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[CANCER RESEARCH 35, 1559-1562,June 1975] Transcription of the Repetitive DNA Sequences in Polyoma-transformed and Nontransformed Mouse Cells in Culture' Leo J. Grady and Wayne P. Campbell Division of Laboratories and Research, New York State Department of Health. Albany, New York 1220/ SUMMARY regular medium and in medium containing 3 @zgof the thymidine analog BUdR2 per ml have been described (6). RNA-DNA saturation hybridization experiments were Radioactive DNA. Secondary cultures of mouse embryo cells were grown in half-gallon roller bottles in the same used to estimate the extent of transcription of repetitive DNA sequences in polyoma-transformed and nontrans medium as above. While still subconfluent, the cultures 15 jzCi/ml (specific formed mouse cells in culture. Measurements were made were exposed to [methy/-3H]thymidine, activity, 50.7 Ci/mmole; New England Nuclear, Boston, with RNA from nontransformed cells in both the subconflu ent and confluent stages of growth, transformed cells in Mass.), for 24 hr. The cells were then harvested and the normal growth medium, and transformed cells grown in DNA was extracted and purified according to published procedures (4). The final DNA preparation had a specific medium containing 5-bromodeoxyuridine, 3 sg/ml, a treat activity of 2.5 x 10@cpm4ig. ment which reduces their tumorigenic potential. No differ ences were observed in the amount of repetitive DNA RNA. The methods for cell culture and for the extraction transcribed or in the families of sequences expressed, except and purification of RNA have been described elsewhere (4). in transformed cells grown in the presence of 5-bromodeox For BUdR, cells were grown in the presence of the analog yuridine, in which case the extent of transcription was for 10 passages before their RNA was extracted. reduced. DNA Fractionation. Tritium-labeled DNA was sus pended in 0.0015 M NaCl-0.000IS M sodium citrate at 300 @zg/mland sheared by passage through a French pressure INTRODUCTION cell at 25,000 psi. Based on sedimentation in alkaline sucrose gradients, DNA sheared in this manner is indistin One of the interests of our laboratory has been to guishable from DNA sheared at 50,000 psi in an American Instrument Co. No. 46-13715 pump. The DNA concentra determine whether virus-induced transformation is accom panied by measurable changes in the proportion of the tion was adjusted to 50 @g/ml,and the buffer was adjusted to 0.12 M PB. The DNA was denatured by immersion in cellular genome that is transcribed. Previously, we reported that, at least in culture, differences in the transcription of boiling water for 5 mm and then incubated at 60°to C0t [nucleotide concentration x time (moles x sec/liter)J = 3 the nonrepetitive DNA sequences do exist between trans x lO_2 (1). At this point, the sample was passed over a formed and nontransformed mouse cells (4). The present hydroxylapatite column (DNA grade; Bio-Rad, Richmond, communication describes experiments designed to measure the degree to which repetitive DNA is transcribed in the Calif.) at 60°,and the column was washed with 5 column same cell lines. Taken together with the earlier studies on volumes of 0.12 M PB + 0.4% SLS to remove single the expression of nonrepetitive DNA, these measurements stranded DNA. Finally, double-stranded DNA was eluted provide a reasonably complete picture of the extent of by washing with 5 column volumes of 0.4 M PB + 0.4% transcription of cellular DNA in cultures of transformed SLS. This procedure served to remove the 10% ofthe mouse and nontransformed cells. genome associated with the very rapidly reassociating satellite DNA. The removal ofthis DNA component should result in lower backgrounds due to DNA-DNA self-reaction MATERIALS AND METHODS in subsequent RNA-DNA hybridization experiments. At the same time, since satellite sequences are not transcribed Cells and Cell Culture. The nontransformed AL/N line of (3), their absence should not alter the results of such mouse cells (1 1) and a polyoma-transformed derivative, PY experiments. AL/N clone 3, were used. The cells were grown in a RNA-DNA Hybridization. Hybridization mixtures con modified Eagle's medium (No. 71035; Grand Island Biolog tamed denatured, fractionated DNA, 0.1 jzg/ml, labeled ical Co., Grand Island, N. Y.) supplemented with 10% fetal with tritiated thymidine and various concentrations of bovine serum (Grand Island Biological Co.). The growth unlabeled RNA in 1.0 ml of 0.12 M PB. Each experiment properties and the tumorigenicity of these cell lines in both also included 2 control samples that contained only 0. 1 jsg of 1 This work was supported in part by USPHS Grant CA the National Cancer Institute. ReceivedJanuary 10, 1975;acceptedMarch 5, 1975. JUNE 13401-02 from 2 The abbreviations used are: BUdR, phate buffer composed of equimolar 5-bromodeoxyuridine; concentrations PB, of NaH,P04 phos and Na,HPO4; SLS, sodium lauryl sulfate. 1975 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1975 American Association for Cancer Research. 1559 L. J. Grady and W. P. Campbell labeled DNA per ml. Reactions were run in 45- x 15-mm glass vials (No. 3-33EA; Fisher Scientific Co., Rochester, N. Y.), and the samples were layered with paraffin oil to prevent evaporation. Incubations were at 60° for 16 hr. Reactions were terminated by diluting samples into 5.0 ml of 0. 12 M PB + 0.4% SLS at @O followed by immediate 0 Ui 4 U 0 12 4 Ui 4 z 0 passage over a column of hydroxylapatite, also at 60@. Single-stranded DNA was washed from the column with 0.12 M PB + 0.4% SLS as above. DNA-DNA and RNA-DNA hybrids were then collected by washing the column with 0.4 MPB + 0.4% SLS. The nucleic acids in the 0.12 M PB and 0.4 M PB fractions were precipitated with trichloroacetic acid (final precipitates were collected 5' concentration, 5%), and the on HAWP filters (Millipore, Bedford, Mass.). Absorption within the precipitate of the low-energy beta particle released by tritium decay can produce serious errors if direct counting of precipitates on filter supports is attempted. To circumvent this problem, filters were placed in glass counting vials and incubated overnight at 68°with cot (moles x sec/liter) Chart 1. Reassociation kinetics of fractionated and unfractionated mouse DNA. Labeled DNA was fractionated as describedunder “Mate rials and Methods.― The reassociation kinetics of this DNA was then compared to that of similarly sheared, but unfractionated, DNA. In order to follow the reactions over a wide range of C0t values, 0. 1 @gof each labeledDNA wasmixed with enoughsheared,unlabeledDNA to give: C0: = l0@ to lO_2,0.9 @g/ml;C0: = lO@ to 6.0, 15 @g/ml;C0: = 6.0 to 10', 600 @g/ml.Samples were denatured and then incubated at 6O@in 0.12 M PB. At various intervals, portions of each samplewere removed and the amount of DNA reassociatedwas determined with columns of hydroxylapatite. •,fractionated DNA; 0, unfractionated DNA. 0.6 ml of 1.0 N HCI (7). After cooling, 15 ml of Aquasol @ (New England Nuclear, Boston, Mass.) were added to each vial, and the samples were counted in a Beckman LS250 scintillation spectrometer. In reconstruction experiments, the counts per sample recovered with this procedure were independent of the amount of unlabeled carrier DNA added to the sample. The amount of DNA in hybrid form (DNA-DNA and RNA-DNA) was taken as the proportion of the total counts recovered that were found in the 0.4 MPB fraction. The quantity of DNA in RNA-DNA hybrids was determined by subtracting from this value the average extent of DNA-DNA self-reaction (2 to 3%) that took place in the controls that contained DNA alone. The hybridization technique for these experiments used unlabeled RNA to drive reactions containing a small amount of labeled DNA. Since the reactions were carried out in solution, DNA-DNA as well as RNA-DNA hybrids were able to form. To keep the background due to the DNA-DNA hybrids to a minimum, the labeled DNA was fractionated prior to use in hybridization experiments so as to remove the very rapidly reassociating satellite DNA sequences. A comparision of the reassociation kinetics of mouse DNA before and after such fractionation is given in Chart I . These data establish that the procedure used to remove the satellite sequences did not alter the reassociation properties of the remainder of the DNA. Inasmuch as Flamm et a!. (3) have shown that satellite DNA is not transcribed, the absence of this fraction should not change the outcome of the hybridization experiments. A summary of RNA-DNA saturation hybridization using RNA from subconfluent and confluent AL/N cells is presented in Chart 2.4. Also the results from an experiment that contained a RNA from the 2 growth states. In every saturation value obtained was about 15%. The range for 2 experiments was 3.3 to 3.9% using RNA from I560 RNA C0N@ENTRATI0N IN ,ug/mI Chart 2. RNA-DNA saturation hybridization experiments.A, results with RNA from: ., a mixture of @, confluent AL/N confluent and cells; V , subconfluent AL/N subconfluent AL/N cells. B, cells; reactions containing RNA from: 0, PY Al/N cells; A, a mixture of PY AL/N and subconfluent AL/N cells; 0, PY AL/N cells grown in medium containing BUdR, 3 @ig/ml. subconfluent cells, 3.3 to 3.8% with RNA from confluent cells, and 3.4 to 3.8% when RNA mixtures were used. These results strongly suggest that there are neither quantitative RESULTS experiments cultures of included are mixture of instance, the EL@0@:@:t nor qualitative differences in the families of repetitive DNA transcribed experiment in the 2 conditions of growth. When an identical was performed using purified nonrepetitive DNA, prepared as described elsewhere (4), no reaction was observed. Thus, it can be concluded that only repetitive DNA formed hybrids under the reaction conditions used. The labeled DNA utilized in all of the experiments described thus far had been fractionated to remove the satellite DNA. Consequently, the 3.5% saturation values obtained are actually equivalent to 3. 1% of the whole mouse genome. In an experiment carried out with subconfluent AL/N RNA and unfractionated mouse DNA, the satura tion value was 2.8% (DNA-DNA self-reaction was 10% in this case). This number is in good agreement with the 3.1% calculated above and supports the contention that removal ofthe satellite DNA does not alter the experimental results. The results of similar experiments using RNA from PY AL/N cells grown either in regular medium or in medium supplemented with BUdR, 3 jsg/ml, are illustrated in Chart 2B. The saturation value obtained with RNA from trans formed cells grown in regular medium is similar to that for nontransformed cells, ranging from 3.2 to 3.7% for 3 experiments. In contrast, experiments with RNA from cells CANCER RESEARCH VOL. 35 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1975 American Association for Cancer Research. Repetitive grown in the presence of BUdR reveal a decrease in the extent of repetitive DNA transcribed, the saturation values being 2.0 and 2.4% for 2 experiments. Chart 2B also includes the data from a reaction that contained a mixture of RNA from subconfluent AL/N cells and PY AL/N cells. No differences were observed in the families of repetitive DNA transcribed in transformed and nontransformed cells. Since repetitive DNA consists of families of closely related, but not identical, base sequences, it is highly improbable that a RNA molecule will hybridize with a region of DNA precisely like that from which it was originally transcribed. The actual extent to which mis matching of base pairs occurs depends largely on the salt concentration and temperature used in the reactions and can be estimated from the thermal stability of the hybrids. An example of the thermal stability of the hybrids formed in this study is shown in Chart 3. The Tm for repetitive DNA-DNA hybrids was 72°, whereas that for repetitive DNA-RNA hybrids was 67°.Even in cases where there are few, if any, mismatched base pairs, there is a 4°difference between the Tm'5 Of DNA-DNA and RNA-DNA hybrids. Therefore, the above values suggest that the extent of mismatching of base pairs in the RNA-DNA hybrids is essentially the same as that in the DNA-DNA hybrids. On the other hand, the Tm'5 for both types of hybrid are approximately 12°lower than those for nonrepetitive DNA hybrids formed under similar conditions (4). Ifthe relation ship determined by Laird et al. (9) that a °decrease in Tm is equivalent to 1.5% mismatching ofbase pairs is applied to our data, it can be estimated that the repetitive DNA hybrids contain 18 to 20% mismatched base pairs. Ui DNA Transcription in Mouse Cells DISCUSSION The 3.5% of the DNA that formed hybrids in these experiments can be viewed in several ways. If this number is corrected for the 10% of the mouse genome removed by the initial fractionation of the DNA, then it represents 3. 1% of the whole mouse genome. However, since only repetitive DNA was able to form hybrids under the experimental conditions used, and since repetitive sequences comprised about 20% of the DNA used, the 3.5% of the DNA that did hybridize is equivalent to 17.5% of the repetitive sequences. These data provide information only with regard to the transcription of families of repetitive DNA; they do not permit conclusions to be drawn regarding the expression of specific sequences. Also, the saturation values obtained are for a given salt concentration and temperature, and reac tions involving repetitive DNA are quite sensitive to changes in these parameters (2). No differences in the families of repetitive DNA ex pressed were found between confluent and subconfluent nontransformed cells in culture. Likewise, there were no differences detected between nontransformed cells and those transformed by polyoma virus. This latter result agrees with the findings of Levine et a!. (10), who worked with SV4O-transformed and nontransformed hamster cells, and supports their conclusion that for established cell lines in culture viral transformation does not lead to changes in transcription of the repetitive DNA sequences of the cell. In the case of transformed cells grown in the presence of the thymidine analog BUdR, we observed a decrease in the transcription of repetitive DNA. Similar results were also found by Kotzin and Baker (8) who studied the effect of BUdR on the transcription of repetitive DNA in the sea urchin. It is also consistent with our previous report of a decrease in the expression of nonrepetitive DNA in PY AL/N cells grown in medium containing BUdR (5). The results obtained thus far in our studies of the transcription of the cell genome in nontransformed and virus-transformed mouse cells in culture can be summarized as follows: (a) no difference is observed in the families of repetitive DNA expressed in the 2 types of cells; (b) there are differences in the transcription of nonrepetitive DNA sequences between transformed and nontransformed cells; (c) growth of transformed cells in medium containing BUdR, 3 j.tg/ml, a treatment that reduces but does not 60 @2 TO TEMPERATURE 80 eliminate tumorigenicity (6), results in a general suppres sion of the transcription of both repetitive and nonrepetitive 90 DNA sequences. (°C) Chart 3. Thermal stability of the RNA-DNA hybrids. After 16 hr incubation at f,@0 the hybridization mixture was diluted into 5.0 ml of 0.12 M PB + 0.4% SLS and passed over a hydroxylapatite column at Single-strandedDNA waswashedfrom the column with 0.12MPB + 0.4% SLS, and the temperatureof the column wasthen raisedin 5°increments. After eachincreasein temperature,the column waswashedwith 3 column volumes of 0. 12 M PB + 0.4% 515 at the new temperature. The dissocia tion of the RNA-DNA hybrids was followed by measuring the percentage of the total trichloroacetic acid precipitable counts, which bound to the column at 600 that eluted at each temperature.•,hybrid of PY AL/N RNA and repetitiveDNA; 0, DNA-DNA hybrid after incubationto C0: = 10. REFERENCES 6O@. 1. Britten, R. J., and Kohne, D. E. Repeated Sequences in DNA. Science. /61:529-540,1968. 2. Church, R. B., and McCarthy, B. J. Related Base Sequences in the DNA of Simpleand ComplexOrganisms.II. The Interpretationof DNA/RNA Hybridization Studies with Mammalian Nucleic Acids. Biochem. Genet., 2: 55-73, 1968. 3. Flamm, W. G., Walker, P. M. B., and McCallum, M. Some Propertiesof the Single Strands from the DNA of the Nuclear Satel lite of the Mouse (Mus musculus). J. Mol. Biol., 40: 423-443, 1969. JUNE 1975 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1975 American Association for Cancer Research. I561 L. J. Grady and W. P. Campbell in Mouse Cells Grown in Tissue Culture. Nature New Biol., 243: 8. Kotzin, B. 1., and Baker, R. F. Selective Inhibition of Genetic Transcription in Sea Urchin Embryos. J. Cell Biol., 55: 74-81, 1972. 195—198, 1973. 9. Laird, C. D., McConaughy, B. L., and McCarthy, B. J. Rate of 4. Grady, L. J., and Campbell, W. P. Non-repetitive DNA Transcription 5. Grady, L. J., and Campbell, W. P. The Distribution of 5-Bromodeox yuridine in the DNA of Polyoma-Transformed Mouse Cells and Some Apparent Effects on Transcription. Exptl. Cell Res., 87: 127-131, 1974. 6. Grady, I. J., and North, A. B. Some Effects of 5-Bromodeoxyuridine on Polyoma-TransformedMouseCells. Exptl. Cell Res.,87: 120-126, 1974. 7. Hattori, T., Aoki, H., Matsuzaki, I., Maruo, B., and Takahashi, H. Liquid Scintillation Counting of H3-Nucleic Acids. Anal. Chem., 37: 159—161, 1965. I562 Fixation of Nucleotide Substitutions in Evolution. Nature, 224: 149-154,1969. 10. Levine, A. S., Oxman, M. N., Eliot, H. M., and Henry, P. H. New Species of Rapidly Hybridizing RNA in Contact-inhibited as Well as Transformed Hamster Cell lines. Cancer Res., 32: 506-510, 1972. I 1. Takemoto, K. K., Ting, R. C. Y., Ozer, H. 1., and Fabisch, P. Establishment of a Cell Line from an Inbred Mouse Strain for Viral Transformation Studies: Simian Virus 40 Transformation and Tumor Production. J. NatI. Cancer Inst., 41: 1401-1409, 1968. CANCER RESEARCH VOL.35 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1975 American Association for Cancer Research. Transcription of the Repetitive DNA Sequences in Polyoma-transformed and Nontransformed Mouse Cells in Culture Leo J. Grady and Wayne P. Campbell Cancer Res 1975;35:1559-1562. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/35/6/1559 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1975 American Association for Cancer Research.