JouRNAL OF VIROLOGY, May 1976, p. 461-472 Copyright 0 1976 American Society for Microbiology

Vol. 18, No. 2

Printed in U-SA.

Intracellular Distribution and Sedimentation Properties of Virus-Specific RNA in Two Clones of BHK Cells Transformed by Polyoma Virus IAN H. MAXWELL' Pollards Wood Research Station, Institute of Cancer Research, Royal Cancer Hospital, Chalfont St. Giles, Buckinghamshire, England, and Department of Molecular Biology, University of Geneva, Geneva, Switzerland

Received for publication 26 November 1975

The virus-specific RNA in two independently derived clones of polyoma virustransformed hamster cells was studied by hybridizing labeled RNA with excess purified polyoma DNA, immobilized on filters. In one clone (PyBHK1), less than 25% of the total labeled virus-specific RNA was found in the cytoplasm, irrespective of the labeling time. In the other clone (PyBHK2), it was estimated that 39% of the total virus-specific RNA was present in the cytoplasm after labeling for 3 h. Both the proportion of radioactive label incorporated into virus-specific RNA and the sedimentation pattern of total virus-specific RNA differed markedly between PyBHK, and PyBHK2. Most of the virus-specific RNA of PyBHK, sedimented in the range 25S-35S, whereas a prominent 188 component was present in PyBHK2. Most of the cytoplasmic virus-specific RNA in both clones sedimented at 1&S-19S. The sedimentation patterns of virus-specific RNA from whole cells and from washed nuclei of PyBHK, were closely similar: it was estimated from sedimentation analysis in dimethyl sulfoxide that the molecular weight of 50% of this RNA was within the range 1.1 x 106 to 2.9 x 106. These results, demonstrating the accumulation of virus-specific RNA within the nucleus in at least one virus-transformed cell line, indicate that the large virusspecific RNA previously described in the nuclei of transformed cells may not have represented precursors of virus-specific mRNA.

The infection of nonpermissive cells with either of the oncogenic DNA viruses, polyoma or simian virus 40 (SV40), results in a fraction of the cells acquiring stably altered growth properties (29). These "transformed" cells contain viral DNA, covalently attached to their nuclear DNA (7, 14, 30), virus-specific RNA (vsRNA) (5), and an intranuclear virus-specific antigen, the T-antigen (6, 13). There is evidence that the T-antigen contains a virus-specified polypeptide (12), implying that at least some of the vsRNA functions as mRNA. The genomes of polyoma virus and SV40 each encode only sufficient information for about five polypeptides of average size (30,000 daltons). These systems therefore provide a means of studying the RNA transcripts, either in mRNA or nuclear RNA, of a small group of genes in growing mammalian cells. Previous reports have shown the presence of virus-specific sequences both in large nuclear RNA molecules and in smaller polysomal RNA in virus-transformed cells (19, 26, 37-39). These

results are consistent with, although not proof of, the concept that virus-specific mRNA is derived from large precursor RNA molecules in the nucleus (19, 39). Studies are presented in this paper of the intracellular distribution of vsRNA and of the sedimentation properties of total, nuclear, and cytoplasmic vsRNA in polyoma virus-transformed hamster cells. These studies indicate that relatively stable vsRNA species, distinct from the cytoplasmic vsRNA, accumulate in the nucleus in at least one cell line. Nuclear vsRNA species, therefore, cannot necessarily be assumed to be precursors of mRNA. These studies also show that substantial differences can exist in the vsRNA species synthesized in different clones arising from the transformation of a single cell line with polyoma virus. MATERIALS AND METHODS Polyoma virus. Large plaque polyoma virus was obtained from Basil Smith. Stocks were prepared either by the method of Winocour (45) or by the in vitro infection of mouse kidney cell cultures at low multiplicity. Isolation and properties of transformed cells.

' Present address: Department of Anatomy, University of Colorado School of Medicine, Denver, Colo. 80220. 461

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Cells of the line BHK-21 C13 (35) were infected with polyoma virus at a multiplicity of 200 to 600 PFU per cell and were plated in soft agar (21). Colonies were picked out and grown to bulk culture, and then clones were selected after plating at high dilution on small pieces of cover slip. Cloning was repeated once. The clones designated PyBHK, and PyBHK2 were obtained from cells infected in separate experiments. Stocks of the cloned cells were stored in liquid N2 and were used to initiate fresh cultures every 4 to 6 weeks. Tests for mycoplasma (3) were negative. Cultures were maintained in 75-cm2 plastic bottles in reinforced Eagle medium (23) with 10% fetal bovine serum, at 37 C, in an atmosphere of air-5% CO2. Under these conditions the mean doubling times of BHK, PyBHK,, and PyBHK2 were approximately 11.5, 10, and 19 h, respectively. Large cultures were grown in glass roller bottles (surface area, 750 cm2), or, in some experiments, PyBHK, cells were grown in suspension culture in spinnermodified Eagle medium with 7% fetal bovine serum and were maintained at 2 x 105 to 8 x 105 cells/ml. The mean doubling time in suspension was usually about 17 h, but longer doubling times were sometimes observed, apparently depending mainly on the batch of serum used. When attached to a solid substrate, PyBHK, and PyBHK2 both grew as fibroblast-like cells with random orientation, in contrast to BHK, which showed characteristic whorls of parallel cells. Control experiments showed that the transformed cells did not produce polyoma virus. Both transformed cell lines were positive for polyoma-specific T-antigen, as determined by the ability of the subcutaneously injected cells to induce the production of antibodies to this antigen in young hamsters. Preparation and hybridization of polyoma viral DNA. Highly purified polyoma DNA component I (46) was obtained by either of two methods. (i) DNA, phenol extracted from partially purified virus, was centrifuged in a sucrose gradient at pH 11.5 to 12. The faster-sedimenting peak (containing reversibly denatured component I) was collected and, after neutralization, applied to a column of benzoylated DEAE-cellulose (18). Pure component I DNA was eluted with 0.9 M NaCl and diluted with water.

(ii) Purified polyoma virus was centrifuged in alkaline (pH 12.5) CsCl (1), and component I DNA was isolated as the 53S random cyclic coil (41), neutralized, and dialyzed to remove CsCl. After heat denaturation, the DNA was applied to nitrocellulose filters (Schleicher and Schuell; B6), which were then dried, baked and cut into 3.5-mm squares, each containing 0.2 ,ug of polyoma DNA (1). Hybridization mixtures (total volume, 0.1 ml, or for fractions taken directly from sucrose gradients, 0.15 ml) contained two filters (for unfractionated RNA) or one filter (for RNA from gradient fractions), each containing 0.2 ,ug of polyoma DNA, and one blank filter (of the same size, but lacking DNA) and the indicated quantities of RNA in a solution of 4 x SSC (lx SSC = 0.15 M NaCl plus 0.015 M sodium citrate)-0.05% sodium dodecyl sulfate. Incubation was for 36 to 44 h at 65 C. The filters were

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washed twice with 4 x SSC and incubated with heattreated RNase A (20 gg/ml in 2x SSC) for 1 h at 37 C. They were then washed 5 times with 4x SSC, containing 0.01% sodium dodecyl sulfate, and once with 4x SSC, dried, and placed in toluene scintillant for determination of radioactivity. Input radioactivity was determined by trichloroacetic acid precipitation of RNA samples on filter paper, with appropriate correction for counting efficiency. The radioactivity remaining on blank filters was usually considerably less than 0.001% of the input; values for blank filters are shown in all sedimentation profiles. Radioactivity bound to filters containing Escherichia coli DNA (0.2 gg) was determined in several experiments and found to be one to two times that bound to blank filters. Control experiments using labeled RNA from the late stage of the lytic infection of mouse kidney cells with polyoma showed that under the above conditions the radioactivity in hybrids was proportional to the RNA input and that little further radioactivity became bound to fresh polyoma DNA filters added after the initial incubation period (1; unpublished observations). Since late lytic RNA contained at least a 50-fold higher concentration of vsRNA than did transformed cell RNA, these results indicated that the amount of polyoma DNA was in large excess of the vsRNA in all the hybridization experiments reported here. Labeling of cells. Cells grown in roller bottles were labeled in suspension, except where otherwise stated. The cells were released from the glass surface by brief incubation with trypsin and EDTA and were suspended at 2 x 106 cells/ml in suspension culture medium. Cells grown in suspension culture were concentrated to 2 x 106 cells/ml or, for labeling periods of 4 h or longer, were used without concentrating (3 x 105 to 6 x 105 cells/ml). [5-3H]uridine and unlabeled uridine were added as indicated in the figure legends, and the suspension was incubated at 37 C for the required period before being chilled by addition to crushed, frozen 0.14 M NaCl10 mM Tris-hydrochloride, pH 7.4. The cells were collected by centrifugation (5 to 10 min at 200 x g) and were washed twice by resuspension in 0.14 M NaCl-10 mM Tris-hydrochloride, pH 7.4 (also containing 10 mM MgCl2 when a cytoplasmic extract was to be prepared). Subcellular fractionation procedure. Cells were suspended at a concentration of 4 x 107 to 10 x 107/ ml in 0.14 M NaCl-10 mM MgCl2-10 mM Tris-hydrochloride, pH 8.0. All operations were at 0 to 4 C. The nonionic detergent Nonidet P-40 was added to a concentration of 0.15%. After gentle mixing and incubation for 10 min, the suspension was centrifuged for 10 min at 300 x g, and the supernatant (cytoplasmic extract) was pipetted from the pellet of crude nuclei. Washed nuclei were prepared by resuspension of crude nuclei in 0.14 M NaCl-10 mM MgCl2-10 mM Tris-hydrochloride, pH 8.0, with six strokes of a Dounce homogenizer and addition of a mixture of Tween 40 and sodium deoxycholate as described by Penman (27). The nuclei were again pelleted by centrifugation for 10 min at 300 x g. Extraction of RNA. Total nucleic acids were ex-

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tracted from whole cells, nuclei, or cytoplasm by a [5-3H]uridine (20 to 30 Ci/mmol) was obtained modification (involving additional chloroform ex- from the Radiochemical Centre, Amersham, or from tractions [manuscript in preparation]) of the method New England Nuclear Corp.; [2-'4C]uridine was of Kirby and Cook (17). This technique was found to from New England Nuclear Corp.; Nonidet P-40 was give substantially higher yields of vsRNA than ex- from Shell Chemical Co. or British Drug Houses; traction with hot phenol. High-molecular-weight actinomycin D was from Calbiochem; fetal bovine RNA was precipitated from a solution of the total serum was from Gibco, Biocult, or Flow Laboratonucleic acids by the addition of NaCl to 3 M and ries. incubation at 0 C overnight. The precipitated RNA RESULTS was separated from DNA by centrifugation through a layer of 6 M NaBr (17) and was then reprecipitated Cell were cultures labeled with [3H]uridine, twice with ethanol. In most experiments (including and RNA extracted either from whole cells was all those where the RNA was to be centrifuged in Me2SO), the RNA was also passed through Sepha- or from cell fractions. vsRNA was detected by dex G75, and the excluded fraction was collected. hybridization with an excess of purified polyPrecipitation of cytoplasmic RNA with 3 M NaCl oma DNA (component I) immobilized on nitrowas omitted except where stated. cellulose filters. Density gradient centrifugation and fractionaProportion of radioactivity in vsRNA after tion. (i) Aqueous conditions. Linear gradients of various labeling periods. The percentage of hysucrose (Schwarz/Mann; RNase free) from 10 to 25% bridization observed with total RNA from (wt/vol) in 50 mM NaCl-1 mM Na2EDTA-10 mM Tris-hydrochloride, pH 7.4, were prepared in total PyBHK, ranged from 0.01 to 0.03, whereas volumes of 4.4 ml or 36 ml and were centrifuged in a much lower values (about 0.002) were obtained Beckman SW56 or SW27 rotor, respectively, at 20 C. with RNA from PyBHK2 (Table 1). The very The volumes of RNA solution (in the same salt low level of hybridization observed with RNA concentrations) layered on the gradients were 0.1 ml from BHK (Table 1) was probably not signifior 1.0 ml. In some experiments (where stated) the cant. Determination of the percentage of hygradients also contained sodium dodecyl sulfate bridization of total RNA, extracted after in(0.05%). The smaller gradients were fractionated by creasing times of labeling, provided a comparicounting drops from the bottom. For hybridization, son of the rate of accumulation of labeled urithese fractions were made up to 4x SSC-0.05% sothat in the major stable dium dodecyl sulfate and incubated directly with dine in vsRNA withRNA of cellular species (mainly rRNA). No filters containing polyoma DNA. The larger graof hybridiin the consistent change percentage dients were displaced with 40% (wt/vol) sucrose solution, and fractions (1 ml) were collected from the zation was detected with labeling periods from top. Carrier yeast RNA (50 Ag per fraction) was* 0.5 h to 9 h (Table 1), indicating that the bulk of added, and the RNA was precipitated overnight at vsRNA did not turn over very rapidly. These -15 C with 2 to 2.5 volumes of ethanol. The RNA observations were consistent with the idea of an from each fraction was redissolved and hybridized mRNA function for most of the vsRNA synthewith polyoma DNA, in a total volume of 0.1 ml. by these transformed cells, since recent (ii) Denaturing conditions. Linear gradients of sized work has shown that the bulk of mammalian chloral hydrate from 0 to 10% (wt/vol) in 1 mM mRNA has a relatively high stability (24, 28, Na2EDTA-1% (vol/vol) water in Me2SO were prepared in a total volume of 36 ml (22). The gradients 34), in contrast to most nuclear RNA (8, 31). were loaded with 1 ml of a solution prepared by However, the results of further experiments, dissolving the RNA sample in 0.25 ml of 1 mM presented below, showed that a high proportion Na2EDTA and mixing with 0.25 ml of Me2SO and 0.5 of the vsRNA accumulated in the cell nuclei ml of dimethylformamide (36). Centrifugation was and therefore did not function as mRNA. carried out at 25 C in an SW27 rotor. Fractionation Proportion of total vsRNA found in subceland hybridization were performed as described lular fractions. Labeled PyBHK, cells were above for sucrose gradients, except that 60% (wt/vol) lysed gently using a nonionic detergent (see sucrose was used for displacement and the RNA pellets were washed with cold 0.1 M sodium acetate- above) and fractionated by differential centrifu70% (vol/vol) ethanol before being dissolved for hy- gation. Nuclei were purified by washing with a double detergent mixture (27). RNA was then bridization. Total acid-insoluble radioactivity in gradient extracted from the cytoplasmic (10,000 x g sufractions was determined by applying samples (20 to pernatant) and other fractions. The proportion 100 ,ul) to 2.5-cm filter paper disks, which were then of the total radioactivity recovered and the perdried and washed with cold 5% (wt/vol) trichloroace- centage of hybridization with polyoma DNA tic acid and ethanol. The dried disks were placed in was determined for the RNA of each fraction, toluene scintillant, and radioactivity was measured and these values were used to calculate the in a liquid scintillation spectrometer. Correction for of the channel overlap was determined using 3H- or 4C- proportion of the total labeled vsRNA labeled RNA, acid precipitated in the same way. cells that was present in the fraction. After 9 h Values shown on the ordinates of the figures are of labeling, 68% of the total vsRNA was recovered in the nuclear fraction, which conthose calculated for whole fractions.

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tained 20% of the total cellular RNA (Table 2). Probably not more than 1/3 of the total RNA in this fraction represented cytoplasmic contamination, since it has been estimated that 14% of the total RNA of mouse L cells is nuclear RNA (8). Only 20% of the total vsRNA was found in the cytoplasmic fraction. Very small amounts of vsRNA were present in the Tween-deoxycholate wash of the nuclei (which contained RNA from perinuclear polysomes [271) and in the 10,000 x g pellet (Table 2). The vsRNA was therefore not concentrated either in membranebound polysomes or in the free polysomes of the cytoplasm but was mostly present within the

nuclei. This conclusion was confirmed by the results of three further experiments in which PyBHK, cells were labeled for 1.5, 2.7, and 17 h. In each experiment less than 25% of the total vsRNA was found in the cytoplasmic fraction. Sedimentation profiles of vsRNA in aqueous sucrose density gradients. RNA preparations were fractionated by centrifugation in sucrose gradients, and individual gradient fractions were hybridized with polyoma DNA. Figure 1 shows the sedimentation profiles of two RNA preparations from PyBHK, cells (experiment II, Table 1). The total 3H radioactivity after 0.5 h of labeling (Fig. la) was present

TABLE 1. Percentage of hybridization with polyoma DNA of total RNA extracted after various labeling periods % Input counts/ Counts/min hybridized Input Length of labelCell line counts/min min (mean) hybridized ing ing o(h) (x 10-) i ii iii

PyBHK,

Expt I

0.5 1.0 2.5 4.25 7.5

4.24 9.42 6.66 15.2 20.7

Expt II

0.5 2.7 9.2

1.55 4.06 2.04

PyBHK,

Expt I

Expt II

BHK

109 299 132 432 461

(0) (0) (0) (2) (0)

116 280 156 386 432

(0) (1) (1) (0) (1)

0.027 0.030 0.022 0.027 0.021

21 (1) 68 (4) 39 (1)

27 (0) 73 (0) 39 (1)

25 38

57 (1) 65 (4)

55 (1) 78 (4)

0.5 3.0

22 63

39 (10) 80 (36)

23 (16) 95 (36)

2.3

14 15 22

2.7

22 (2) 65 (1) 40 (0)

0.015 0.017 0.019

0.0022

0.0019 44 (12) 160 (16)

0.0016 0.0018

4 (4) 5 (0) 0.0003 2 (0) 3 (0) 0.0002 5 (0) 7 (1) 0.0003 a The cells were labeled in suspension for the lengths of time indicated, and total RNA was extracted and hybridized with polyoma DNA. Columns i, ii and iii show replicate determinations of counts per minute hybridized; radioactivity bound to blank filters (values given in parentheses) has been subtracted.

TABLE 2. Proportion of the total vsRNA found in subcellular fractions of PyBHK,' Yield of RNA S Yieldllular ofactionNA counSP cells % Totalp(counts/mini act c

Subcellular fraction

% Hybridization with

polyoma DNA

% Total

vsRNA

cells

Cytoplasm 1,570 58 44,100 0.0012 (51; 2)b 20 10,000 x g pellet 188 7 42,100 0.0017 (23; 2) 3 Tween-deoxycholate wash 410 15 41,400 0.0019 (57; 2) 8 Washed nuclei 546 20 71,100 0.0072 (353; 2) 68 PyBHK, cells (9 x 107), grown in suspension culture, were labeled for 9 h with [3H]uridine (2.5 mCi) in the presence of unlabeled uridine (1.7 juM). Cell fractionation and RNA extraction were then carried out as described in Materials and Methods. ° Values in parentheses are mean counts per minute hybridized; number of replicates.

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3H in

465

Total

3H14C

0co I

IC

0

9

c m

0= IS

I

a

M

1

c0

10

FRACTION NO. FIG. 1. Centrifugation in aqueous sucrose gradients of total RNA from PyBHK, cells, labeled with PHiuridine for (a) 0.5 h and (b) 9.2 h. A suspension culture of PyBHK, cells was labeled for 20 h with ['4C]uridine (0.02 tuCi/ml). Samples containing 3 x 107 cells were then treated as follows: (a) the cells were concentrated to 2 x 106 per ml in fresh medium and were labeled with [3H]uridine (30 pCi/ml) for 0.5 h, or (b) the cell suspension, initially containing 4.6 x 105 cells/ml, was labeled without concentrating by the addition of [3H]uridine (7.7 pfXi/ml) and unlabeled uridine (1.7 pM) for 92 h. After labeling, the cells were chilled and washed, and RNA was extracted as described in Materials and Methods. Centrifugation of the sucrose gradients was carried out in an SW56 rotor at 50,000 rpm for 90 min. The quantity of RNA loaded on each gradient was 75 to 80 pg. Sedimentation is from right to left. Symbols: A, 14C; 0, 3H total; *, 3H hybridized; *, 3H bound to blank filters in the hybridization reactions.

largely as prominent peaks of precursor tRNA as fast-sedimenting heterogeneous RNA. After 9.2 h most of the radioactivity was present in mature 28S and 18S rRNA (Fig. lb). In contrast, the sedimentation of vsRNA showed very little difference between the two labeling times. Also, the sedimentation pattern of and

vsRNA after 2.7 h of labeling (data not shown) virtually identical with that observed after 9.2 h. Thus, the vsRNA that accumulated in PyBHK, cells during 9 h of labeling appeared to be of the same size as the vsRNA observed after a label of only 0.5 h. Figure 2 shows the sedimentation profile of was

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10 FRACTION NO.

30

FIG. 2. Centrifugation in an aqueous sucrose gradient of RNA from the nuclei of PyBHK1 cells labeled with [3H]uridine for 9 h. The RNA was prepared in the experiment described in Table 2. The gradient was centrifuged in an SW56 rotor at 50,000 rpm for 90 min. The quantity of RNA loaded was approximately 100 pg. Sedimentation is from right to left. Symbols: 0, 3H total; *, 3H hybridized; *, 3H bound to blank filters.

RNA extracted from purified nuclei of PyBHK,. The pattern of vsRNA was very similar to that observed in total cell RNA (Fig. 1), in accordance with the observation that the bulk of the vsRNA was present within the nuclei (Table 2). Sedimentation profiles of vsRNA in density gradients prepared in Me2SO. Further sedimentation experiments were conducted under denaturing conditions, in the presence of a high concentration of Me2SO, with the intention of minimizing aggregation and variations in the secondary structure of RNA species (33, 36). Figure 3 shows the sedimentation of RNA extracted from crude (i.e., unwashed) nuclei and from total cytoplasm of PyBHK,. The cytoplasmic vsRNA (Fig. 3b) was observed to sediment considerably more slowly than most of the vsRNA of the crude nuclei. Using the rRNA species as standards, it was estimated that the molecular weight of 50% of the crude nuclear vsRNA was within the range 1.1 x 106 to 2.9 x 106 (fractions 15 to 21, Fig. 3a), whereas the

remainder of this RNA was distributed approximately equally between the lower and the upper parts of the gradient. The cytoplasmic vsRNA sedimented mainly as a broad band slightly faster than 18S rRNA, and it was estimated that less than 30% of the cytoplasmic vsRNA had a molecular weight greater than 1.1 x 106. The above estimates were obtained from a plot (not shown) of the logarithm of sedimentation rate against the logarithm of molecular weight (36), assuming molecular weights of 0.7 x 106 and 1.75 x 106 for 18S and 288 rRNA (20). Properties of the vsRNA of PyBHK2 cells. The low vsRNA content of PyBHK2 cells (Table 1) made quantitative studies more difficult than with PyBHK,. However, the radioactivity obtained in hybrids was sufficient to enable sedimentation analysis of vsRNA to be made in aqueous sucrose gradients (Fig. 4). Closely similar sedimentation profiles were obtained for vsRNA in four different preparations of total

FIG. 3. Density gradient centrifugation in Me2SO of (a) crude nuclear RNA and (b) cytoplasmic RNA from PyBHK,. (a) Cells growing in a roller bottle were labeled for 24 h with ['4C]uridine (10 ,uCi) and were then incubated overnight with unlabeled medium. Cells (108) were then labeled in suspension, in fresh medium for 1.5 h with [3H]uridine (40 .uCi/ml) in the presence of actinomycin D (0.05 pg/ml, added 30 min before the [3H]uridine). Crude nuclei were isolated, and RNA was extracted as described in Materials and Methods. RNA (50 pg) was centrifuged in an Me2SO-chloralhydrate gradient in an SW27 rotor at 27,000 rpm for 17.5 h. (b) Cytoplasmic RNA was prepared from 1.5 x 108 cells that had been labeled, in suspension, for 20 h with ['4C]uridine (0.02 uCi/ml) and then for 2.7 h with [3H]uridine (30 puCilml). RNA (170 i'g) was centrifuged in each of two Me2SO-chloralhydrate gradients in an SW27 rotor at 27,000 rpm for 22 h. Corresponding fractions of the two gradients were pooled, before the addition ofcarrier yeast RNA and ethanol precipitation, prior to hybridization. Sedimentation is from right to left. Symbols: A, 14C; 0, 3H total; 0, 3H hybridized; *, 3H bound to blank filters.

3H in

Total

hrid

3HC

?~8S

(a)

~~~63

A

14

188

01~

100

~

~

c)

*0

Ab

42

500

2

1

A-~~~~0

O

1003

10 FRACT ION NO. 467

30

0

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MAXWELL

RNA from PyBHK2 cells, two labeled for 0.5 h and two labeled for 3 h. Two of these profiles are shown in Fig. 4. There was no marked difference between the sedimentation patterns of vsRNA from PyBHK2 cells labeled for 0.5 or 3 h (Fig. 4a and b). This observation was similar to that made with PyBHK, (Fig. 1), as was the observation of vsRNA sedimenting around the 28S region, probably as several components. In addition, a prominent band of vsRNA sedimenting at 18S was observed in the total RNA preparations from PyBHK2 (Fig. 4a and b). This was in contrast to the results obtained with PyBHK,, where only a small proportion of the vsRNA of total cell RNA was observed to sediment in the 188 region (Fig. 1). Cytoplasmic RNA from PyBHK2 also showed a prominent peak of vsRNA sedimenting at approximately 18S (Fig. 4c). The cytoplasmic RNA probably also contained smaller quantities of faster-sedimenting vsRNA. A very prominent band of precursor-ribosomal RNA was observed in the total 3H pattern shown in Fig. 4a, probably corresponding to the 45S precursor in HeLa cells (43). In the total 3H-labeled RNA sedimentation pattern of PyBHK, cells, also labeled for 0.5 h, this peak was much less prominent (Fig. la), and a higher proportion of the total 3H was present in a slower-sedimenting precursor (probably corresponding to HeLa 32S [43]) and in 28S and 18S rRNA. The processing of precursor tRNA evidently occurred more slowly in PyBHK2 cells than in PyBHK,. This may be related to the slower growth rate of PyBHK2 (see above). The 188 peak of vsRNA observed in the total RNA from PyBHK2 cells, labeled for 0.5 h (Fig. 4a), might represent a primary transcript or a product of the processing of larger RNA molecules. If the latter possibility were correct, this processing would have to occur rapidly compared with the processing of precursor tRNA. The total radioactivity recovered in hybrids (i.e., the total vsRNA) in the gradient fractions of total and cytoplasmic RNA (Fig. 4b and c) was 1093 and 497 counts/min, respectively. The quantity of each type of RNA that had been loaded on the gradients was 500 ,ug. The propor-

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tion of intranuclear RNA in mouse L cells has been reported to be 14% of the total cellular RNA (8). Assuming this figure to be also true for PyBHK2 cells, 500 ,ug of total cellular RNA would contain 430 jig of cytoplasmic RNA, and the latter would account for 497 x 430 . 500 = 427 counts/min in vsRNA. Expressing the latter value as a percentage of 1,093 counts/min (i.e., the total vsRNA found in the total cellular RNA) gives an estimate of 39% for the proportion of the total labeled vsRNA present in the cytoplasm. This value was somewhat higher than that found for PyBHK, cells (Table 2; see above). Thus, although PyBHK2 cells synthesized much less vsRNA than PyBHK, (Table 1), a higher proportion of the vsRNA was transported to the cytoplasm in PyBHK2. The effect of longer labeling periods with PyBHK2 cells has not been investigated. The above results show that, after labeling this cell line for 3 h, the majority of the vsRNA was present in the nuclei.

DISCUSSION The general properties of the cells studied in this report (see above) were typical of other virus-transformed cell lines. The cells were isolated by their ability to form colonies in soft agar suspension. They grew with random orientation on solid substrates. They contained the polyoma-specific T-antigen, but no detectable capsid antigen or infectious virus. vsRNA was detected in both cell lines, although in markedly different quantity. Although the incorporation of [3H]uridine into vsRNA by each cell line varied somewhat from experiment to experiment, the values obtained for PyBHK1 were, on average, about 10-fold higher than for PyBHK2 (see Table 1). The results presented in Table 1 showed that, during continuous labeling with L3H]uridine, radioactivity accumulated in vsRNA. The percentage of hybridization was an approximate measure of the accumulation of radioactivity in vsRNA compared with that in rRNA. This percentage did not change with labeling periods ranging from 0.5 to 9 h, indicating that most of the vsRNA did not turn over rapidly. This ki-

FIG. 4. Centrifugation in aqueous sucrose gradients of RNA from PyBHK2. (a) and (b) Total RNA from cells labeled for 0.5 h and 3 h, respectively; (c) cytoplasmic RNA. The cells were grown in roller bottles and were labeled in suspension. (a) Cells (3.3 x 107) were labeled with [3H]uridine (40 pXi/ml) for 0.5 h, in suspension medium containing dialyzed serum, and total RNA was extracted. (b) and (c) Cells (108) were labeled with [3H]uridine (40 Adiml) for 3 h, and then total RNA was extracted from half the culture, and cytoplasmic RNA was extracted from the remaining half. Precipitation with 3 M NaCl was included in the preparation of the cytoplasmic RNA. Sucrose gradient centrifugation was carried out in an SW27 rotor for 6 h at 27,000 rpm at20 C. The quantities ofRNA loaded on the gradients were (a) 850 pg and (b) and (c) 500 pg. The 28S and 18S positions in gradient (a) (which was centrifuged separately from [b] and [ci) were determined by reference to rRNA from PyBHK,. Symbols: 0, 3H total; 0, 3H hybridized; *, 3H bound to blank filters.

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netic behavior was similar to that reported for the bulk of the mRNA of mammalian cells (24, 28, 34) and quite different from that of heterogeneous nuclear RNA (8, 31). However, the results presented in Table 2 showed that most of the vsRNA that accumulated in PyBHK, remained within the nuclei and therefore did not function as mRNA. The presence of polyomaspecific T-antigen (12) in the nuclei and the detection of polyadenylated vsRNA in the polysomes (unpublished observation) of PyBHK, indicated that some of the vsRNA functioned as mRNA; this was evidently only a minor fraction of the total vsRNA. The sedimentation analysis of total or nuclear RNA from PyBHK, under aqueous conditions (Fig. 1 and 2) or in Me2SO (Fig. 3) indicated the existence of several components of vsRNA, some of which sedimented faster than 288 rRNA. These vsRNA components accumulated in the cells, as shown by the absence of any significant change in the sedimentation pattern of vsRNA with increase in labeling time (Fig. 1) or in a pulse-chase experiment (not shown). In contrast to these observations with total RNA, sedimentation analysis of the cytoplasmic RNA of PyBHK, showed a broad band of vsRNA, sedimenting slightly faster than 188 rRNA (Fig. 3). Similarly, the major component of vsRNA in the cytoplasm of PyBHK2 cells was found to sediment at approximately 18S (Fig. 4c). However, the sedimentation pattern of vsRNA in the total RNA of these two cell lines differed markedly in that a prominent 18S component was observed in PyBHK2 but not in PyBHK, (Fig. 1 and 4). This observation can be explained by the synthesis of much larger amounts of faster-sedimenting vsRNA components in PyBHK, than in PyBHK2 cells, which would have masked the minor component of 188 vsRNA in the total RNA sedimentation pattern of PyBHK,. This interpretation is also consistent with the observation that a lower proportion of the total vsRNA was present in the cytoplasm of PyBHK, cells than of PyBHK2. The presence of 18 to 19S vsRNA has previously been reported in polysomes or total cytoplasm during productive and abortive infections with polyoma and SV40 (9, 37, 40), as well as in the cytoplasm and polysomes of SV40-transformed cells (37, 40). The 18S vsRNA component was prominent in the sedimentation pattern of total RNA from PyBHK2 even when these cells had been labeled for only 0.5 h. Since the labeling of the cytoplasmic mRNA of mammalian cells (with the exception of that for histones) shows a lag of 20 to 30 min (2, 32), it is probable that most of

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the 18S vsRNA labeled for 0.5 h was present in the nuclei. These results therefore suggest that the cytoplasmic 18S vsRNA was derived from nuclear vsRNA having about the same molecular weight. It is not known whether the 18S vsRNA represented a primary transcription product or was derived from the processing of a larger RNA molecule. However, any such processing must have been rapid compared with the processing of precursor tRNA, since the latter was present as a very prominent peak after 0.5 h of labeling (Fig. 4a). The sedimentation properties of the vsRNA of polyoma-transformed cells have not previously been studied in detail. Sedimentation patterns reported for the total vsRNA of SV40transformed cells show considerable variation (37, 40), in some cases resembling the vsRNA of PyBHK, cells and in other cases being more similar to that of PyBHK2. There have been very few previous studies of the effect of the length of labeling time on the sedimentation properties of the vsRNA of transformed cells. The average sedimentation coefficient of the labeled vsRNA that accumulated in PyBHK, cells was considerably higher than that of the cytoplasmic vsRNA. However, the accumulation of smaller species of labeled total vsRNA (probably largely comprising the cytoplasmic vsRNA) after long labeling times has been reported in two different lines of SV40-transformed cells (37, 47). After briefer labeling of the latter cells, much of the labeled vsRNA consisted of larger molecules (37, 47). The latter may have represented precursors of virus-specific mRNA, or, alternatively, nuclear vsRNA species that turned over rapidly compared with the relatively stable, nucleus-restricted vsRNA found in PyBHK,. Studies of the sedimentation distribution (but not the labeling kinetics) of nuclear and cytoplasmic vsRNA in other cell lines transformed by SV40 (19) or adenovirus 2 (26, 39) showed the presence of much larger molecules of vsRNA in the nuclei than in the cytoplasm, an observation consistent with the idea that the large nuclear species might represent precursors of virus-specific mRNA (19, 39). These results have frequently been quoted in support of the concept that eukaryotic mRNA, in general, may be derived from very large nuclear RNA precursors (10, 15, 44). These arguments are weakened by the present demonstration of nucleus-restricted vsRNA in PyBHK, cells. Furthermore, as discussed above, the finding of 188 vsRNA in the total RNA of PyBHK2 cells, labeled for only 0.5 h, raises the possibility that the transcripts which give rise to virus-specific

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mRNA may not be larger than the mRNA it- Jacqueline Amiguet for drawing the figures. This work was supported by grants to the Institute of self. This possibility is also suggested by the Research from the Medical Research Council and virtual absence of vsRNA sedimenting faster Cancer the Cancer Research Campaign, by a Royal Society Eurothan 18 to 19S in the total RNA of an SV40- pean Exchange Fellowship, and by funds from the Swiss transformed hamster cell line after 0.5 h of National Science Foundation. labeling (40). LITERATURE CITED Recent experiments involving the hybridizaN. H., E. Buetti, K. Scherrer, and R. Weil. tion of RNA from PyBHK, cells with the la- 1. Acheson, 1971. Transcription of the polyoma virus genome: beled, separated strands of polyoma DNA (4) synthesis and cleavage of giant late polyoma-specific have shown that the nuclear and cytoplasmic RNA. Proc. Natl. Acad. Sci. U.S.A. 68:2231-2235. vsRNA species are complementary to about 60 2. Adesnik, M., and J. E. Darnell. 1972. Biogenesis and characterisation of histone messenger RNA in HeLa and 40%, respectively, of the early strand. (The cells. J. Mol. Biol. 67:397-406. early strand of viral DNA is the strand which is 3. Barile, M. F., and R. T. Schimke. 1963. A rapid chemicomplementary to the viral mRNA, present in cal method for detecting PPLO contamination of tissue cell cultures. Proc. Soc. Exp. Biol. Med. 114:676polysomes during the early period of lytic infec679. tion.) Thus, a substantial fraction, not only of 4. Beard, P., N. H. Acheson, and I. H. Maxwell. 1976. the mass of vsRNA but also of the sequences Strand-specific transcription of polyoma virus DNA represented in vsRNA, was restricted to the early in productive infection and in transformed cells. J. Virol. 17:20-26. nuclei in this cell line. In common with many T. L. 1966. Virus-specific RNA in cells proother transformed cell lines (11, 16, 25), 5. Benjamin, ductively infected or transformed by polyoma virus. PyBHK, contains several viral genome equivaJ. Mol. Biol. 16:359-373. lents of integrated viral DNA per cell (H. 6. Black, P. H., W. P. Rowe, H. C. Turner, and R. J. Huebner. 1963. A specific complement-fixing antigen Turler and I. Maxwell, unpublished observapresent in SV40 tumor and transformed cells. Proc. tion). It may be that the vsRNA sequences Natl. Acad. Sci. U.S.A. 50:1148-1156. transcribed from only certain of the integration 7. Botchan, M., and G. McKenna. 1973. Cleavage of intesites ultimately function as mRNA, whereas grated SV40 by R1 restriction endonuclease. Cold Spring Harbor Symp. Quant. Biol. 38:391-395. those from other sites remain nucleus reB. P., and E. H. McConkey. 1974. Stabilstricted. The mechanism determining nucleus 8. Brandhorst, ity of nuclear RNA in mammalian cells. J. Mol. Biol. restriction or transport to the cytoplasm of 85:451-463. vsRNA is unknown. It has been shown that the 9. Buetti, E. 1974. Characterization of late polyoma mRNA. J. Virol. 14:249-260. virus-specific mRNA of cells transformed by J. E., W. R. Jelinek, and G. R. Molloy. 1973. SV40 and polyoma is polyadenylated (42; un- 10. Darnell, of mRNA: genetic regulation in mammaBiogenesis published observations). Evidence will be prelian cells. Science 181:1215-1221. sented elsewhere (manuscript in preparation) 11. Gelb, L. D., D. E. Kohne, and M. A. Martin. 1971. Quantitation of simian virus 40 sequences in African that most of the nuclear vsRNA of PyBHK, green monkey, mouse and virus-transformed cell geis present in molecules that are polyadenylnomes. J. Mol. Biol. 57:129-145. ated and therefore that nucleus restriction of 12. Graessmann, A., M. Graessmann, H. Hoffmann, and J. vsRNA in this cell line is not due to a failure in Niebel. 1974. Inhibition by interferon of SV40 tumor antigen formation in cells injected with SV40 cRNA polyadenylation. transcribed in vitro. FEBS Lett. 39:249-251. It is a reasonable hypothesis that an interac- 13. Habel, K. 1965. Specific complement-fixing antigens in a tion between a virus-specified component and polyoma tumors and transformed cells. Virology cellular control element in virus-transformed25:55-61. cells, resulting in the stimulation of the replica- 14. Hirai, K., D. Henner, and V. Defendi. 1974. Hybridization of simian virus 40 complementary RNA with tion of the cellular genome, may be responsible nucleolus-associated DNA isolated from simian virus for the maintenance of the altered growth prop40-transformed Chinese hamster cells. Virology erties of these cells. Although it is likely that 60:588-591. the postulated virus-specified component might 15. Imaizumi, T., H. Diggelmann, and K. Scherrer. 1973. Demonstration of globin messenger sequences in be a polypeptide (40), it is also conceivable that giant nuclear precursors of messenger RNA of avian the vsRNA of transformed cells might have a erythroblasts. Proc. Natl. Acad. Sci. 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bonucleic acid from mammalian tissues. Biochem. J. 104:254-257. 18. Komano, T., and R. L. Sinsheimer. 1968. Preparation and purification of 4OX-RF component I. Biochim. Biophys. Acta 155:295-298.

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