J. & Sharp, P. A. (1982) Mol. Cell. Biol. 2, 13041319. 32. Sassone-Corsi, P., Corden, J., Kddinger, C. & Chambon, P. (1981) Nucleic Acids Res. 9, 3941-3958. 33. Wong, T., Nicolau, C. & Hofschneider, P. H. (1980) Gene 10, 87-94. 34. Roggenkamp, R., Kustermann-Kuhn, B. & Hollenberg, C. P. (1981) Proc. Natl. Acad. Sci. USA 78, 4466-4470.
