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

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 rol...
387KB Sizes 0 Downloads 0 Views