Preliminary 9. Jackson. V. Shires. A, Chalklev, R & Granner, D K, J biol &em ZSO’(1975)4856.10. Allfrey, V G, Faulkner, R & Mirsky, A E, Proc natl acad sci US 5 1 (1964) 786. 11. Adler, A J, Fasman, G D, Wangh, L J & Allfrey, V G, J biol them 249 (1974) 2911. 12. Wangh, L A, Ruiz-Carrillo & Allfrey, V G, Arch biochem biophys 150 (1972) 44. 13. Berlowitz, L & Palotta, D, Exp cell res 71 (1972) 45. 14. Gorovsky, M A, Pleger, G L, Kleevert, J B & Johmann, C A, J cell bio157 (1973) 773. 15. Joachim, H. Cell differ 4 (1975) 123. 16. Sarkander. H I. Fleischer-Lambroooulos. H & Brade, W P, FEBS lett 52 (1975) 40. 1 17. Poeo. B G T. Allfrev. V G & Mirskv. A E. Proc na; acad sci US 55 (1966) 805. ’ 18. Pogo, B G T, Pogo, A 0, Allfrey, V G & Mirsky, A E, Proc natl acad sci US 59 (1%8) 1337. 19. Hynes, R 0 &Gross, P R, Dev biol21 (1970) 383. 20. Thaler, M M, Cox, C L & Villee, C A, J cell bio142 (1969) 846. 21. Tidwell, T, Allfrey, V G & Mirsky, A E, J biol them 243 (1968) 707. 22. Lumbers, J, Threlfall, C J & Stoner, H B, Anal biochem 31 (1969) 21. 23. Johnson, A‘W, Wilhelm, J A & Hnilica, L S, Biochim bionhvs acta 295 (1973) 150. 24. Yukawa, O’&-Koshihara,‘ H, Dev biol 33 (1973) 477. 25. Byvoet, P, Biochim biophys acta 160(1968) 217. 26. Shepherd, G R, Biochim biophvs _ - acta 299 (1973) 485: 27. Orengo, A & Hnilica, L A, Exp cell res 62 (1970) 331. 28. Moav, B & Nemer, M, Biochemistry 10(1971)881. 29. Seale, R L & Aronson, A I, J mol biol 75 (1973) 633. 30. Gallwitz, D & Mueller, G C, J biol them 244 (1969) 5947. 31. Kedes, L H, Gross, P R, Cognetti, G & Hunter, A L, J mol bio145 (1969) 337. 32. Harvey, E B, The American Arbacia and other sea urchins. Princeton University Press, Princeton, NY (1956). Received March 10, 1976 Accepted April 7, 1976

Stimulation of cellular RNA synthesis in mouse-kidney cell cultures infected with SV40 virus P. MAY, E. MAY and J. BORDE, Znstifut de Recherches Scientifiques France

sur le Cancer, 94800 Villejuif*

In confluent primary mouse-kidney cell cultures, abortive infection with SV40 has been demonstrated to cause an increase in the bulk of cel-

Summary.

notes

433

lular RNA (mainly rRNA). However, the increase in the rate of rRNA synthesis is not involved in the initiation of the virus-induced cellular DNA replication since after actinomycin D treatment (0.05 Fg/ml, from 6 to 9 h p.i.) the onset of cellular DNA replication takes place at a time when the rate of rRNA synthesis is still depressed.

Simian virus 40 (SV40) induces in confluent, “contact-inhibited” primary mousekidney cell cultures an abortive infection that leads to the replication of the cell chromatin and mitosis while no detectable amounts of viral progeny DNA or capsid protein are produced [l-4]. In this paper, we show that infection of confluent primary mouse-kidney cell cultures with SV40 stimulates the synthesis of the. bulk of cellular RNA (mainly rRNA). To decide whether the activation of rRNA is involved in the virus-induced initiation of cellular DNA replication, an analysis of recovery times of the macromolecular syntheses after actinomycin D treatment has been performed. Materials

and Methods

Primary mouse-kidney cell cultures, prepared from lo-day-old CR-l mice, were grown in large lOO-mm plastic Petri dishes or, when indicated,-in 30-mm plastic Petri dishes containing 22 X 22 mm glass coverslips (coverslip cultures). The cultures were infected with 0.4 ml (or 0.15 ml in 30-mm Petri dishes) of plaque purified non-defective wild-type SV40 [ 1, 21. All viral preparations contained about 2X 108PFU/ml. The results were the same whether crude viral lysates or highly purified viral preparations were used for infection. The rate of [5-3H]TdR incorporation was determined bv labellina coverslio cultures for 1 h at the times indicated inResults as described in ref. [ 11. SV40-infected and mock-infected mouse-kidney cells were incubated for 1 h at the appropriate times with 10 &i [5-3H]uridine ([3H]U, CEA, France, 20 Ci/ mmol) and 5 pg/ml unlabelled uridine (Calbiochem, A Grade) in 5 ml of medium. The cultures were then washed consecutively with ice-cold phosphate buffer saline (PBS) and cold 5 % trichloroacetic acid (TCA). After centrifugation at 16000 g for 5 min, DNA and RNA were extracted from the pellet according to Schmidt & Thannhauser r51 and the RNA content was determined by the orcinoi method with yeast RNA (grade, XI, Sigma) used as standard. A 100~~1aliquot of the alkaline hydrolysate containing the nucleotides derived from the RNA was mixed in 2 ml of ethanol and its radioactivity was determined. Exp Cell Res 100 (1976)

434 Preliminary notes Table 1. Rate of incorporation of [3HJuridine into RNA at varying times in SV40-infected primary mouse kidney cell cultures with or without exposure to actinomycin D [3H]Uridine incorporated into cellular RNA (cpmx 10-3/cu1ture) Cultures not treated with actinomycin D

Actinomycin-treated cultures

Labelling period

Mock-infected

SV40-infected

Mock-infected

SV40-infected

8-9 h pi. 18-19 h p.i. 23-24 h p.i.

129 t11.2 12l.lf 2.3 147 f 0.7

124k2.4 222f3.9 362f7.1

22.4* l.Oa 22.7f 1.6 47.3f0.2

24.0f0.3a 40.9k2.0 103.622.2

Confluent mouse kidney cell cultures were infected at 37°C (2x lo8 PFU/ml) and exposed (or not) from 6 to 9 h; pi. to actinomycin D (0.05 pg/ml). At varying times after infection, the cultures were labelled with [3H]U (10 &i/ml) for 1 h in the presence of 5 pg/ml of unlabelled uridine. At the end of the pulse, the amount of r3H]U incorporated into RNA (cpm/culture) was determined. Each value represents the mean (+S.E.M.) of duplicate or triplicate cultures. ’ During the pulse with [SH]U from 8 to 9 h p.i., actinomycin D was present in the medium covering the cultures.

Radiolabelling of cellular RNA and analysis in sucrose density gradient. Mouse-kidney cell cultures

were infected with SV40 as described above and exposed to actinomycin D (0.05 pg/ml) from 6 to 9 h p.i. At 18 h pi. r3H]U (plus 5 @g/ml of unlabelled uridine) was added to the culture medium (final concentration

Table 2. The effect of infection with SV40 on the amount of total cellular RNA in confluent primary mouse kidney cultures Amount of total RNA per culture SV40-infected Exptb 1

2 3 4 5 6 Mean+S.E.M.

Mock-infected (control) (pg/culture)O

CLglculture” % of control

14.5 125 203 175 207 122

166 147 228 204 235 135

114.5 117.6 112.3 116.5 113.5 110.7 114.2f0.96

Confluent primary mouse kidney cell cultures in lOO-mm Petri dishes were infected (2x lo8 PFU/ml) at 37°C. In all experiments the virus was adsorbed to the cells at 37°C for 1 h. After the adsorption the cultures were washed once and then covered with serumfree reinforced Eagle’s medium. At 24 h p.i. the amount of total RNA extracted per culture was determined. a Each figure is an average of determinations on two replicate cultures and the individual determinations agree within +4 % of the mean. b All cell cultures used in the same experiment were prepared in parallel. Exp Cell Res 100 (1976)

10 &i/ml). At 24 h p.i., the cultures were washed once with 5 ml/Petri dish of PBS and the cells were scraped off into 1 ml of PBS. The RNA was extracted and treated with DNAse according to Scherrer [6]. After ethanol precipitation, the RNA samples were dissolved in 1 vol of 1 mM EDTA, pH 7.1, and 4 vol of dimethylsulfoxide (DMSO, puriss, Fluka AG, Buchs S.G.). The mixture was heated for 2 min at 70°C. Samples in DMSO were then diluted 1 : 5 with the same buffer used for preparing sucrose gradients (10 mM triethanolamine, pH 7.4, 50 mM NaCl, 1 mM EDTA). RNA was layered on top of a 16 ml linear (15-30% w/v) sucrose density gradient and centrifuged in a Spinco SW 27.1 rotor at 23000 rpm for 16 h at 20°C. Fractions of 0.4 ml were collected from the top of the tube. For each fraction, two aliquots of 100 ~1 were put on glass fiber filters. The filters were extracted with cold 10% TCA, then with cold 5 % TCA, washed with ethanol, dried and their radioactivity was counted.

Results and Discussion The effect of the abortive infection with SV40 on [3H]U incorporation into cellular RNA. The stimulation of incorporation of [3H]U into cellular RNA observed with the SV40-infected cultures not treated with actinomycin D, is seen in table 1. Virus-induced net increase in total cellular RNA. Table 2, containing the results from six independent experiments, illustrates the net increase in the amount of total (mainly ribosomal) RNA per infected culture observed 24 h p.i. as compared with parallel mock-infected cultures. (It was

Preliminary notes 435 [3H]TdR into cellular DNA (fig. 1) and of [3H]U into cellular RNA (table 1) was determined at various times. The typical experiment represented in fig. 1 shows that after actinomycin treatment, SV40-induced cellular DNA replication began at 18 h p.i. and was fully active at 24 h p.i. Cellular RNA synthesis during the period from 18 to 24 h p.i. resumed at a relatively slow rate (table 1). The RNA labelled from 18 to 24 h p.i. was analysed in sucrose density gradients (fig. 2). Its radioactivity exhibited a broad polydispersed distribution in time after infection (hours); ordinate: E3H]TdR incorporation (cpmx 10-3/coverslip). 0, Infected (-act.); A, mock-infected (-act.); 0, infected (+act.); A, mock-infected (+act.). Effect of actinomvcin D on the time course of cellular DNA synthesis: Confluent mouse kidney cell cultures (coverslip cultures) were infected (2x lOa PFU/ ml) at 37°C exposed (or not) to actinomycin D (0.05 &ml) from 6 to 9 h p.i. The cultures were then labilled at the indicated times for 1 h with [3H]TdR (5 &i/ml) and the incorporation into DNA (cpm/ coverslip) was determined: Each point represents’the average of values from duplicate cultures.

Fig. I. Abscissa:

found that the number of cells in infected and parallel mock-infected cultures was the same (-t3 %) at 24 h p.i.). A similar increase in total cellular RNA has been observed in confluent primary mouse-kidney cells productively infected with polyoma [3, 41. However, Oda & Dulbecco [7] have reported stimulation of only cellular mRNA synthesis during the productive infection of BCS-1 cells by SV40. Treatment with actinomycin D from 6 to 9 h p.i. Since synthesis of rRNA appeared to be stimulated in SV40-infected mouse kidney cell cultures, we were interested to determine whether this stimulation was involved in the initiation of SV40-induced cellular DNA replication. The cell cultures were exposed to actinomycin (0.05 pglml) from 6 to 9 h p.i. and the incorporation of

28s

J-

ias 1

4s -1

fraction no.; ordinate: 3H cpmx lOm3/ fraction. 0, Drug-treated; 0, not drug-treated. Sucrose gradient analysis of RNA synthesized by SV40-infected cultures treated or not with actinomycin D. Two parallel sets (two 100 mm Petri dish cultures per setj of confluent primary mouse kidney cell cultures were infected at 37°C with SV40 as described in Materials and Methods. One set of cultures was exposed to actinomycin D (0.05 @g/ml) from 6 to 9 h p.i. At 18 h p.i. we added 5 ml per culture of fresh medium containing 10 &i/ml [3H]uridine and 5 pg/ml unlabelled uridine. At 24 h o.i. cells were harvested and the RNA was extracted. After a DMSO treatment, RNA was centrifugated on a sucrose gradient as described under Methods.

Fig. 2. Abscissa:

Exp CdRrs

100 (1976)

436 Preliminary

notes

the gradient while a fraction sedimented in a peak corresponding to the low molecular weight RNA species (7-4s). In contrast, when parallel cultures were infected without exposure to the drug, the sedimentation pattern showed the typical peaks of radioactivity corresponding to the 28s and 18s rRNA. Thus after actinomycin treatment, the rate of synthesis of rRNA was still depressed between 18 h and 24 h p.i., which was confirmed by fractionation of RNA on Oligo-TdR-cellulose (unpublished results). In conclusion the results of these experiments show that after actinomycin D treatment, the onset of cellular DNA replication takes place at a time when the rRNA synthesis is still inhibited to a large extent. In our system, the activation of rRNA synthesis does not form a controlling step in G 1 (or GO) for the subsequent initiation of S phase, as has been suggested for other cell systems [8, 93.

Adenovirus 12 uncoiler regions of human chromosome 1 in relation to the 5s rRNA genes D. M. STEFFENSEN,’ P. SZAB02 and J. K. McDOUGALL,$* ‘Department of Genetics and Development, University of Illinois, Urbana, IL 61801, ‘SloanKettering Institute for Cancer Research, New York, NY 10021 USA, and 3Department of Cancer Studies, University of Birmingham, Birmingham Bl5 ZTJ, UK

Two uncoiler regions, induced by adenovirus 12, have been identified on human chromosome 1 at lq42 and 1~36. In situ hybridization with [rz51-5S]rRNA places the 5s genes more accurately at lq4243 immediately distal to the uncoiled site, lq42.

Summary.

Adenovirus 12 has been reported to cause a gap or an uncoiling of human chromosome 17 and an associated increase in thymidine kinase activity [ 11.As well as the occasional chromatid and chromosome aberration, there is another uncoiling region near the end of the long arm of chromosome 1 at lq41-43 [2] at the same location that the 5s rRNA genes have been mapped [3, 4, 51. This study attempts to resolve these mapping overlaps by combining two techWe would like to thank Dr Roger Weil for his helpful suggestions, Drs George Khoury, Robert Perry and niques with the same cells. The data will Norman Salzman for critical discussion and review of show that the 5s rRNA genes at lq4243 the manuscript. are immediately distal to the uncoiled site at lq42. The procedures permit the identiReferences fication of another adenovirus 12 gap at 1. May, E, May, P & Weil, R, Proc natl acad sci US 68 1~36. (1971) 1208. 2. -Ibid 70 (1973) 1654. Materials and Methods 3. Weil, R, Salomon, C, May, E & May, P, Viruses, evolution and cancer (ed E Kurstak & K Iodinated 5s RNA from HeLa cells was a gift from Dr Maramorosh) p. 455. Academic Press, New York Wolf Prensky. The specific activity of the RNA (1974). samples used for hybridization was approx. 3~ 10’ 4. Weil, R, May, E & May, P, Cold Spring Harbor domlve. Prior to hvbridization. the RNA was loaded symp 39 (1975) 381. on a.&AE cellulose column, extensively washed with 5. Schmidt, G & Thannhauser, S J, J biol them 161 0.3 M NaCl, 0.01 M Tris (pH 7.5), eluted with 1 M (1945) 83. NaCl, and precipitated with 2 vol of ethanol after car6. Scherrer. K, Fundamental techniques in virology rier E. coli RNA (1 ma/ml) had been added. The (ed K Habel & N P Salzman) p.-413. Academic precipitate was collected-by centrifugation, dried, and Press, New York (1969). dissolved in 50% formamide, 2xSSC (pH 7.0). 7. Oda, K & Dulbecco, R, Virology 35 (1968) 439. Chromosome preparations were hybridized as de8. Ellem. K A & Mironescu, S, J cell physio179 (1972) scribed [6] with some modifications; the preincubation 389. step at high temperature was omitted since it was 9. Green, H, Control of proliferation in animal cells detrimental to cytological quality. The hybridization (ed B Clarkson & R Baserga) p. 473. Cold Spring time was 10 h at a [lz51-5S)RNA concentration of 0.1 Harbor lab (1974). Received March 16, 1976 Accepted April 6, 1976 Exp Ceil Res 100 (1976)

* Present address: Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.