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The initiation of transcription of sv40 DNA at late time after infection
VIROLOGY
92, 310-323
The initiation
(1979)
of Transcription
of SV40 DNA at Late Time after Infection
ORGAD LAUB, SUSAN BRATOSIN, MIA HOROWITZ, AND YOSEF ALONI’ Department of Genetics, The Weizmann Institute
of Science, Rehovot, Israel
Accepted September 29, 1978
In vivo labeled RNA was purified from productively infected cells and in, vitro labeled RNA was purified from transcriptional complexes of SV40. The purified RNAs were denatured and fractionated by sedimentation through sucrose gradients. Labeled RNAs of various lengths were hybridized with restriction fragments of SV40 DNA of a known order. In both cases the shortest RNAs hybridized with a fragment which spans between 0.67 and 0.76 map units and the hybridization with this fragment decreased with successively longer RNAs indicating that transcription initiates within this fragment or very close to it. Similar enrichment for this fragment was obtained using nascent RNA chains labeled in vitro with a short pulse. Electron microscopic analysis of transcriptional complexes of SV40 has revealed a substantial fraction with one short nascent RNA chain. The initiation site of the nascent chains was mapped at coordinate 0.67 2 0.02. The accumulation of transcriptional complexes with short nascent chains, initiated at coordinate 0.67 2 0.02, and the abundance of labeled nascent RNAs complementary to a fragment spanning between 0.67 and 0.76 map units could indicate the existence of an attenuator site in which RNA chain elongation is blocked, unless a stimulating factor is present which allows transcription to continue into a complete transcript. INTRODUCTION
stand the mechanism for the joining of the leader sequences to the body of the message The molecular biology of SV40 has been under intensive investigation for a number depends on where transcription initiates of years. These studies have provided con- and whether or not the mRNA is processed siderable information regarding the regula- from a larger precursor as has been suggested for Ad-2 mRNA (Goldberg et tion of gene expression and, in particular, al., 1977). the controls of transcription and post tranIn the present studies, we have localized scriptional processing of mRNA (Acheson, on the conventional map of SV40 the ini1976; Kelly and Nathans, 1977). In spite of tiation site for late transcription at coordithe massive information that has been acnate 0.67 t 0.02. The significance of this cumulated, the basic question of where does transcription of SV40 DNA start and stop finding to the understanding of the mechanism of the initiation of transcription and remains unsolved. A novel observation-splicing-has re- splicing of SV40 mRNAs is discussed. cently been made for the maturation of SV40 mRNAs (Aloni et al., 1977a, b; Hsu MATERIALS AND METHODS and Ford, 1977; Lavi and Groner, 1977; BraCells and viruses. Growth of plaque-puritosin et al., 1978). The 5’ leader sequences fied SV40 on BSC-1 cells as well as concenof these viral mRNAs are not transcribed tration’and purification of the virus from the adjacent to the coding sequences. A similar observation was also reported for Ad-2 late tissue culture lysates and preparation of SV40 DNA component I have been described mRNAs (Berget et al., 1977; Chow et al., (Laub and Aloni, 1975). In these experi1977). A central point necessary to underments BSC-1 cells were infected with 50 to 1 To whom requests for reprints should be ad- 100 plaque forming units (PFU) per cell of stock 777 sv40. dressed. 0042~6822/79/020310-14$02.00/0 Copyright
All rights
0 1979 by Academic Press, Inc. of reproductionin any form reserved.
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RNA extraction and sedimentation. Cytoplasmic and nuclear fractions were prepared by lysing the cells in phosphate-buffered saline containing 0.5% Nonidet P-40. The cytoplasmic lysate was carefully removed by low-speed centrifugation at 4”. The nuclear fraction was washed further with 2 ml of phosphate-buffered saline containing 1.0% of Nonidet P-40 and 0.5% of sodium deoxycholate, and nuclei were collected by low-speed centrifugation (Penman, 1966). RNA was extracted with phenol-chloroform-isoamyl alcohol at room temperature and collected by ethanol precipitation (Penman, 1966). The precipitate was resuspended in TKM buffer (0.05 M Tris pH 6.7, 0.025 M KCl, and 0.0025 M MgCl,) and digested with 25 to 50 pg of DNase/ml (Worthington, RNase-free electrophoretically purified) at 2” for 60 min. The digest was extracted with SDS-phenol and the extract was denatured in 90% formamide before sedimentation through 15-30% sucrose gradient in SDS buffer (Aloni and Attardi, 1971). complexes Isolation of transcriptional for electron microscopy. The infected cultures were washed with phosphate-buffered saline and treated with Nonident P-40 detergent (0.5%) to give the nuclear fraction (Penman, 1966). The nuclei were disrupted with 0.25% Triton X-100 and 0.25% Sarkosyl in 0.2 M NaCl, 10 mM Tris, pH 6.7, and 1 m&f EDTA. The cellular chromatin was pelleted by centrifugation at 30,000 g for 20 min at 2” and the transcriptional complexes were purified from the nucleoplasmic supernatant by velocity sedimentation in a neutral sucrose gradient. Cleavage of the transcriptional complexes with Bgll and BamHl restriction endonucleases was carried out in 60 mM NaCl, 6 mM MgC&, 6 m&f Tris, pH 7.9, and 6 mM mercaptoethanol. Cleavage with EcoR, endonuclease was carried out in 100 m&f NaCl, 100 m&f Tris, pH 7.5, and 6 mM MgCl,. The reactions were carried out at 37” for 15 min and they were stopped by dialysis against 200 mM NaCl, 10 mM Tris, pH 6.7, and 1 mM EDTA at 4” for 3 hr. In vitro RNA sunthesis. The standard
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reaction mixture contained 1.0-2.0 ml of Sarkosyl supernatant as well as a final concentration of 0.15 M (NH&SO,, 5 mM KCl, 30 m&ZHEPES-NaOH, pH 8.0,l mM CaCl,, 1.5 mM MnC&, 1 mM DTT, 25 pg/ml each of CTP, ATP, GTP, [cz-~~P]UTP (80-250 Ci/mmol, AmershamlSearle Corp., Arlington Heights, Ill.), and 0.25% Sarkosyl. Incubation was at 26”. Extraction and analysis of RNA from transcriptional complexes. RNA was extracted with SDS-phenol-chloroform-isoamyl alcohol (Penman, 1966) at room temperature and fractionated by chromatography on Sephadex G-50 and the void volume was collected by ethanol precipitation. The precipitate was resuspended in TKM buffer and digested with 25 pug/mlof DNase (Worthington, RNase-free electrophoretically purified) at 2” for 60 min. The digest was extracted with SDS-phenol and collected by ethanol precipitation and centrifugation. Filter hybridization. SV40 DNA was cleaved with the desired enzyme, and the fragments were separated by 1.4% agarosegel electrophoresis (Sharp et al., 1973). The DNA was transferred from the gel onto nitrocellulose paper (Schleicher and Schuell unless otherwise specified) with 6x SSC using the technique of Southern (1975). Each filter (10 cm width) contained 25 pg of SV40 DNA. Nitrocellulose strips cut from these filters were incubated with the 32P-or 3H-labeled RNA in 1.5 ml of 4 x SSC, 0.1% SDS for 24 hr at 68”. After hybridization the strips were washed with 2x SSC treated with 10 pg/ml RNase in 2x SSC for 30 min at 22”, washed again with 2x SSC, and dried. Pieces, 1.5 mm, were cut and the radioactivity was counted in toluenebased scintillation fluid. Bands of radioactivity were also analyzed after exposure of the nitrocellulose strips to an X-ray film. Visualization of transcriptional complexes under the electron microscope. In order to visualize transcriptional complexes (TC) by electron microscopy (EM) we modified the benzyldimethylalkylammonium chloride (BAC) spreading method from that described by Vollenweider et al. (1975). Samples (15 ~1) purified by sedimentation through neutral sucrose gradients were
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diluted into a mixture of 5 ~1 of 0.7 M triethanolamine, pH 8.5, 40 ,ul of H,O, and 40 ~1 of formamide (Fluka, purissima grade). Five microliters of BAC from a stock solution of 1 mg/ml in formamide was added immediately before spreading. Samples were spread onto a hypophase of 0.035 M triethanolamine, pH 8.5. The molecules were picked up on Parlodion-coated grids, stained with m-any1 acetate, and shadowed with Pt-Pd (8020) at an angle of 9” (Davis et al., 1971). A Philips EM300 electron microscope using a 50-pm objective aperture and 60 kV accelerating voltage was employed. Negatives (40,000x) were enlarged 20x with a Kodak enlarger and traced onto paper. Lengths were measured on these tracings with a map measurer.
ET AL.
size up to five complete SV40 transcripts (Darnell et al., 1967). The labeled RNA was denatured in 90% formamide and was sedimented through sucrose-SDS gradients. Fractions were pooled as indicated in Fig. lA, collected by ethanol precipitation, and hybridized with nitrocellulose paper strips each containing the five fragments of SV40 DNA (Southern, 1976)produced by cleavage of Form I DNA with restriction endonucleases EcoRI Hpal, and Bgll(Fig. 2). The nitrocellulose paper was treated with RNase, washed, and dried and the strips were cut and counted. Figure 1B shows the distributions, in sucrose gradient, of viral RNA sequences complementary to the five restriction fragments. Figure 1C summarizes the proportions of RNA sequences complementary to each of the fragments in the various pooled RESULTS fractions. Note that RNA sequences comLocalization of the Major Initiation Site plementary to fragment e (0.67-0.76) were found in all the pooled fractions from the for Late Trans&ption top to the bottom of the gradient. However, In order to localize the initiation site for the highest proportion of sequences comlate transcription we have adapted the plementary to fragment e was in the shortest Dintzis principles (1961) as described re- RNA and this proportion decreased with cently by Bachenheimer and Dame11(1975). increase in the length of the RNA. In the It is assumed that after a short pulse with largest RNAs (fractions 4, 5, and 6), the a radioactive precursor of RNA, RNA mole- proportion of sequences complementary to cules would contain labeled sequences com- fragment e was almost constant. Similar plementary to some or all regions of the kinetics were obtained for each fragment DNA. However, labeled sequences comple- depending on its position on the physical mentary to a fragment of DNA which in- map of SV40 DNA in which larger RNA cludes the initiation site for transcription was enriched for fragments more remote would be in the shortest chains while la- from fragment e. In the largest RNA (fracbeled sequences complementary to a DNA tions 5 and 6) the hybridization with each fragment far from the initiation site would fragment was almost proportional to its be in successively larger chains. Two sys- length. Thus it appears that the major starttems were studied: In the first we pulse- ing points for transcription on the late strand labeled infected cells for a short period of is in fragment e and at the distal end of time in order to enrich the ratio of newly fragment a (see also Horowitz et al., 1978) synthesized labeled RNA to processed la- and that the L-SV40 DNA strand is combeled RNA. In the second, we elongated pletely or almost completely transcribed. in vitro the unprocessed nascent RNA Similar results were obtained with 2-min attached to transcriptional complexes, puri- pulse-labeled RNA indicating that “processed” RNA did not disturb the analysis. fied the RNA, and analyzed it. (a) Analysis of in vivo labeled nuclear The hybridization of the shortest RNAs SV.40 RNA. Nuclear RNA was prepared with fragments a and c may represent tranfrom SV40-infected cells labeled for 5 min scripts of the E-SV40 DNA strand, in the counterclockwise direction, as 5- 10% of the with [3H]uridine at 48 hr postinfection. This labeling time is long enough to synthe- nuclear viral RNA is transcribed at late
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number
FIG. 1. Analysis of 3H-labeled nuclear RNA of various sedimentation rates by hybridization to restriction fragments. (A) BSC-1 cells were infected with SV40 and labeled for 5 min with [5,6-3H]uridine (0.1 mCi/ml, 41 Ci/mmol) at 48 hr postinfection. [3H]RNA was prepared from the nuclear fraction, denatured in 90% formamide at 37”, and centrifuged through 15-30% (w/w) sucrose in SDS buffer in a SW 27.1 rotor for 20 hr at 25,000 rpm at 20”. Aliquots of 20 ~1 from each fraction were counted. (B) Pooled fractions as indicated in A were collected by ethanol precipitation and centrifugation and annealed to filter blots containing EcoRI, Hpal, and Bgll restriction enzyme fragments. The map positions of these fragments are shown in Fig. 2. (C) The percentage radioactivity bound to each fragment was determined from the total radioactivity bound to the five restriction fragments for each hybridization mixture. (D) BSC-1 cells were infected with SV40 and labeled with [5,6-3H]uridine for 4 hr. [3H]RNA was prepared from the cytoplasmic fraction and chromatographed on an oligo(dT)cellulose column. The poly(A)+ RNA was collected by ethanol precipitation and centrifuged through a sucrose gradient as in A. The 16 S region of the gradient was collected by ethanol precipitation and centrifugation and annealed with a filter blot as in B.
time after infection from the E-strand (Laub and Aloni, 1975). It is interesting to note that RNA found in fraction 3 of the gradient, which contains RNA species of about 16 S size, was not enriched for sequences complementary to fragment d (0.0-o. 17), which contains the majority of the template sequences for the 16 S viral RNA. Figure 1D shows that cytoplasmic RNA sedimenting in the same region of the gradient is highly enriched for sequences complementary to fragment d. These results are consistent with our previous studies (Aloni et al., 1975) which have shown that the 16 S viral RNA is processed in the cytoplasm.
conclusion is valid only if there is no significant RNA processing during the labeling period. If processing occurs rapidly then the best time to study the organization of the transcript is during its formation, that is, while the molecule is nascent. To achieve sufficient radioactivity in nascent RNA we have labeled active transcriptional complexes (Laub and Aloni, 1976) for 3 min using [cx-~~P]UTP. During this period less than 100 nucleotides are added per RNA molecule (Shani et al., 1977; and our unpublished results). The 32P-labeled RNA was extracted from the transcriptional complexes, purified, denatured as for in vivo (b) Analysis of nascent RNA of tranlabeled RNA, and sedimented through a scriptional complexes of SV.40. The above neutral sucrose-SDS gradient. Figure 3
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shows that the RNA had a mean S value of about 10 S with some components sedimenting faster than the 18 S r-RNA marker. Fractions were collected and pooled as indicated. The 32P-labeledRNA of the pooled fractions was hybridized with the five fragments produced by cleavage of Form I SV40 DNA with EcoRI, Hpal, and Bgll restriction endonucleases (Fig. 2) and then blotted on nitrocellulose paper (Southern, 1976). The nitrocellulose paper was treated with RNase, washed, dried, and exposed to an X-ray film. The results in Fig. 3 show that the slowest sedimenting RNA hybridized predominantly with fragment e (0.6’7-0.76) and the proportion of hybridization with this fragment decreased with the successively faster sedimenting RNA components. These results strongly suggest that the initiation site for late transcription is localized between coordinates 0.67 and 0.76 or very close to it on the physical map of SV40 DNA. Thus, labeled RNA from SV40infected cells and from transcriptional complexes of SV40 gave similar results. It is interesting to note that the radioA
e
dcba
FIG. 2. Map positions of restriction fragments produced by cleavage of Form I SV40 DNA with EcoRI, &al, and Bgll restriction endonucleases.The cleavage sites of the restriction enzymes are: EcoRI 0.0; Hpal 0.17, 0.37, 0.76; Bgll 0.67. The strip below the map shows the result of a fractionation of the restriction fragments on 1.4% agarose, blotted on a nitrocellulose membrane filter and hybridized with 32P-labeled nicktranslated SV40 DNA. At the end of the hybridization the membrane filter was exposed to an X-ray film. Note that the intensity of the band is proportional to the length of the fragment. Similar results were obtained with both Schleicher and Schuell and Millipore membrane filters.
eL
Fraction
number
FIG. 3. Hybridization of in vitro synthesized 32Plabeled RNA to restriction fragments. Transcriptional complexes isolated from 2 x 10’ cells were used to synthesize RNA for 3 min in the presence of [(u-Pa*]UTP. The 32P-labeled RNA was purified, denatured for 10 min in 90% formamide at 37”, diluted in SDS buffer, and run through a 15-30% sucrose gradient in SDS buffer at 25,000 rpm for 18 hr in the Spinco SW 27.1 rotor at 20”. The 28 S and 18 S markers are the positions of [3H]rRNAs run in the same tube. Inset: Pooled fractions as indicated were annealed with a nitrocellulose filter blot containing the five EcoRI, Hpal, and Bgll restriction fragments (see Fig. 2). The nitrocellulose filter was exposed to an X-ray film and the bands developed are shown.
activity bound to fragments a and c, as monitored by the intensity of the bands, after exposure to an X-ray film, depended on the brand of nitrocellulose paper on which the restriction fragments were blotted (Southern, 1975). Figure 4 shows a comparison between two brands of membrane filters; one was purchased from Schleicher and Schuell and the other from Millipore. It could be seen in Fig. 4 that the level of hybridization with fragments a and c blotted on the Schleicher and Schuell membrane filter is higher than the level of hybridization obtained with the same restriction fragments blotted on the Millipore membrane filter. Since within a series we have hybridized RNAs of various lengths with the same paper under identical conditions and obtained reproducible results, we believe that the pattern of hybridization with fragment e is not an artifact of the technique.
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0
a
OF SV40
Electron Microscopic Examination DNAIRNA Complexes
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of SV40
We have recently described the isolation of SV40 DNA/RNA complexes from cells productively infected with SV40 (Laub and Aloni, 1976). The complexes sediment in neutral sucrose gradient at about 23-25 S. We have screened several spreading techniques for analyzing the TC present in the 23-25 S region of the gradient in the EM and have adopted the protein free BAC technique in the presence of formamide (Vollenweider et al., 1975). Examinations in the EM have revealed, in most cases,
e
FIG. 4. A comparison between the availability of restriction fragments blotted on Millipore and Scheicher and Schuell filters to hybridize with in vitro synthesized 32P-labeled viral RNA. 32P-Labeled RNA was synthesized in vitro for 20 min using [c~-~~P]UTP. The 3ZP-labeled RNA was purified and hybridized with the restriction fragments of SV40 DNA shown in Fig. 2. In A the restriction fragments were blotted on a Millipore (0.45 pm) membrane filter and in B they were blotted on a Schleicher and Schuell membrane filter. At the end of the hybridization the blots were washed, dried, and exposed to an X-ray film.
Figure 5 shows the results of an experiment in which the transcriptional complex was labeled with a 2-min pulse in vitro using [cx-~~P]UTP. RNA from this complex was purified and hybridized to EcoRI, Hpal, Bgll restriction fragments blotted on a Schleicher and Schuell membrane filter. It can be seen that fragment e is enriched relative to its size. Identical results were obtained using a Millipore membrane filter.
FIG. 5. Hybridization between 32P-labeled RNA synthesized in vitro for 2 min using [a-32P]UTP and restriction fragments of SV40 DNA, as in Fig. 2, blotted on a Schleicher and Schuell membrane filter. After the hybridization the blot was washed, dried, and exposed to an X-ray film.
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TC with only a few nascent RNAs per DNA molecule. Circular DNAs with a great number of nascent RNA chains, distributed all along the molecule, were occasionally seen. We observed different types of SV40 DNA molecules complexed with RNA: completely or partially relaxed circular DNA (Fig. 6A), superhelical DNA (Fig. 6B), and oligomers (Fig. SC). The proportion of relaxed DNA was correlated with the concentration of formamide in the sample. Lowering the formamide concentration led to observation of relatively less relaxed molecules and more superhelical DNA templates. We suggest, therefore, that Form I DNA is the major template for transcription. A similar conclusion based on biochemical studies was recently reported by Birkenmeier et al. (1977). When the TC were cleaved with restriction endonucleases most of the nascent chains remained attached to their DNA template, suggesting that in addition to the superhelicity of the DNA the RNA is also being held to the DNA by the RNA polymerase (Birkenmeier et al., 19’7’7). Figure 7 shows the appearance of the cleaved
ET AL.
TC. The distribution of the nascent RNA chains along the DNA molecule is consistent with the conclusion that at least in some molecules there is complete or almost complete transcription of the DNA strands. E3H]Thymidine pulse-labeled SV40 DNA (replicating intermediates) isolated immediately after labeling is also found in the 23-25 S region of the sucrose gradient (Fareed et al., 1972). Under the electron microscope no nascent RNA chains were observed attached to replicating intermediates, indicating that this form of DNA is not transcribed. Moreover, the presence of replicating intermediates in the preparation provided an excellent control indicating the absence of nonspecific attachment of RNA to DNA. The Major Initiation Site for Late Transcription as Determined by Electron Microscopic Analysis Among circular DNA molecules carrying nascent RNA chains a substantial fraction (about 10%) had one short but extended nascent molecules. Examples of these com-
FIG. 6. TC spread by the protein-free BAC technique. (A, B) Monomers respectively. (C) Dimer spread in 40% formamide. Bars = 0.5 pm.
spread in 40 and 30% formamide,
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OF SV40
FIG. 7. Linear SV40 DNA with nascent RNA chains. TC were cleaved with restriction (A) and EcoRI (B) and prepared for EM as in Fig. 6. Each bar = 0.5 pm.
endonucleases Bgll
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plexes are shown in Fig. 8. The short nascent chains are either RNA or single-stranded DNA. The second alternative could be true only if one of the two DNA strands was nicked allowing nascent RNA to hybridize with its complementary DNA strand and so displacing the secondDNA strand (Thomas et al., 1976). The RNA/DNA complexes may originate from the hybridization of free RNA with the viral DNA, but this seems to be unlikely since the DNA was never exposed to denaturing conditions which is a prerequisite for RNA/DNA hybridization. Additional support against this supposition is the absence of nascent chains attached to replicating intermediates. In order to determine whether the initiation of the short nascent chains maps at a specific site we cleaved the DNA with restriction endonucleases and obtained linear molecules of unit-length SV40 DNA with the small nascent chains still attached to them. Examples of such molecules cleaved with Bgll, BamHl, and EcoRI are shown in Figs. 9A, B, and 9C, respectively. The possibility of DNA strand displacement by RNA, as discussed above, is more likely with linear molecules than with circular DNA, in particular if the cleavage site is close to the hybridization site of the RNA due to the presence of a free end. However, the high sensitivity of the nascent chains to RNase (5 Fg for 30 min at 22’) tends to indicate that the majority of the singlestranded chains are nascent RNAs. The localization of the initiation site for tran-
scription is independent of whether the measured chains were RNA or displaced single-stranded DNA. It should also be mentioned that the molecules shown in Figs. 9A, B, and C are not replicating intermediates, because replicating intermediates would have forks at both ends of the molecule (in the case of a Bgll cut) or a bubble at an internal position in the molecule (in the cases ofBamH1 andEcoR cuts) typical for bidirectional replication (Fareed et al., 1972). Figures lOA, B, and C show examples of replicating intermediates cleaved with Bgll, BamHl, and EcoRI, respectively. The lengths of the nascent chains were measured and aligned at the attachment point to each side on the DNA templates and the distances of the 5’ termini of the RNA molecules from the near end of the DNA were determined and expressed as the fractional length of the DNA. The analysis gave two sets of numbers: In one set the numbers were at random and depended on the length of the RNA while in the second set they showed a high level of consistency. The statistical analysis was carried out on the second set of numbers. Figure 11 summarizes the results of the analysis in the form of four histograms. Three of the histograms show the location, on the physical map of SV40 DNA, of the 5’ end of the RNA, as calculated from the distances between the 5’ termini of the nascent chains (see above) and the cleavage sites of the restriction endonucleases:BgZl in A, BamHl in B, and EcoRI in C. The fourth histogram
FIG. 8. Circular SV40 DNA with short nascent chain. The spreading was by the BAC technique in 40% formamide. Bar = 0.5pm.
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+ IFIG. 9. Linear SV40 DNA with short nascent chain. TC were cleaved with restriction endonucleases Bgll (A), BamHl (B), and EcoRI (C). In the tracings arrow indicates the initiation site of transcription for the nascent chain.
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FIG. 10. Replicating ; intermediates cleaved with restriction endonucleases Bgll (A), BumHI (B), and EcoRI
is a summation of these three histograms. It could be seen that independent of the restriction enzyme used to cleave the DNA only one reproducible peak was obtained. This peak maps at position 0.67 + 0.02 on the physical map of SV40 DNA. We suggest that this location represents the major initiation site for late transcription. The EM analysis thus confirmed the results obtained by the biochemical approach. DISCUSSION
One of the basic questions in the understanding of how cells and viruses transcribe and produce their mRNAs is where and how the primary transcripts initiate and terminate. The best approach to determine the initiation site for transcription is by analyzing the nucleotides near the 5’ end of the newly synthesized RNA. However, for unknown reasons, we (unpublished results) and others (Ferdinand et al., 19’77) have failed to detect any labeled 5’ termini of SV40-specific RNAs. In the present paper we describe three independent studies which were undertaken in order to localize the initiation site for transcription of SV40 DNA at late time after infection. Two of the studies were based on the Dintzis principles (1961) as described recently by Bachenheimer and Darnell (1975) and in the third study we measured
(Cl.
nascent RNA chains attached to transcriptional complexes under the EM. Localization of the Initiation Site(s) Based on the Din&is Principles The rationale of this approach is that after short pulses with radioactive precursors, RNA molecules would contain some labeled sequences complementary to each region of the DNA, but the labeled RNA complementary to a fragment of DNA which includes the initiation site for transcription would be in the shortest chains, while labeled RNA complementary to a DNA fragment far from the initiation site would be in successively longer chains. Similar analyses were performed previously in other systems only with in viva labeled RNA or with isolated nuclei (Goldberg et al., 1977). The use of transcriptional complexes to label the RNA has two main advantages: First, higher levels of radioactivity can be incorporated into SV40 nascent chains, and second, no physiological processing occurs in the in vitro system which may complicate the analysis of the in vivo labeled RNA. The results obtained with both in vivo and in vitro RNAs have indicated that the major initiation site for late transcription is within a fragment which spans between 0.67 and 0.76 on the physical map of SV40 DNA and at the distal end of fragment a (Horowitz et al., 1978).
INITIATION
s
Frcctiaal
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fmm Bgl, cut
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a new conformation of the structure of the viral DNP. In this case the role of the 5’ leader sequences would be to regulate the levels of the viral mRNAs at early and late times after infection. It is interesting to note that the 160-nucleotide leader region of E. coli Trp operon mRNA, like the leader of SV40 late mRNAs, has the potentiality to codefor a small peptide (Dharet al., 1977). There is a less likely possibility which can explain the same results. The enrichment of RNA complementary to fragment e may result from a decrease of the stability of attachment of long RNA chains to the template as compared with the stability of attachment of short RNA chains which are closer to the initiation site of transcription.
Froctionol length from Eco RI cut
FIG. 11. Histograms indicating the initiation site for transcription. The nascent chains in molecules such as shown in Fig. 9 were measured and aligned on the DNA and the distances of their 5’ ends from the near end of the restriction enzyme cleavage site were measured and plotted as the fractional length of the DNA. The histograms are oriented to each other with respect to the restriction endonuclease cleavage site. The inset shows the physical map of SV40 DNA with the location on the map of the Bgll, BamHl, and EcoRI cleavage sites.
Electron Microscopic Visualization Transcriptional Complexes
of
Both relaxed and superhelical molecules as well as oligomers of SV40 DNA carrying nascent RNA chains have been observed but no nascent molecules were found on replicating intermediates, suggesting that once a molecule starts to replicate it cannot serve as template for transcription until the replication is completed. It is possmle that both the relaxed and superhelical molecules serve as templates for transcription Attenuator-A Site in Which Transcription in vivo in different stages of the physiois Blocked logical process, however, one may be an The accumulation of TC with short na- artifact of the procedures. The presence scent chains and the abundance of labeled of transcriptional complexes with many RNA complementary to the e fragment after growing RNA chains indicates that many a short label in vitro (Fig. 5) could indicate RNA polymerase molecules can be active at a structure similar to the Trp attenuator any given moment on the same DNA molein Escherichia coli (for review, see Rabus- cule. Up to 10 RNA molecules could be say and Geiduschek 1977). The Trp atten- counted in a single transcriptional complex. uator is a site at which RNA chain elonga- This is probably an underestimate of the tion is blocked. In vitro short RNA tran- maximum number of growing chains occurscripts are produced and are not released ring in vivo on the same template, since from the template. In viva the transcrip- it is conceivable that at least part of the tional block at the attenuator appears to RNA was detached from the template durbe subject to regulation. If the accumulation ing the preparation. of the short nascent chains on SV40 DNA The largest RNA bushes found to be attemplates arises from the existence of an tached to SV40 DNA in the present work attenuator on SV40 DNA (at approximately were as large as a complete transcript, coordinate 0.75) then in vivo the tran- suggesting that at least one of the two SV40 scriptional block at the attenuator could DNA strands is completely transcribed. respond to modified RNA polymerase, However because we were unable to extend T-Ag, other viral proteins(s)(VP-3?), or the large nascent RNA chains, we could
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not determine whether there was any size occur both in the nucleus and in the cytopattern of the nascent RNA chains along plasm. A similar mechanism of splicing has the SV40 DNA molecule. We were unable been suggested for Ad-2 mRNAs (Berget to conclude from the present study the pro- et al., 1977). portion of molecules which are completely transcribed. ACKNOWLEDGMENTS Models for the Joining of the Leader to the Coding Sequences We have recently shown (Aloni et al., 197’7a,b; Bratosin et al., 1978) that the 5’ leader sequences of the two major cytoplasmic viral RNA components are transcribed from a segment of the genome which is not adjacent to the coding sequences and which maps from 0.70 + 0.01 to 0.75 + 0.01 map units. This leader was estimated to contain about 200 nucleotides. Similar observations were made for mRNAs extracted from adenovirus 2-infected cells (Berget et al., 1977; Chow et al., 1977; Klessig, 1977). Several models for such splicing of SV40 late mRNAs could be suggested. These include: (1) intermolecular ligation of RNA, (2) deletion of intervening DNA sequences, (3) looping out of intervening DNA sequences so that the RNA polymerase could skip over short distances, and (4) deletion of the appropriate intervening RNA sequences. The observations made in the present work which show that there is one major initiation site for late transcription and that the template is mainly Form I SV40 DNA of unit length exclude the first two alternatives. The third alternative is excluded because analysis of R-loop structures formed between nuclear viral RNA and linear SV40 DNA has shown the presence in nuclear RNA of sequences complementary to the intervening DNA segment (Horowitz et al., 1978). We therefore conclude that the joining of the leader sequences to the coding region occurs via the fourth alternative of looping out intervening RNA sequences and then covalent joining of the leader to the coding sequences via an intramolecular digestion and ligation. The observation that the 16 S RNA is processed from the 19 S in the cytoplasm (Aloni et al., 1975; Groner, Carmi and Aloni, unpublished results) suggests that splicing of RNA can
This research was supported by a Public Health Service Grant from The National Cancer Institute. We thank E. Jakobovits for critical reading of the manuscript. REFERENCES ACHESON,N. H. (19’76).Transcription during productive infection with polyoma virus and simian virus 40. Cell 8, 1-12. ALONI, Y. (1974). Biogenesis and characterization of SV40 and polyoma RNAs in productively infected cells. Cold Spring Harbor Symp. Quant. Biol. 39, 165-1’78. ALONI, Y., and ATTARDI, G. (1971). Expression of the mitochondrial genome in HeLa cells. II. Evidence for complete transcription of mitochondrial DNA. J. Mol. Biol. 55, 251-270. ALONI, Y., BRATOSIN, S., DHAR, R., LAUB, O., HOROWITZ,M., and KHOURY, G. (1977a). Splicing of SV40 mRNA: A novel mechanism for the regulation of gene expression in mammalian cells. Cold Spring Harbor
Symp. Quant. Biol. 42, 559-570.
ALONI, Y., DHAR, R., LAUB, O., HOROWITZ,M., and KHOURY, G. (197713).Novel mechanism for RNA maturation: The leader sequences of simian virus 40 mRNA are not transcribed adjacent to the coding sequences. Proc. Nat. Acad. Sci. USA 74, 36363690.
ALONI, Y., SHANI, M., and REUVENI, Y. (1975). RNAs of simian virus 40 in productively infected monkey cells: Kinetics of formation and decay in enucleate cells. Proc. Nat. Acad. Sci. USA 72, 2537-2591. BACHENHEIMER, S., and DARNELL, J. E. (1975). Adenovirus-2 mRNA is transcribed as part of highmolecular weight precursor RNA. Proc. Nat. Acad. Sci. USA 72, 4445-4449.
BERGET, S. M., MOORE,C., and SHARP, P. A. (1977). Spliced segments at the 5’ terminus of adenovirus 2 late mRNA. Proc. Nat. Acad. Sci. USA 74, 31713175. BIRKENMEIER,E. H., RADONOVICH,M. F., SHANI, M., and SALZMAN, N. P. (1977). The SV40 DNA template for transcription of late mRNA in viral nucleoprotein complexes. Cell 11, 495-504. BRATOSIN,S., HOROWITZ,M., LAUB, O., and ALONI, Y. (1978). Electron microscopic evidence for splicing of SV40 late mRNAs. Cell 13, 783-790. CHOW, L. T., GELINAS, R. E., BROKER, T. R., and
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ROBERTS, R. J. (1977). An amazing sequence arrangement at the 5’ ends of adenovirus 2 messenger RNA. Cell 12, l-8. DARNELL, J. E., GIRARD, M., BALTIMORE, D., SUMMERS, D. F., and MAIZEL, J. V. (1967). The synthesis and translation of poliovirus RNA. In “Molecular Biology of Viruses” (J. Colter, ed.), pp. 3’75-401. Academic Press, New York. DAVIS, R. M., SIMON, M., and DAVIDSON, N. (1971). Electron microscope hetroduplex methods for mapping regions of base sequence homology in nucleic acids. In “Methods in Enzymology” (L. Grossman and Moldave, eds.), Vol. 21, pp. 413-428. Academic Press, New York. DHAR, R., SUBRAMANIAN,K. N., PAN, J., and WEISSMAN, S. M. (1977). Nucleotide sequence of a fragment of SV40 DNA that contains the origin of DNA replication and specifies the 5’ ends of “early” and “late” viral RNA. IV. Localization of the SV40 DNA complementary to the 5’ ends of viral RNA. Proc. Nat. Acad. Sci. USA 74, 827-831. DINTZIS, H. M. (1961). Assembly of the peptide chains of hemoglobin. Proc. Nat. Acad. Sci. USA 47, 247-261. FAREED, G. C., SEBERING,E. D., and SALZMAN,N. P. (1972). Cleavage of replicative intermediates of simian virus 40 deoxyribonucleic acid by restriction endonuclease of Escherichia coli B. J. Biol. Chem. 247, 5872-5879.
FERDINAND, F. J., BROWN, M., and KHOURY, G. (1977). Synthesis and characterization of late lytic simian virus 40 RNA from transcriptional complexes. Virology 78, 150-161. GOLDBERG, S., WEBER, J., and DARNELL, J. E. (1977). The definition of a large viral transcription unite late in Ad2 infection of HeLa cells: Mapping by effects of ultraviolet irradiation. Cell 10,617-622. HOROWITZ,M., LAUB, O., BRATOSIN, S., and ALONI, Y. (1978). Splicing of SV,, late mRNA is a posttranscriptional process. Nature (London) 275, 558-559. HSU, M. T., and FORD, J. P. (1977). Sequence of arrangement of the 5’ ends of simian virus 40 16s and 19s mRNAs. Proc. Nat. Acad. Sci. USA 74, 4982-4985.
KELLY, T. J., and NATHANS, D. (1977). The genome of simian virus 40. In “Advances in Virus Research”
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(M. A. Lauffer, F. B. Bang, R. Maramorosch, and K. M. Smith, eds.), Vol. 21, pp. 85-174. Academic Press, New York. KLESSIG, D. F. (1977). Two adenovirus mRNAs have a common 5’ terminal leader sequence encoded at least 10 Kb upstream from their main coding region. Cell 12, 9-21. LAUB, O., and ALONI, Y. (1975). Transcription of simian virus 40. V. Regulation of simian virus 40 gene expression. J. Viral. 16, 1171-1183. LAUB, O., and ALONI, Y. (1976). VII. SV40 DNARNA polymerase complex isolated from productively infected cells transcribed in vitro. Virology 75, 346-354.
LAVI, S., and GRONER, Y. (1977). 5’-Terminal sequences and coding region of late simian virus 40 are derived from noncontiguous segments of the viral genome. Proc. Nat. Acad. Sci. USA 74,53235327.
PENMAN, S. (1966). RNA metabolism in the HeLa cell nucleus. J. Mol. Biol. 17, 117-130. RABUSSAY, D., and GEIDUSCHEK, P. E. (1977). In “Comprehensive Virology” (H. Frankel-Conrat and R. R. Wagner, eds.), Vol. 8, pp. 1- 196. Plenum, New York. SHANI, M., BIRKENMEIER, E., MAY, E., and SALZMAN, N. P. (1977). Properties of simian virus 40 transcriptional intermediates isolated from nuclei of permissive cells. J. Viral. 23, 20-28. SHARP, P. A., SUGDEN,B., and SAMBROOK,J. (1973). Detection of two restriction endonuclease activities in haemophilus parainfiuenzae using analytical agarose-ethidium bromide electrophoresis. Biochemistry 13, 3055-3063. SOUTHERN, E. M. (1975). Detection of sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503-517. THOMAS,M., WHITE, R. L., and DAVIS, R. W. (1976). Hybridization of RNA to double stranded DNA: Formation of R-loops. Proc. Nat. Acad. Sci. USA 73, 2294-2298.
VOLLENWEIDER, H. J., SOGO,J. M., and KOLLER, T. H. (1975). A routine method for protein-free spreading of double- and single-stranded nucleic acid molecules. Proc. Nat. Acad. Sci. USA 72, 83-87.
92, 310-323
The initiation
(1979)
of Transcription
of SV40 DNA at Late Time after Infection
ORGAD LAUB, SUSAN BRATOSIN, MIA HOROWITZ, AND YOSEF ALONI’ Department of Genetics, The Weizmann Institute
of Science, Rehovot, Israel
Accepted September 29, 1978
In vivo labeled RNA was purified from productively infected cells and in, vitro labeled RNA was purified from transcriptional complexes of SV40. The purified RNAs were denatured and fractionated by sedimentation through sucrose gradients. Labeled RNAs of various lengths were hybridized with restriction fragments of SV40 DNA of a known order. In both cases the shortest RNAs hybridized with a fragment which spans between 0.67 and 0.76 map units and the hybridization with this fragment decreased with successively longer RNAs indicating that transcription initiates within this fragment or very close to it. Similar enrichment for this fragment was obtained using nascent RNA chains labeled in vitro with a short pulse. Electron microscopic analysis of transcriptional complexes of SV40 has revealed a substantial fraction with one short nascent RNA chain. The initiation site of the nascent chains was mapped at coordinate 0.67 2 0.02. The accumulation of transcriptional complexes with short nascent chains, initiated at coordinate 0.67 2 0.02, and the abundance of labeled nascent RNAs complementary to a fragment spanning between 0.67 and 0.76 map units could indicate the existence of an attenuator site in which RNA chain elongation is blocked, unless a stimulating factor is present which allows transcription to continue into a complete transcript. INTRODUCTION
stand the mechanism for the joining of the leader sequences to the body of the message The molecular biology of SV40 has been under intensive investigation for a number depends on where transcription initiates of years. These studies have provided con- and whether or not the mRNA is processed siderable information regarding the regula- from a larger precursor as has been suggested for Ad-2 mRNA (Goldberg et tion of gene expression and, in particular, al., 1977). the controls of transcription and post tranIn the present studies, we have localized scriptional processing of mRNA (Acheson, on the conventional map of SV40 the ini1976; Kelly and Nathans, 1977). In spite of tiation site for late transcription at coordithe massive information that has been acnate 0.67 t 0.02. The significance of this cumulated, the basic question of where does transcription of SV40 DNA start and stop finding to the understanding of the mechanism of the initiation of transcription and remains unsolved. A novel observation-splicing-has re- splicing of SV40 mRNAs is discussed. cently been made for the maturation of SV40 mRNAs (Aloni et al., 1977a, b; Hsu MATERIALS AND METHODS and Ford, 1977; Lavi and Groner, 1977; BraCells and viruses. Growth of plaque-puritosin et al., 1978). The 5’ leader sequences fied SV40 on BSC-1 cells as well as concenof these viral mRNAs are not transcribed tration’and purification of the virus from the adjacent to the coding sequences. A similar observation was also reported for Ad-2 late tissue culture lysates and preparation of SV40 DNA component I have been described mRNAs (Berget et al., 1977; Chow et al., (Laub and Aloni, 1975). In these experi1977). A central point necessary to underments BSC-1 cells were infected with 50 to 1 To whom requests for reprints should be ad- 100 plaque forming units (PFU) per cell of stock 777 sv40. dressed. 0042~6822/79/020310-14$02.00/0 Copyright
All rights
0 1979 by Academic Press, Inc. of reproductionin any form reserved.
310
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RNA extraction and sedimentation. Cytoplasmic and nuclear fractions were prepared by lysing the cells in phosphate-buffered saline containing 0.5% Nonidet P-40. The cytoplasmic lysate was carefully removed by low-speed centrifugation at 4”. The nuclear fraction was washed further with 2 ml of phosphate-buffered saline containing 1.0% of Nonidet P-40 and 0.5% of sodium deoxycholate, and nuclei were collected by low-speed centrifugation (Penman, 1966). RNA was extracted with phenol-chloroform-isoamyl alcohol at room temperature and collected by ethanol precipitation (Penman, 1966). The precipitate was resuspended in TKM buffer (0.05 M Tris pH 6.7, 0.025 M KCl, and 0.0025 M MgCl,) and digested with 25 to 50 pg of DNase/ml (Worthington, RNase-free electrophoretically purified) at 2” for 60 min. The digest was extracted with SDS-phenol and the extract was denatured in 90% formamide before sedimentation through 15-30% sucrose gradient in SDS buffer (Aloni and Attardi, 1971). complexes Isolation of transcriptional for electron microscopy. The infected cultures were washed with phosphate-buffered saline and treated with Nonident P-40 detergent (0.5%) to give the nuclear fraction (Penman, 1966). The nuclei were disrupted with 0.25% Triton X-100 and 0.25% Sarkosyl in 0.2 M NaCl, 10 mM Tris, pH 6.7, and 1 m&f EDTA. The cellular chromatin was pelleted by centrifugation at 30,000 g for 20 min at 2” and the transcriptional complexes were purified from the nucleoplasmic supernatant by velocity sedimentation in a neutral sucrose gradient. Cleavage of the transcriptional complexes with Bgll and BamHl restriction endonucleases was carried out in 60 mM NaCl, 6 mM MgC&, 6 m&f Tris, pH 7.9, and 6 mM mercaptoethanol. Cleavage with EcoR, endonuclease was carried out in 100 m&f NaCl, 100 m&f Tris, pH 7.5, and 6 mM MgCl,. The reactions were carried out at 37” for 15 min and they were stopped by dialysis against 200 mM NaCl, 10 mM Tris, pH 6.7, and 1 mM EDTA at 4” for 3 hr. In vitro RNA sunthesis. The standard
OF SV40
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reaction mixture contained 1.0-2.0 ml of Sarkosyl supernatant as well as a final concentration of 0.15 M (NH&SO,, 5 mM KCl, 30 m&ZHEPES-NaOH, pH 8.0,l mM CaCl,, 1.5 mM MnC&, 1 mM DTT, 25 pg/ml each of CTP, ATP, GTP, [cz-~~P]UTP (80-250 Ci/mmol, AmershamlSearle Corp., Arlington Heights, Ill.), and 0.25% Sarkosyl. Incubation was at 26”. Extraction and analysis of RNA from transcriptional complexes. RNA was extracted with SDS-phenol-chloroform-isoamyl alcohol (Penman, 1966) at room temperature and fractionated by chromatography on Sephadex G-50 and the void volume was collected by ethanol precipitation. The precipitate was resuspended in TKM buffer and digested with 25 pug/mlof DNase (Worthington, RNase-free electrophoretically purified) at 2” for 60 min. The digest was extracted with SDS-phenol and collected by ethanol precipitation and centrifugation. Filter hybridization. SV40 DNA was cleaved with the desired enzyme, and the fragments were separated by 1.4% agarosegel electrophoresis (Sharp et al., 1973). The DNA was transferred from the gel onto nitrocellulose paper (Schleicher and Schuell unless otherwise specified) with 6x SSC using the technique of Southern (1975). Each filter (10 cm width) contained 25 pg of SV40 DNA. Nitrocellulose strips cut from these filters were incubated with the 32P-or 3H-labeled RNA in 1.5 ml of 4 x SSC, 0.1% SDS for 24 hr at 68”. After hybridization the strips were washed with 2x SSC treated with 10 pg/ml RNase in 2x SSC for 30 min at 22”, washed again with 2x SSC, and dried. Pieces, 1.5 mm, were cut and the radioactivity was counted in toluenebased scintillation fluid. Bands of radioactivity were also analyzed after exposure of the nitrocellulose strips to an X-ray film. Visualization of transcriptional complexes under the electron microscope. In order to visualize transcriptional complexes (TC) by electron microscopy (EM) we modified the benzyldimethylalkylammonium chloride (BAC) spreading method from that described by Vollenweider et al. (1975). Samples (15 ~1) purified by sedimentation through neutral sucrose gradients were
312
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diluted into a mixture of 5 ~1 of 0.7 M triethanolamine, pH 8.5, 40 ,ul of H,O, and 40 ~1 of formamide (Fluka, purissima grade). Five microliters of BAC from a stock solution of 1 mg/ml in formamide was added immediately before spreading. Samples were spread onto a hypophase of 0.035 M triethanolamine, pH 8.5. The molecules were picked up on Parlodion-coated grids, stained with m-any1 acetate, and shadowed with Pt-Pd (8020) at an angle of 9” (Davis et al., 1971). A Philips EM300 electron microscope using a 50-pm objective aperture and 60 kV accelerating voltage was employed. Negatives (40,000x) were enlarged 20x with a Kodak enlarger and traced onto paper. Lengths were measured on these tracings with a map measurer.
ET AL.
size up to five complete SV40 transcripts (Darnell et al., 1967). The labeled RNA was denatured in 90% formamide and was sedimented through sucrose-SDS gradients. Fractions were pooled as indicated in Fig. lA, collected by ethanol precipitation, and hybridized with nitrocellulose paper strips each containing the five fragments of SV40 DNA (Southern, 1976)produced by cleavage of Form I DNA with restriction endonucleases EcoRI Hpal, and Bgll(Fig. 2). The nitrocellulose paper was treated with RNase, washed, and dried and the strips were cut and counted. Figure 1B shows the distributions, in sucrose gradient, of viral RNA sequences complementary to the five restriction fragments. Figure 1C summarizes the proportions of RNA sequences complementary to each of the fragments in the various pooled RESULTS fractions. Note that RNA sequences comLocalization of the Major Initiation Site plementary to fragment e (0.67-0.76) were found in all the pooled fractions from the for Late Trans&ption top to the bottom of the gradient. However, In order to localize the initiation site for the highest proportion of sequences comlate transcription we have adapted the plementary to fragment e was in the shortest Dintzis principles (1961) as described re- RNA and this proportion decreased with cently by Bachenheimer and Dame11(1975). increase in the length of the RNA. In the It is assumed that after a short pulse with largest RNAs (fractions 4, 5, and 6), the a radioactive precursor of RNA, RNA mole- proportion of sequences complementary to cules would contain labeled sequences com- fragment e was almost constant. Similar plementary to some or all regions of the kinetics were obtained for each fragment DNA. However, labeled sequences comple- depending on its position on the physical mentary to a fragment of DNA which in- map of SV40 DNA in which larger RNA cludes the initiation site for transcription was enriched for fragments more remote would be in the shortest chains while la- from fragment e. In the largest RNA (fracbeled sequences complementary to a DNA tions 5 and 6) the hybridization with each fragment far from the initiation site would fragment was almost proportional to its be in successively larger chains. Two sys- length. Thus it appears that the major starttems were studied: In the first we pulse- ing points for transcription on the late strand labeled infected cells for a short period of is in fragment e and at the distal end of time in order to enrich the ratio of newly fragment a (see also Horowitz et al., 1978) synthesized labeled RNA to processed la- and that the L-SV40 DNA strand is combeled RNA. In the second, we elongated pletely or almost completely transcribed. in vitro the unprocessed nascent RNA Similar results were obtained with 2-min attached to transcriptional complexes, puri- pulse-labeled RNA indicating that “processed” RNA did not disturb the analysis. fied the RNA, and analyzed it. (a) Analysis of in vivo labeled nuclear The hybridization of the shortest RNAs SV.40 RNA. Nuclear RNA was prepared with fragments a and c may represent tranfrom SV40-infected cells labeled for 5 min scripts of the E-SV40 DNA strand, in the counterclockwise direction, as 5- 10% of the with [3H]uridine at 48 hr postinfection. This labeling time is long enough to synthe- nuclear viral RNA is transcribed at late
INITIATION
OF TRANSCRIPTION
Fractton
OF SV40
313
number
FIG. 1. Analysis of 3H-labeled nuclear RNA of various sedimentation rates by hybridization to restriction fragments. (A) BSC-1 cells were infected with SV40 and labeled for 5 min with [5,6-3H]uridine (0.1 mCi/ml, 41 Ci/mmol) at 48 hr postinfection. [3H]RNA was prepared from the nuclear fraction, denatured in 90% formamide at 37”, and centrifuged through 15-30% (w/w) sucrose in SDS buffer in a SW 27.1 rotor for 20 hr at 25,000 rpm at 20”. Aliquots of 20 ~1 from each fraction were counted. (B) Pooled fractions as indicated in A were collected by ethanol precipitation and centrifugation and annealed to filter blots containing EcoRI, Hpal, and Bgll restriction enzyme fragments. The map positions of these fragments are shown in Fig. 2. (C) The percentage radioactivity bound to each fragment was determined from the total radioactivity bound to the five restriction fragments for each hybridization mixture. (D) BSC-1 cells were infected with SV40 and labeled with [5,6-3H]uridine for 4 hr. [3H]RNA was prepared from the cytoplasmic fraction and chromatographed on an oligo(dT)cellulose column. The poly(A)+ RNA was collected by ethanol precipitation and centrifuged through a sucrose gradient as in A. The 16 S region of the gradient was collected by ethanol precipitation and centrifugation and annealed with a filter blot as in B.
time after infection from the E-strand (Laub and Aloni, 1975). It is interesting to note that RNA found in fraction 3 of the gradient, which contains RNA species of about 16 S size, was not enriched for sequences complementary to fragment d (0.0-o. 17), which contains the majority of the template sequences for the 16 S viral RNA. Figure 1D shows that cytoplasmic RNA sedimenting in the same region of the gradient is highly enriched for sequences complementary to fragment d. These results are consistent with our previous studies (Aloni et al., 1975) which have shown that the 16 S viral RNA is processed in the cytoplasm.
conclusion is valid only if there is no significant RNA processing during the labeling period. If processing occurs rapidly then the best time to study the organization of the transcript is during its formation, that is, while the molecule is nascent. To achieve sufficient radioactivity in nascent RNA we have labeled active transcriptional complexes (Laub and Aloni, 1976) for 3 min using [cx-~~P]UTP. During this period less than 100 nucleotides are added per RNA molecule (Shani et al., 1977; and our unpublished results). The 32P-labeled RNA was extracted from the transcriptional complexes, purified, denatured as for in vivo (b) Analysis of nascent RNA of tranlabeled RNA, and sedimented through a scriptional complexes of SV.40. The above neutral sucrose-SDS gradient. Figure 3
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shows that the RNA had a mean S value of about 10 S with some components sedimenting faster than the 18 S r-RNA marker. Fractions were collected and pooled as indicated. The 32P-labeledRNA of the pooled fractions was hybridized with the five fragments produced by cleavage of Form I SV40 DNA with EcoRI, Hpal, and Bgll restriction endonucleases (Fig. 2) and then blotted on nitrocellulose paper (Southern, 1976). The nitrocellulose paper was treated with RNase, washed, dried, and exposed to an X-ray film. The results in Fig. 3 show that the slowest sedimenting RNA hybridized predominantly with fragment e (0.6’7-0.76) and the proportion of hybridization with this fragment decreased with the successively faster sedimenting RNA components. These results strongly suggest that the initiation site for late transcription is localized between coordinates 0.67 and 0.76 or very close to it on the physical map of SV40 DNA. Thus, labeled RNA from SV40infected cells and from transcriptional complexes of SV40 gave similar results. It is interesting to note that the radioA
e
dcba
FIG. 2. Map positions of restriction fragments produced by cleavage of Form I SV40 DNA with EcoRI, &al, and Bgll restriction endonucleases.The cleavage sites of the restriction enzymes are: EcoRI 0.0; Hpal 0.17, 0.37, 0.76; Bgll 0.67. The strip below the map shows the result of a fractionation of the restriction fragments on 1.4% agarose, blotted on a nitrocellulose membrane filter and hybridized with 32P-labeled nicktranslated SV40 DNA. At the end of the hybridization the membrane filter was exposed to an X-ray film. Note that the intensity of the band is proportional to the length of the fragment. Similar results were obtained with both Schleicher and Schuell and Millipore membrane filters.
eL
Fraction
number
FIG. 3. Hybridization of in vitro synthesized 32Plabeled RNA to restriction fragments. Transcriptional complexes isolated from 2 x 10’ cells were used to synthesize RNA for 3 min in the presence of [(u-Pa*]UTP. The 32P-labeled RNA was purified, denatured for 10 min in 90% formamide at 37”, diluted in SDS buffer, and run through a 15-30% sucrose gradient in SDS buffer at 25,000 rpm for 18 hr in the Spinco SW 27.1 rotor at 20”. The 28 S and 18 S markers are the positions of [3H]rRNAs run in the same tube. Inset: Pooled fractions as indicated were annealed with a nitrocellulose filter blot containing the five EcoRI, Hpal, and Bgll restriction fragments (see Fig. 2). The nitrocellulose filter was exposed to an X-ray film and the bands developed are shown.
activity bound to fragments a and c, as monitored by the intensity of the bands, after exposure to an X-ray film, depended on the brand of nitrocellulose paper on which the restriction fragments were blotted (Southern, 1975). Figure 4 shows a comparison between two brands of membrane filters; one was purchased from Schleicher and Schuell and the other from Millipore. It could be seen in Fig. 4 that the level of hybridization with fragments a and c blotted on the Schleicher and Schuell membrane filter is higher than the level of hybridization obtained with the same restriction fragments blotted on the Millipore membrane filter. Since within a series we have hybridized RNAs of various lengths with the same paper under identical conditions and obtained reproducible results, we believe that the pattern of hybridization with fragment e is not an artifact of the technique.
INITIATION
A
OF TRANSCRIPTION
0
a
OF SV40
Electron Microscopic Examination DNAIRNA Complexes
315
of SV40
We have recently described the isolation of SV40 DNA/RNA complexes from cells productively infected with SV40 (Laub and Aloni, 1976). The complexes sediment in neutral sucrose gradient at about 23-25 S. We have screened several spreading techniques for analyzing the TC present in the 23-25 S region of the gradient in the EM and have adopted the protein free BAC technique in the presence of formamide (Vollenweider et al., 1975). Examinations in the EM have revealed, in most cases,
e
FIG. 4. A comparison between the availability of restriction fragments blotted on Millipore and Scheicher and Schuell filters to hybridize with in vitro synthesized 32P-labeled viral RNA. 32P-Labeled RNA was synthesized in vitro for 20 min using [c~-~~P]UTP. The 3ZP-labeled RNA was purified and hybridized with the restriction fragments of SV40 DNA shown in Fig. 2. In A the restriction fragments were blotted on a Millipore (0.45 pm) membrane filter and in B they were blotted on a Schleicher and Schuell membrane filter. At the end of the hybridization the blots were washed, dried, and exposed to an X-ray film.
Figure 5 shows the results of an experiment in which the transcriptional complex was labeled with a 2-min pulse in vitro using [cx-~~P]UTP. RNA from this complex was purified and hybridized to EcoRI, Hpal, Bgll restriction fragments blotted on a Schleicher and Schuell membrane filter. It can be seen that fragment e is enriched relative to its size. Identical results were obtained using a Millipore membrane filter.
FIG. 5. Hybridization between 32P-labeled RNA synthesized in vitro for 2 min using [a-32P]UTP and restriction fragments of SV40 DNA, as in Fig. 2, blotted on a Schleicher and Schuell membrane filter. After the hybridization the blot was washed, dried, and exposed to an X-ray film.
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TC with only a few nascent RNAs per DNA molecule. Circular DNAs with a great number of nascent RNA chains, distributed all along the molecule, were occasionally seen. We observed different types of SV40 DNA molecules complexed with RNA: completely or partially relaxed circular DNA (Fig. 6A), superhelical DNA (Fig. 6B), and oligomers (Fig. SC). The proportion of relaxed DNA was correlated with the concentration of formamide in the sample. Lowering the formamide concentration led to observation of relatively less relaxed molecules and more superhelical DNA templates. We suggest, therefore, that Form I DNA is the major template for transcription. A similar conclusion based on biochemical studies was recently reported by Birkenmeier et al. (1977). When the TC were cleaved with restriction endonucleases most of the nascent chains remained attached to their DNA template, suggesting that in addition to the superhelicity of the DNA the RNA is also being held to the DNA by the RNA polymerase (Birkenmeier et al., 19’7’7). Figure 7 shows the appearance of the cleaved
ET AL.
TC. The distribution of the nascent RNA chains along the DNA molecule is consistent with the conclusion that at least in some molecules there is complete or almost complete transcription of the DNA strands. E3H]Thymidine pulse-labeled SV40 DNA (replicating intermediates) isolated immediately after labeling is also found in the 23-25 S region of the sucrose gradient (Fareed et al., 1972). Under the electron microscope no nascent RNA chains were observed attached to replicating intermediates, indicating that this form of DNA is not transcribed. Moreover, the presence of replicating intermediates in the preparation provided an excellent control indicating the absence of nonspecific attachment of RNA to DNA. The Major Initiation Site for Late Transcription as Determined by Electron Microscopic Analysis Among circular DNA molecules carrying nascent RNA chains a substantial fraction (about 10%) had one short but extended nascent molecules. Examples of these com-
FIG. 6. TC spread by the protein-free BAC technique. (A, B) Monomers respectively. (C) Dimer spread in 40% formamide. Bars = 0.5 pm.
spread in 40 and 30% formamide,
INITIATION
OF TRANSCRIPTION
OF SV40
FIG. 7. Linear SV40 DNA with nascent RNA chains. TC were cleaved with restriction (A) and EcoRI (B) and prepared for EM as in Fig. 6. Each bar = 0.5 pm.
endonucleases Bgll
318
LAUB ET AL.
plexes are shown in Fig. 8. The short nascent chains are either RNA or single-stranded DNA. The second alternative could be true only if one of the two DNA strands was nicked allowing nascent RNA to hybridize with its complementary DNA strand and so displacing the secondDNA strand (Thomas et al., 1976). The RNA/DNA complexes may originate from the hybridization of free RNA with the viral DNA, but this seems to be unlikely since the DNA was never exposed to denaturing conditions which is a prerequisite for RNA/DNA hybridization. Additional support against this supposition is the absence of nascent chains attached to replicating intermediates. In order to determine whether the initiation of the short nascent chains maps at a specific site we cleaved the DNA with restriction endonucleases and obtained linear molecules of unit-length SV40 DNA with the small nascent chains still attached to them. Examples of such molecules cleaved with Bgll, BamHl, and EcoRI are shown in Figs. 9A, B, and 9C, respectively. The possibility of DNA strand displacement by RNA, as discussed above, is more likely with linear molecules than with circular DNA, in particular if the cleavage site is close to the hybridization site of the RNA due to the presence of a free end. However, the high sensitivity of the nascent chains to RNase (5 Fg for 30 min at 22’) tends to indicate that the majority of the singlestranded chains are nascent RNAs. The localization of the initiation site for tran-
scription is independent of whether the measured chains were RNA or displaced single-stranded DNA. It should also be mentioned that the molecules shown in Figs. 9A, B, and C are not replicating intermediates, because replicating intermediates would have forks at both ends of the molecule (in the case of a Bgll cut) or a bubble at an internal position in the molecule (in the cases ofBamH1 andEcoR cuts) typical for bidirectional replication (Fareed et al., 1972). Figures lOA, B, and C show examples of replicating intermediates cleaved with Bgll, BamHl, and EcoRI, respectively. The lengths of the nascent chains were measured and aligned at the attachment point to each side on the DNA templates and the distances of the 5’ termini of the RNA molecules from the near end of the DNA were determined and expressed as the fractional length of the DNA. The analysis gave two sets of numbers: In one set the numbers were at random and depended on the length of the RNA while in the second set they showed a high level of consistency. The statistical analysis was carried out on the second set of numbers. Figure 11 summarizes the results of the analysis in the form of four histograms. Three of the histograms show the location, on the physical map of SV40 DNA, of the 5’ end of the RNA, as calculated from the distances between the 5’ termini of the nascent chains (see above) and the cleavage sites of the restriction endonucleases:BgZl in A, BamHl in B, and EcoRI in C. The fourth histogram
FIG. 8. Circular SV40 DNA with short nascent chain. The spreading was by the BAC technique in 40% formamide. Bar = 0.5pm.
INITIATION
OF TRANSCRIPTION
OF SV40
+ IFIG. 9. Linear SV40 DNA with short nascent chain. TC were cleaved with restriction endonucleases Bgll (A), BamHl (B), and EcoRI (C). In the tracings arrow indicates the initiation site of transcription for the nascent chain.
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FIG. 10. Replicating ; intermediates cleaved with restriction endonucleases Bgll (A), BumHI (B), and EcoRI
is a summation of these three histograms. It could be seen that independent of the restriction enzyme used to cleave the DNA only one reproducible peak was obtained. This peak maps at position 0.67 + 0.02 on the physical map of SV40 DNA. We suggest that this location represents the major initiation site for late transcription. The EM analysis thus confirmed the results obtained by the biochemical approach. DISCUSSION
One of the basic questions in the understanding of how cells and viruses transcribe and produce their mRNAs is where and how the primary transcripts initiate and terminate. The best approach to determine the initiation site for transcription is by analyzing the nucleotides near the 5’ end of the newly synthesized RNA. However, for unknown reasons, we (unpublished results) and others (Ferdinand et al., 19’77) have failed to detect any labeled 5’ termini of SV40-specific RNAs. In the present paper we describe three independent studies which were undertaken in order to localize the initiation site for transcription of SV40 DNA at late time after infection. Two of the studies were based on the Dintzis principles (1961) as described recently by Bachenheimer and Darnell (1975) and in the third study we measured
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nascent RNA chains attached to transcriptional complexes under the EM. Localization of the Initiation Site(s) Based on the Din&is Principles The rationale of this approach is that after short pulses with radioactive precursors, RNA molecules would contain some labeled sequences complementary to each region of the DNA, but the labeled RNA complementary to a fragment of DNA which includes the initiation site for transcription would be in the shortest chains, while labeled RNA complementary to a DNA fragment far from the initiation site would be in successively longer chains. Similar analyses were performed previously in other systems only with in viva labeled RNA or with isolated nuclei (Goldberg et al., 1977). The use of transcriptional complexes to label the RNA has two main advantages: First, higher levels of radioactivity can be incorporated into SV40 nascent chains, and second, no physiological processing occurs in the in vitro system which may complicate the analysis of the in vivo labeled RNA. The results obtained with both in vivo and in vitro RNAs have indicated that the major initiation site for late transcription is within a fragment which spans between 0.67 and 0.76 on the physical map of SV40 DNA and at the distal end of fragment a (Horowitz et al., 1978).
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a new conformation of the structure of the viral DNP. In this case the role of the 5’ leader sequences would be to regulate the levels of the viral mRNAs at early and late times after infection. It is interesting to note that the 160-nucleotide leader region of E. coli Trp operon mRNA, like the leader of SV40 late mRNAs, has the potentiality to codefor a small peptide (Dharet al., 1977). There is a less likely possibility which can explain the same results. The enrichment of RNA complementary to fragment e may result from a decrease of the stability of attachment of long RNA chains to the template as compared with the stability of attachment of short RNA chains which are closer to the initiation site of transcription.
Froctionol length from Eco RI cut
FIG. 11. Histograms indicating the initiation site for transcription. The nascent chains in molecules such as shown in Fig. 9 were measured and aligned on the DNA and the distances of their 5’ ends from the near end of the restriction enzyme cleavage site were measured and plotted as the fractional length of the DNA. The histograms are oriented to each other with respect to the restriction endonuclease cleavage site. The inset shows the physical map of SV40 DNA with the location on the map of the Bgll, BamHl, and EcoRI cleavage sites.
Electron Microscopic Visualization Transcriptional Complexes
of
Both relaxed and superhelical molecules as well as oligomers of SV40 DNA carrying nascent RNA chains have been observed but no nascent molecules were found on replicating intermediates, suggesting that once a molecule starts to replicate it cannot serve as template for transcription until the replication is completed. It is possmle that both the relaxed and superhelical molecules serve as templates for transcription Attenuator-A Site in Which Transcription in vivo in different stages of the physiois Blocked logical process, however, one may be an The accumulation of TC with short na- artifact of the procedures. The presence scent chains and the abundance of labeled of transcriptional complexes with many RNA complementary to the e fragment after growing RNA chains indicates that many a short label in vitro (Fig. 5) could indicate RNA polymerase molecules can be active at a structure similar to the Trp attenuator any given moment on the same DNA molein Escherichia coli (for review, see Rabus- cule. Up to 10 RNA molecules could be say and Geiduschek 1977). The Trp atten- counted in a single transcriptional complex. uator is a site at which RNA chain elonga- This is probably an underestimate of the tion is blocked. In vitro short RNA tran- maximum number of growing chains occurscripts are produced and are not released ring in vivo on the same template, since from the template. In viva the transcrip- it is conceivable that at least part of the tional block at the attenuator appears to RNA was detached from the template durbe subject to regulation. If the accumulation ing the preparation. of the short nascent chains on SV40 DNA The largest RNA bushes found to be attemplates arises from the existence of an tached to SV40 DNA in the present work attenuator on SV40 DNA (at approximately were as large as a complete transcript, coordinate 0.75) then in vivo the tran- suggesting that at least one of the two SV40 scriptional block at the attenuator could DNA strands is completely transcribed. respond to modified RNA polymerase, However because we were unable to extend T-Ag, other viral proteins(s)(VP-3?), or the large nascent RNA chains, we could
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not determine whether there was any size occur both in the nucleus and in the cytopattern of the nascent RNA chains along plasm. A similar mechanism of splicing has the SV40 DNA molecule. We were unable been suggested for Ad-2 mRNAs (Berget to conclude from the present study the pro- et al., 1977). portion of molecules which are completely transcribed. ACKNOWLEDGMENTS Models for the Joining of the Leader to the Coding Sequences We have recently shown (Aloni et al., 197’7a,b; Bratosin et al., 1978) that the 5’ leader sequences of the two major cytoplasmic viral RNA components are transcribed from a segment of the genome which is not adjacent to the coding sequences and which maps from 0.70 + 0.01 to 0.75 + 0.01 map units. This leader was estimated to contain about 200 nucleotides. Similar observations were made for mRNAs extracted from adenovirus 2-infected cells (Berget et al., 1977; Chow et al., 1977; Klessig, 1977). Several models for such splicing of SV40 late mRNAs could be suggested. These include: (1) intermolecular ligation of RNA, (2) deletion of intervening DNA sequences, (3) looping out of intervening DNA sequences so that the RNA polymerase could skip over short distances, and (4) deletion of the appropriate intervening RNA sequences. The observations made in the present work which show that there is one major initiation site for late transcription and that the template is mainly Form I SV40 DNA of unit length exclude the first two alternatives. The third alternative is excluded because analysis of R-loop structures formed between nuclear viral RNA and linear SV40 DNA has shown the presence in nuclear RNA of sequences complementary to the intervening DNA segment (Horowitz et al., 1978). We therefore conclude that the joining of the leader sequences to the coding region occurs via the fourth alternative of looping out intervening RNA sequences and then covalent joining of the leader to the coding sequences via an intramolecular digestion and ligation. The observation that the 16 S RNA is processed from the 19 S in the cytoplasm (Aloni et al., 1975; Groner, Carmi and Aloni, unpublished results) suggests that splicing of RNA can
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