- Email: [email protected]
Strand-specific transcription of polyoma virus DNA late during productive infection
J. XoZ. Biol.
(1977) 115, 237-242
LETTERS To THE EDwon
Strand-specific Transcription of Polyoma Virus DNA Late during Productive Infection Thr
rolativc rates of transcription from the E and L st,rands of polyoma virus have been measured by hybridization of pulse-labelled RNA extracted from polyoma-infected secondary mouse. embryo cells to a.n txcess of sepamted polyoma DNA strands in solution. L DNA strand t8ranscripts comprise 91 to 97”; of the newly synthesized polyoma RNA mwlc at late times during productjive infection.
DXA
Late during productive infection by polyoma virus, RKA which is complementary to the entirety of both viral DXA strands is present’ in the nuclei of the infected cells (Aloni & Locker, 1973; Kamen et al., 1974). Only one-half of the sequences which are t,ranserjbed from each DNA strand, however, are ultimately found in cytoplasmic polyadenylat,ed mRNA (Kamen & Shure, 1976; Tiirler et al.. 1976). This suggest,s that post-transcriptional processing is important for viral gene expression. Studies using radioactively labelled, single-stranded polyoma DNA as a probe to measure the abunda’nce of total unlabelled, virus-specific RNA further showed that nuclear transcripts of one of the DNA strands (the L strand) are present in at least a tenfold excess over corresponding transcriptIs of the complementary (or E) DNA strand (Kamen et al., 1974). Similar data have been obtained for t,he closely related papova virus SV40 (see Acheson (1976) for a comparative review of SV40 and polyoma transcription). These observations led to the proposal that t)emplate strand selechion is cont’rolled at the transcriptional level, while subsequent RNA processing select’s which portions of each primary transcript become mRNS. A direct demonstration of transcriptional control at. the level of template strand selection demands the analysis of newly synthesized rather than of total viral RNB. To measure the proportion of newly synthesized RNA which is complementary to eit,her polyoma DNA strand, it. is necessary to use a hybridization assay that prevent,s t’he competitive self-annealing of viral RNA sequences. Accordingly, we have hybridized pulse-labelled RNA extracted from the nuclei of infected cells to excess amounts of either E or L strand polyoma DP;A in solution. Separated strands of polyoma DNA, labelled at a low specific activity (100 to 1000 cts/min per pg) with [32P]ortjhophosphate, were made by essentially the same method previously used for the preparation of highly radioactive polyoma DNA strands (Kamen et al., 1974). The 10~ levels of 32P were used to facilitate DNA quantitation during strand separation and did not, interfere with subsequent hybridization assays. Radioactive RNA was annealed to excess amounts of DNA strands in solution and bhe resulting hybrids were recovered by adsorption ho nitrocellulose filters (Nygaard & Hall, 1964). Cont,aminat;ing REA was removed by digestion with ribonuclease A combined with ext.ensive washing (see Table 1 for experimental details), 237
238
.
I
I
0.02
0
-
o-04
1
0.06
0.08
Polyoma DNA (pg) PIG. 1. Hybridization of asymmetric polyoma cRNA to E and L strand polyoma DNA. Asynimetric 32P-labelled cRNA was prepared at a spec. act. of 5 x 107 cts/min per pg as described by Kamen et al. (1976), except that the concentration of [32P]UTP (40 Ci/mmol) used was 0.1 mm and that of the other nucleosido triphosphates was 0.5 mu. Input radioactivity/hybridization 850 cts/min for L DNA strand hybridizations (-•-[.I--) and 9000 cts/min for E strantl hybridizations (-a-e--). Data are corrected for counter background of 10 cts/min.
TABLE
Hybridization
of asymw&ic
3H inputt (cts/min)
cRNA cRNA
polyoma cRNA
Strand
1510 921
Input bg)
E L
0.05
0.05
to E or L strand polyowza DNA Radioactivity recovered (cts/min)
DNA
RNA
Sample
1
32P inputt (cts/min) 613 921
L3Hl RNA? 2 666
Yield
w?
DNA? 506 751
(‘;/o)
RNA
[V] DNA
0.1 12
53 82
13Hl
Asymmetric sH-labelled cRNA was prepared at a spec. act. of 1.0 X lo6 cts/min per pg as preof each nucleoside triphosviously described (Kamen et al., 1976), except that the concentration i mmol) was used. Separated strands of polyoma phate was lowered to 0.5 mM and [3H]UTP (2 C'/ DNA, either at lower (100 to 1000 cts/min per pg) or higher (1 x IO6 to 5 r: lo6 cts/min per pg) 32P spec. act. were prepared frcm polyoma superhelical DNA after digestion with restriction endonuclease EcoRI using the method described by Kamen et al. (1974). [3H]cRNA was heated with sap-labelled E or L strand DNA (a mixture of low and high spec. act. DNA containing the indicated quantity of radioactivity) at 107°C for 15 min in 0.1 ml of hybridization buffer (40 mMTris (pH 7.5), 1 mM-EDTA, 1 ix-NaCl) prior to annealing at 70°C for 15 h. The annealing reactions were then diluted into 10 ml of loading buffer (100 mivr-Tris (pH 7.5), 300 mM-NaCl, 2 mm-EDTA, 1 M-KCl) which was then passed under suction through nitrocellulose filters (Schleicher & Schull, 0.46 pm), which had been presoaked in loading buffer. Each filter was washed with 50 ml of loading buffer and digested with 20 pg pancreatic ribonuclease/ml (Sigma type X-A, preheated at 100°C for 5 min) in 5 ml of 2 x SSC (SSC is 0.15 iv-Nacl, 0.015 nr-trisodium citrate, pH 7.5) for 1 h. After a further wash under suction with 50 ml of loading buffer, the filters were dried and assayed for radioactivity by liquid scintillation counting. Input radioactivities were determined by spotting portions of the hybridization mixes onto presoaked nitroccllulose filters, drying and counting as described above. t Corrected
for counter
background
of 10 cts/min.
LETTERS
TO
THE
EDITOR
539
In preliminary experiments 3H-labelled asymmetric (L strand-specific) polyoma cRNA (the product of transcription in vitro of polyoma superhelical DNA by Escherichia coli RNA polymerase at high ionic strengt’h) was used to measure the efficiency of the hybridization assay and to tesb the DNA strand preparations for cross-contamination. Figure 1 shows that maximally 70(y0 of t)he cRNA was recovered as RNAase-resistant hybrid after exhaust’ive annealing to excess amounts of L strand. DNA, whereas less than 0.1 o/owas recovered when t’he cRNA was annealed mit’h E strand DNA. The 700/, recovery of the cRNA largely reflects the loss of
3.2
I 0
0.02
0.04
0.06
0.08
0.10
Polyoma DNA (pg )
FIG. 2. Hybridization of RNA extracted from polyoma-infected mouse embryo cells late in infection to E and L strand polyoma DNA. 3H-labelled nuclear RNA was prepared from cells pulse-labelled at 32 h post-infection as described in Table 2. Input radioactivity/hybridization = 24,000 cts/min for L strand hybridizations (-n-m--) and 160,000 cts/min for E strand hybridizations (-@-a-). [3H]RNA extracted from uninfected control cells showed less than 0.03% hybridization to either DNA strand. The experimental points are shown without subtraction of this background hybridization but have been corrected for counter background of 10 cts/min and normalized for DNA rec0ver.y as described in the text.
adsorbed DNA from the filters during treatment, with RNAase and subsequent, washing, as shown in Table 1. The efficiency of the assay varied between 60% and 70%, depending on the DNA preparation used, hut, was invariant between experiments for any given preparation. RNA which had been extracted from the nuclei of mouse embryo cells pulselabelled with [3H]uridine for 30 minutes at various times late during polyoma infection was annealed to E or L strand DNA and assayed for formation of ribonuclease A resistant hybrids. Viral DNA synthesis was first observed between 16 and 20 hours post-infection in the cell system used (see Table 2 footnote for experimental 16
36 36 (ZUock)
27 30 32 33
24
(h)
RNA extracted post-infection
E
radioactivity
84,500 116,000 164,000 142,000 15,000 6100 83,000 9000 165,000
hybridizations
Input
hybridizations ~- __ ~..___ 84,500 47,000 28,000 22,000 15,900 6100 16,000 9000 165,000
L
(cts/min)
of RNA
Plus strand DNA 1578 907 92x 819 471 315 619 297 34
Plus E strand DNA 141 136 355 234 32 30 341 32 54
62 68 31 31 14 2 22 3 15
(cts/min)
recovered
L
E or L
MiIlUS polyoma DNA
to
Radioactivity
extracted late in infection polyoma
0.09 0.06 0.20 0.14 0.12 0.46 0.38 0.32 0.02
hybridizations
E
O/n Radioactivity
strand
L
recoveredt
1.79 1.78 3.27 3.70 3.07 5.10 3.80 3.38 0.01
hybridizations
DNA
-~~ %E
+
4.8 3.3 5.8 3.6 3.8 8.1 9.1 8.6
‘YJ
%E
x 100
i Corrected
for minus
polyoma
DNA
control
hybridizations.
Primary mouse embryo cells, grown to confluence in Dulbecco’s modified Eagle’s medium (DME medium) supplemented with 10% calf serum, were sub1968). The cells cultured at 4 x IO6 cells/go-mm dish and maintained at 37°C in DME medium containing 0.50,; foetal calf serum for 3 days (Fried & Pitts, were infected with plaque-purified polyoma virus (AZ st.rain) at 100 plaque-forming units/cell in 1 ml of DME medium containing 59<, foet,al calf serum for 96 min. Additional medium (5 ml) was then added and the infected cells were kept at 37°C. At varying times post-infection the cells were labelled for 30 min with 200 @i [5,6-3H]uridine (50 Ci/mmol; Radiochemical Centre, Amersham) in 1 ml of fresh medium. The cells were washed on the dish with ice-cold TD policeman. The cell suspensions were then centrifuged at buffer (Fried, 1970) and harvested by scraping the cell sheet off the dishes with a silicon e rubber 1000 g for 10 min at 0°C and cell pellets were resuspended in 5 ml of Go-Hi pH (10 m&r-Tris (pH 8.5), 0.14 nr-NaCl, 1.5 mM-MgCl,, 0.5% NP40: Lindberg & (Schwartz-iMann; RNAase-free), 0.5% (v/v) NP40 (B.D.H.) and then Darnell, 1970). Nuclei were isolated by sedimentation through 24% ( w ! v ) sucrose resuspended in 5 ml of a buffer containing 2% sodium dodecyl sulphate, 10 m&r-Tris (pH 7.5), 10 mar-EDTA, 25 pg polyvinyl sulphonate/ml and 200 pg Proteinase K/ml (Merck) by repeated passage through a 19.gauge hypodermic needle. After 3 h at 3i0i! the solutions were diluted with 5 ml of distilled water and extracted with an equal volume of phenol/chloroform/isoamyl alcohol (50 : 50 : 1). Nucleic acids were recovered from the aqueous phase by precipitation with ethanol. DNA was hydrolysed by digestion for 2 h at 37°C in a buffer containing 10 mx-Tris (pH 7.5), 50 pg deoxyribonualease I/ml (Worthington; ribonuclease-free grade), 16 mx.MgCl,. After addition of sodium dodecy sulphate to 0.1% and EDTA to 20 mM, the extraction, precipitation and digestion were repeated once. The final RNA pellets were resuspended in 0.4 ml of 10 m&r-Tris (pH 7.5), I mxr-EDTA, 0.1 ok1 sodium dodecyl sulphate. Typical yields of radioactivity from one 90 mm dish were 1.5 x lo6 to 2 x 10s cts/min sH at approx. 105 cts/min per pg. Hybridizations were done as described in Table 1 using 0.05 pg E or L polyoma DNA strands. DNA synthesis was measured by labelling of parallel cell cultures with 200 pCli of js2P]orthophosphate (3000 Ci/mmol; Radiochemirnl Centre, Amersham) in 1 ml DME medium minus phosphate for 4-h periods. After Hirt extraction (Hut. 1967) and agarose gel electrophoresis of deprot.einized Hirt supernatant fractions, radioactive polyoma form I DNA was detected by autoradiography. The first detectable viral DNA replicat,ion occurred in the 16 to 20-h post-infection labelling period. All radioactivity values (cts/min) shown are corrected for counter hackground of 10 cts/min.
-~.
Time
Hybridization
TABLE 2
LETTERS
TO
THE
241
EDITOR
details). Table 2 shows that the vast majority (91 to 97%) of newly synthesized viral RNA, pulse-labelled at times ranging from 24 to 36 hours post-infection, anneals to the L DNA strand in DNA strand excess. This is confirmed by the DNA saturation curves shown in Figure 2, which demonstrate that only about 5% of the viral RNA harvested 32 hours after infection hybridizes to the E DNA strand in DNA excess. The fraction of t*otal RNA synthesis which is polyoma-specific increased during the late period (Table 2); at 24 hours post-infection, about 0.1% is complementary to the E DNA4 strand and about 2.7% is complementary to the L DNA strand (after correction of thrx data shown in Table 2 for the efficiency of the hybridization assay), while at 33 hours the corresponding values are 0.65% and 6.7%. The proportion of virusspecitic RNA that was transcribed from the E DNA strand varied from approximately 4% to 9 “/b during the same period. However, this variation is probably caused h,v t,he inaccuracy inherent in quantitating the low levels of E DNA strand transcription observed at the earliest time points shown. Similarly, it proved difficult to IIHP the assay described here to study the early phase of t,he lyt’ic cycle, since the amounts of polyoma transcription detected were too close to the non-specific background keel of bhe assay (O$l to 0.03% of input radioactivity). In conclusion, analysis of pulse-labelled polyoma RNA strongly suggests that viral RNA is predominantly hranscribed from the L DEA strand at late t’imes after infection. Similar results have recently been reported for SV40 virus (Laub $ Aloni, 1975; Reed et ul., 1976; Gilboa & Aviv, 1976). Since t’he ratio of E t’o L strand transcripts measured with the pulse-labelled material is very similar to that previously report’ed for unlabelled viral RNA (Kamen et uZ., 1974), we infer that control at the transcriptional rather than at the post-transcriptional level is primarily responsible for the bias towards L DNA strand transcripts which is found in polyoma mRP;A. One objection to t’his inference is that rapid processing by cellnlar nucleases account,s for the results obtained. The results of experiments in vitro using transcript,ion complexes purified from infected cells (described in the accompanying paper, Condit d nl. (1977)) exclude this objection. We conchldc that t,hr expression of polyoma virus DNA is subject, t,o transcriptional control. Impekl Cancer Rcscarch Fnntl Laborat#ories, P.O. Box 123 Lincoln’s Inn Fields, London WC2A 3PX, England Rwr~i\~c~d
17 Fc~brrmry
1977, md
ANDREW J. FLAVELL ROBERTKAMEX
in revised form 16 .Jrlnr 1977
REFERENCES Acheson, N. (1976). CeZE,8, 1-12. Aloni, Y. & Locker, H. (1973). I’irology, 54, 495-505. Con&t, R. C., Cowie, A., Kamen, R. & Birg, F. (1977). .J. Xol. Biol. 115, 233-253. Fried, M. (1970). Virology, 40, 605-617. Fried, M. & Piths, J. D. (1968). Virology, 34, 761-770. Cilbo+ E. & Aviv, H. (1976). Cell, 7, 567-573. Hirt, B. (1967). J. Mol. BioZ. 26, 365-369. Kamm, R. & Shure, H. (1976). Cell, 7, 361-371. Kamen, R., Lindstrom, D. M., Shure, H. & Old, R. IV. (1974). CoZdSpring HarborSymp. G)ctnnt. BioZ. 39, 187-198.
(1977) 115, 237-242
LETTERS To THE EDwon
Strand-specific Transcription of Polyoma Virus DNA Late during Productive Infection Thr
rolativc rates of transcription from the E and L st,rands of polyoma virus have been measured by hybridization of pulse-labelled RNA extracted from polyoma-infected secondary mouse. embryo cells to a.n txcess of sepamted polyoma DNA strands in solution. L DNA strand t8ranscripts comprise 91 to 97”; of the newly synthesized polyoma RNA mwlc at late times during productjive infection.
DXA
Late during productive infection by polyoma virus, RKA which is complementary to the entirety of both viral DXA strands is present’ in the nuclei of the infected cells (Aloni & Locker, 1973; Kamen et al., 1974). Only one-half of the sequences which are t,ranserjbed from each DNA strand, however, are ultimately found in cytoplasmic polyadenylat,ed mRNA (Kamen & Shure, 1976; Tiirler et al.. 1976). This suggest,s that post-transcriptional processing is important for viral gene expression. Studies using radioactively labelled, single-stranded polyoma DNA as a probe to measure the abunda’nce of total unlabelled, virus-specific RNA further showed that nuclear transcripts of one of the DNA strands (the L strand) are present in at least a tenfold excess over corresponding transcriptIs of the complementary (or E) DNA strand (Kamen et al., 1974). Similar data have been obtained for t,he closely related papova virus SV40 (see Acheson (1976) for a comparative review of SV40 and polyoma transcription). These observations led to the proposal that t)emplate strand selechion is cont’rolled at the transcriptional level, while subsequent RNA processing select’s which portions of each primary transcript become mRNS. A direct demonstration of transcriptional control at. the level of template strand selection demands the analysis of newly synthesized rather than of total viral RNB. To measure the proportion of newly synthesized RNA which is complementary to eit,her polyoma DNA strand, it. is necessary to use a hybridization assay that prevent,s t’he competitive self-annealing of viral RNA sequences. Accordingly, we have hybridized pulse-labelled RNA extracted from the nuclei of infected cells to excess amounts of either E or L strand polyoma DP;A in solution. Separated strands of polyoma DNA, labelled at a low specific activity (100 to 1000 cts/min per pg) with [32P]ortjhophosphate, were made by essentially the same method previously used for the preparation of highly radioactive polyoma DNA strands (Kamen et al., 1974). The 10~ levels of 32P were used to facilitate DNA quantitation during strand separation and did not, interfere with subsequent hybridization assays. Radioactive RNA was annealed to excess amounts of DNA strands in solution and bhe resulting hybrids were recovered by adsorption ho nitrocellulose filters (Nygaard & Hall, 1964). Cont,aminat;ing REA was removed by digestion with ribonuclease A combined with ext.ensive washing (see Table 1 for experimental details), 237
238
.
I
I
0.02
0
-
o-04
1
0.06
0.08
Polyoma DNA (pg) PIG. 1. Hybridization of asymmetric polyoma cRNA to E and L strand polyoma DNA. Asynimetric 32P-labelled cRNA was prepared at a spec. act. of 5 x 107 cts/min per pg as described by Kamen et al. (1976), except that the concentration of [32P]UTP (40 Ci/mmol) used was 0.1 mm and that of the other nucleosido triphosphates was 0.5 mu. Input radioactivity/hybridization 850 cts/min for L DNA strand hybridizations (-•-[.I--) and 9000 cts/min for E strantl hybridizations (-a-e--). Data are corrected for counter background of 10 cts/min.
TABLE
Hybridization
of asymw&ic
3H inputt (cts/min)
cRNA cRNA
polyoma cRNA
Strand
1510 921
Input bg)
E L
0.05
0.05
to E or L strand polyowza DNA Radioactivity recovered (cts/min)
DNA
RNA
Sample
1
32P inputt (cts/min) 613 921
L3Hl RNA? 2 666
Yield
w?
DNA? 506 751
(‘;/o)
RNA
[V] DNA
0.1 12
53 82
13Hl
Asymmetric sH-labelled cRNA was prepared at a spec. act. of 1.0 X lo6 cts/min per pg as preof each nucleoside triphosviously described (Kamen et al., 1976), except that the concentration i mmol) was used. Separated strands of polyoma phate was lowered to 0.5 mM and [3H]UTP (2 C'/ DNA, either at lower (100 to 1000 cts/min per pg) or higher (1 x IO6 to 5 r: lo6 cts/min per pg) 32P spec. act. were prepared frcm polyoma superhelical DNA after digestion with restriction endonuclease EcoRI using the method described by Kamen et al. (1974). [3H]cRNA was heated with sap-labelled E or L strand DNA (a mixture of low and high spec. act. DNA containing the indicated quantity of radioactivity) at 107°C for 15 min in 0.1 ml of hybridization buffer (40 mMTris (pH 7.5), 1 mM-EDTA, 1 ix-NaCl) prior to annealing at 70°C for 15 h. The annealing reactions were then diluted into 10 ml of loading buffer (100 mivr-Tris (pH 7.5), 300 mM-NaCl, 2 mm-EDTA, 1 M-KCl) which was then passed under suction through nitrocellulose filters (Schleicher & Schull, 0.46 pm), which had been presoaked in loading buffer. Each filter was washed with 50 ml of loading buffer and digested with 20 pg pancreatic ribonuclease/ml (Sigma type X-A, preheated at 100°C for 5 min) in 5 ml of 2 x SSC (SSC is 0.15 iv-Nacl, 0.015 nr-trisodium citrate, pH 7.5) for 1 h. After a further wash under suction with 50 ml of loading buffer, the filters were dried and assayed for radioactivity by liquid scintillation counting. Input radioactivities were determined by spotting portions of the hybridization mixes onto presoaked nitroccllulose filters, drying and counting as described above. t Corrected
for counter
background
of 10 cts/min.
LETTERS
TO
THE
EDITOR
539
In preliminary experiments 3H-labelled asymmetric (L strand-specific) polyoma cRNA (the product of transcription in vitro of polyoma superhelical DNA by Escherichia coli RNA polymerase at high ionic strengt’h) was used to measure the efficiency of the hybridization assay and to tesb the DNA strand preparations for cross-contamination. Figure 1 shows that maximally 70(y0 of t)he cRNA was recovered as RNAase-resistant hybrid after exhaust’ive annealing to excess amounts of L strand. DNA, whereas less than 0.1 o/owas recovered when t’he cRNA was annealed mit’h E strand DNA. The 700/, recovery of the cRNA largely reflects the loss of
3.2
I 0
0.02
0.04
0.06
0.08
0.10
Polyoma DNA (pg )
FIG. 2. Hybridization of RNA extracted from polyoma-infected mouse embryo cells late in infection to E and L strand polyoma DNA. 3H-labelled nuclear RNA was prepared from cells pulse-labelled at 32 h post-infection as described in Table 2. Input radioactivity/hybridization = 24,000 cts/min for L strand hybridizations (-n-m--) and 160,000 cts/min for E strand hybridizations (-@-a-). [3H]RNA extracted from uninfected control cells showed less than 0.03% hybridization to either DNA strand. The experimental points are shown without subtraction of this background hybridization but have been corrected for counter background of 10 cts/min and normalized for DNA rec0ver.y as described in the text.
adsorbed DNA from the filters during treatment, with RNAase and subsequent, washing, as shown in Table 1. The efficiency of the assay varied between 60% and 70%, depending on the DNA preparation used, hut, was invariant between experiments for any given preparation. RNA which had been extracted from the nuclei of mouse embryo cells pulselabelled with [3H]uridine for 30 minutes at various times late during polyoma infection was annealed to E or L strand DNA and assayed for formation of ribonuclease A resistant hybrids. Viral DNA synthesis was first observed between 16 and 20 hours post-infection in the cell system used (see Table 2 footnote for experimental 16
36 36 (ZUock)
27 30 32 33
24
(h)
RNA extracted post-infection
E
radioactivity
84,500 116,000 164,000 142,000 15,000 6100 83,000 9000 165,000
hybridizations
Input
hybridizations ~- __ ~..___ 84,500 47,000 28,000 22,000 15,900 6100 16,000 9000 165,000
L
(cts/min)
of RNA
Plus strand DNA 1578 907 92x 819 471 315 619 297 34
Plus E strand DNA 141 136 355 234 32 30 341 32 54
62 68 31 31 14 2 22 3 15
(cts/min)
recovered
L
E or L
MiIlUS polyoma DNA
to
Radioactivity
extracted late in infection polyoma
0.09 0.06 0.20 0.14 0.12 0.46 0.38 0.32 0.02
hybridizations
E
O/n Radioactivity
strand
L
recoveredt
1.79 1.78 3.27 3.70 3.07 5.10 3.80 3.38 0.01
hybridizations
DNA
-~~ %E
+
4.8 3.3 5.8 3.6 3.8 8.1 9.1 8.6
‘YJ
%E
x 100
i Corrected
for minus
polyoma
DNA
control
hybridizations.
Primary mouse embryo cells, grown to confluence in Dulbecco’s modified Eagle’s medium (DME medium) supplemented with 10% calf serum, were sub1968). The cells cultured at 4 x IO6 cells/go-mm dish and maintained at 37°C in DME medium containing 0.50,; foetal calf serum for 3 days (Fried & Pitts, were infected with plaque-purified polyoma virus (AZ st.rain) at 100 plaque-forming units/cell in 1 ml of DME medium containing 59<, foet,al calf serum for 96 min. Additional medium (5 ml) was then added and the infected cells were kept at 37°C. At varying times post-infection the cells were labelled for 30 min with 200 @i [5,6-3H]uridine (50 Ci/mmol; Radiochemical Centre, Amersham) in 1 ml of fresh medium. The cells were washed on the dish with ice-cold TD policeman. The cell suspensions were then centrifuged at buffer (Fried, 1970) and harvested by scraping the cell sheet off the dishes with a silicon e rubber 1000 g for 10 min at 0°C and cell pellets were resuspended in 5 ml of Go-Hi pH (10 m&r-Tris (pH 8.5), 0.14 nr-NaCl, 1.5 mM-MgCl,, 0.5% NP40: Lindberg & (Schwartz-iMann; RNAase-free), 0.5% (v/v) NP40 (B.D.H.) and then Darnell, 1970). Nuclei were isolated by sedimentation through 24% ( w ! v ) sucrose resuspended in 5 ml of a buffer containing 2% sodium dodecyl sulphate, 10 m&r-Tris (pH 7.5), 10 mar-EDTA, 25 pg polyvinyl sulphonate/ml and 200 pg Proteinase K/ml (Merck) by repeated passage through a 19.gauge hypodermic needle. After 3 h at 3i0i! the solutions were diluted with 5 ml of distilled water and extracted with an equal volume of phenol/chloroform/isoamyl alcohol (50 : 50 : 1). Nucleic acids were recovered from the aqueous phase by precipitation with ethanol. DNA was hydrolysed by digestion for 2 h at 37°C in a buffer containing 10 mx-Tris (pH 7.5), 50 pg deoxyribonualease I/ml (Worthington; ribonuclease-free grade), 16 mx.MgCl,. After addition of sodium dodecy sulphate to 0.1% and EDTA to 20 mM, the extraction, precipitation and digestion were repeated once. The final RNA pellets were resuspended in 0.4 ml of 10 m&r-Tris (pH 7.5), I mxr-EDTA, 0.1 ok1 sodium dodecyl sulphate. Typical yields of radioactivity from one 90 mm dish were 1.5 x lo6 to 2 x 10s cts/min sH at approx. 105 cts/min per pg. Hybridizations were done as described in Table 1 using 0.05 pg E or L polyoma DNA strands. DNA synthesis was measured by labelling of parallel cell cultures with 200 pCli of js2P]orthophosphate (3000 Ci/mmol; Radiochemirnl Centre, Amersham) in 1 ml DME medium minus phosphate for 4-h periods. After Hirt extraction (Hut. 1967) and agarose gel electrophoresis of deprot.einized Hirt supernatant fractions, radioactive polyoma form I DNA was detected by autoradiography. The first detectable viral DNA replicat,ion occurred in the 16 to 20-h post-infection labelling period. All radioactivity values (cts/min) shown are corrected for counter hackground of 10 cts/min.
-~.
Time
Hybridization
TABLE 2
LETTERS
TO
THE
241
EDITOR
details). Table 2 shows that the vast majority (91 to 97%) of newly synthesized viral RNA, pulse-labelled at times ranging from 24 to 36 hours post-infection, anneals to the L DNA strand in DNA strand excess. This is confirmed by the DNA saturation curves shown in Figure 2, which demonstrate that only about 5% of the viral RNA harvested 32 hours after infection hybridizes to the E DNA strand in DNA excess. The fraction of t*otal RNA synthesis which is polyoma-specific increased during the late period (Table 2); at 24 hours post-infection, about 0.1% is complementary to the E DNA4 strand and about 2.7% is complementary to the L DNA strand (after correction of thrx data shown in Table 2 for the efficiency of the hybridization assay), while at 33 hours the corresponding values are 0.65% and 6.7%. The proportion of virusspecitic RNA that was transcribed from the E DNA strand varied from approximately 4% to 9 “/b during the same period. However, this variation is probably caused h,v t,he inaccuracy inherent in quantitating the low levels of E DNA strand transcription observed at the earliest time points shown. Similarly, it proved difficult to IIHP the assay described here to study the early phase of t,he lyt’ic cycle, since the amounts of polyoma transcription detected were too close to the non-specific background keel of bhe assay (O$l to 0.03% of input radioactivity). In conclusion, analysis of pulse-labelled polyoma RNA strongly suggests that viral RNA is predominantly hranscribed from the L DEA strand at late t’imes after infection. Similar results have recently been reported for SV40 virus (Laub $ Aloni, 1975; Reed et ul., 1976; Gilboa & Aviv, 1976). Since t’he ratio of E t’o L strand transcripts measured with the pulse-labelled material is very similar to that previously report’ed for unlabelled viral RNA (Kamen et uZ., 1974), we infer that control at the transcriptional rather than at the post-transcriptional level is primarily responsible for the bias towards L DNA strand transcripts which is found in polyoma mRP;A. One objection to t’his inference is that rapid processing by cellnlar nucleases account,s for the results obtained. The results of experiments in vitro using transcript,ion complexes purified from infected cells (described in the accompanying paper, Condit d nl. (1977)) exclude this objection. We conchldc that t,hr expression of polyoma virus DNA is subject, t,o transcriptional control. Impekl Cancer Rcscarch Fnntl Laborat#ories, P.O. Box 123 Lincoln’s Inn Fields, London WC2A 3PX, England Rwr~i\~c~d
17 Fc~brrmry
1977, md
ANDREW J. FLAVELL ROBERTKAMEX
in revised form 16 .Jrlnr 1977
REFERENCES Acheson, N. (1976). CeZE,8, 1-12. Aloni, Y. & Locker, H. (1973). I’irology, 54, 495-505. Con&t, R. C., Cowie, A., Kamen, R. & Birg, F. (1977). .J. Xol. Biol. 115, 233-253. Fried, M. (1970). Virology, 40, 605-617. Fried, M. & Piths, J. D. (1968). Virology, 34, 761-770. Cilbo+ E. & Aviv, H. (1976). Cell, 7, 567-573. Hirt, B. (1967). J. Mol. BioZ. 26, 365-369. Kamm, R. & Shure, H. (1976). Cell, 7, 361-371. Kamen, R., Lindstrom, D. M., Shure, H. & Old, R. IV. (1974). CoZdSpring HarborSymp. G)ctnnt. BioZ. 39, 187-198.