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Transcription of viral sequences in cells transformed by adenovirus type 5
VIKOI,OGY
89, 347-359
Transcription
(1978)
of Viral
Sequences
in Cells Type
E. I. FROLOVA Institute
of Molecular
Biology,
Academy
by Adenovirus
5 E. S. ZALMANZON
AND
Accepted
Transformed
of Sciences May
of the
USSR,
Moscou~,
USSR
8, 1978
‘I’he kinetics of renaturation of ‘“P-labeled restriction endonuclease fragments of adenovirus 5 DNA were measured in the presence of calf thymus DNA and DNA extracted from five lines of adenovirus 5-transformed cells. The number of copies of different fragments per diploid quantity of cell DNA was calculated. All five transformed cell lines contained sequences homologous to the HpaI fragment E, but varied in the other adenovirus 5 DNA sequences. Thus, the sequences which lie on the left-hand end of adenovirus 5 DNA must specify any viral functions required for the maintenance of the transformed cell phenotype. Transcription of viral sequences in three lines of adenovirus 5-transformed cells was studied by hybridization of [ “I’]RNA from transformed cell nuclei with restriction endonuclease fragments of adenovirus 5 DNA which were attached to a nitrocellulose filter. Almost all viral DNA sequences present in transformed cells were transcribed into nuclear RNA. Preferential digestion of transcribed viral sequences with DNase I was analyzed using kinetics of renaturation of “P-labeled specific fragments of adenovirus 5 DNA in the presence of DNA extracted from DNase I treated nuclei of transformed cells. Only part of the sequences of HpaI fragment E comprising the left 47’ of the viral genome was sensitive to DNase I digestion in all lines of transformed cells which were studied. All viral sequences prcscnt in the DFKl ccl1 line were partially sensitive to DNase I digestion. INTRODUCTION
Knowledge of the structure and transcription of the integrated viral genomes in transformed cells is necessary for understanding of the mechanisms of cell transformation and regulation of gene expression in eukaryotic cells. Viral genomes in transformed cell DNA usually are investigated using restriction fragments of viral DNA by measuring the increase in their reassociation rates by t,ransformed DNA (Gelb et al., 1971). Using this technique it has been shown that no line of rat and hamster cells transformed by group C adenoviruses examined contains a complete copy of the viral genome and the extent of viral sequences and the number of copies present per diploid quantity of transformed cell DNA varies in different cell lines (Gallimore et al., 1974; Sambrook et al., 1974; Sharp et al., 1974b; Flint et al., 1976). The segment of viral DNA comprising the left hand 12% of the viral DNA was
common to all adenovirus transformed cells. Studies on the transcription of adenovirus genes in transformed cells demonstrated that only a portion of the viral sequences which represent the early adenoviral genes are transcribed (Flint et al., 1976). From this type of analysis it is not clear whether all the copies of a certain sequence are active in transcription, however, with a few exceptions, the number of RNA transcripts of a given sequence closely parallels the number of copies of viral DNA, from cell line to cell line (Flint and Sharp, 1976). In this paper we described the experiments which have been performed using five lines of Ad5transformed cells. Four of them (DFKl, FKAdl, FKAd5, and FKAd6) were independently isolat,ed after infection of rat primer embryonic fibroblasts by Ad5 and one was obtained after transformation of rat embryo cells by Ad5 DNA. The biological characteristics of these lines (growth properties, sensitivity to the repetitive Ad5 0042~6822/78/0892-0347$02.00/O Copyright 0 19% hy Academic
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348
FHOLOVA
AND
ZALMANZON
were isolated, digested with DNase I (Worthington) as described by Weintraub and Groudine (1976) and DNA from native and DNase I digested nuclei was isolated (Weintraub and Groudine, 1976). Transformed cell DNA was isolated as described by Gallimore et al. (1974). RNA. [““PIRNA was extracted from isolated nuclei of Ad5-transformed cells and purified by the method of Scherrer (1969). Restriction endonucleases. Endonuclease Eco RI was isolated from Escherichia coli RY-13 (Yoshimory, 1971). HpaI was prepared from Hemophilusparainfluenzae according to Sharp et al. (1974a). Preparation of specific fragments of Ad 5 DNA. HpaI restriction fragments of Ad5 DNA were separated by electrophoresis on 0.7 x 30 cm cylindrical gels of 0.7% agarose (Flint et al., 1976) and prepared as described by Gallimore et al. (1974). Combined restriction of Ad5 DNA by HpaI and Eco RI was made in incubation mixture containing 10 mM Tris-HCl, pH 7.4, 10 mM MgC12,6 n&f KCl, 1 mMdithiothreito1, and 5 mM NaCI. Resulting fragments were sepMATERIALS AND METHODS arated by electrophoresis on a 0.4 x 12 x Cells. Lines of rat embryo cells trans- 15 cm slab gel containing 1% agarose. formed by Ad5 were grown in glass roller Radioactive labeling of DNA. HpaI rebottles (New Brunswick) in Eagle’s mini- striction fragments of Ad5 DNA were lamal essential medium (MEM), supple- beled to specific activities of l-8 x 10’ mented with 10% calf serum. KB cells for cpm/pg using four deoxy[n-“‘Plnucleotide cultivation of Ad5 virus were grown in sus- triphosphates (250 Ci/mmol, Amersham) pension cultures in MEM supplemented and DNA-polymerase I (Fraction VII, with 5% calf serum. Boehringer Mannheim Biochemicals) (Bot‘QP-labeled cells were prepared in the than et al., 1976). The reaction was termifollowing manner: cells were washed three nated by the addition of sodium dodecyl times during 1 hr and one time during 12 hr sulfate (SDS) and EDTA to final concenwith phosphate-free MEM, containing 25 trations of 0.5% and 0.01 M, respectively. mM Hepes and 1% dialyzed calf serum and The radioactive Ad5 DNA HpaI fragments then incubated for 4 hr in the same medium were freed from proteins by two extractions with phenol and separated from unincorwith 500 &i per ml of H:,‘“PO, (carrier-free, porated deoxynucleotide triphosphates by Amersham). Virus. Ad5 was propagated in suspension passagethrough a column (0.5 x 20 cm) of G-50 Sephadex equilibrated with 0.01 M culture of KB cells at an input multiplicity of IO plaque-forming units (PFU) per cell. Tris-HCl, pH 7.7, 0.001 M EDTA. Further Virus was purified by a modification of the separation was obtained by chromatogramethod of Green and Pina (1963) (Lonberg- phy on hydroxylapatite. Eluates from hyHolm and Philipson, 1969). droxylapatite column (in 0.4 M Na-phosphate, pH 6.8) were then used for reassociaViral DNA. Ad5 DNA was extracted from purified virions as described (Petters- tion kinetic experiments. Blotting. A gel containing DNA fragson and Sambrook, 1973). Cell DNA. Nuclei from transformed cells ments was immersed in 0.2 M NaOH and infection, T-antigen content, oncogenicity for newborn rats, etc.) were described in more detail elsewhere (Zalmanzon et al., 1978). In the present work, we examined viral DNA sequences in these cell lines by reassociation kinetics and determined the number of copies of different fragments of Ad5 DNA per diploid quantity of cell DNA. Transcribed viral sequences in these cell lines were investigated by analysis of labeled nuclear RNA transcripts. As it has been shown for globin gene (Weintraub and Groudine, 1976), ovalbumin gene (Garel and Axel, 1976) and total structural genes (Lewy and Dixon, 1977) the actively transcribed sequences have an altered chromatin conformation that reflected by preferential digestion of these DNA sequences by DNAse I. We used mild DNAse I digestion of transformed cell nuclei to study chromatin conformation, possibly reflecting transcriptional activity, of different regions of the integrated viral genomes in four lines of Ad5-transformed cells.
TRANSCRIPTION
OF
0.6 A4 NaCl for 45 min at room temperature and neutralized in 1.0 M Tris-HCl, pH 7.4, and 0.6 M NaCl for a further period of 45 min (Botchan et al., 1976). The DNA was then transferred onto a sheet of nitrocellulose (B6, Schleicher and Schull) using the method described by Southern (1975). The filter containing the DNA was washed with 2 x SSC, allowed to dry in air for 1-2 hr and heated in vacua at 80” for 4 hr. HLybridization conditions. DNA:RNA filter hybridization was carried out at 65” for 16 hr in 6 X SSC, 0.5% SDS, 0.001 M EDTA. Before hybridization a strip of nitrocellulose filter (4 mm x 11 cm) with HpaI + Eco RI Ad5 restriction fragments was soaked in a solution containing 0.02% FicoIl, 0.02% bovine serum albumine, and 0.02% polyvinylpyrrolidone dissolved in 2 x SSC (Denhardt, 1966) for a period of 4-5 hr at 65”. The filter was then incubated under the above conditions with 10 x 10” cpm of [“‘P]RNA from the nuclei of transformed cells in a volume 0.2 ml. Following hybridization the nitrocellulose filter was washed with 2 x SSC, incubated in 7 M urea, 2 x SSC, 0.5% SDS at 42” for a period of 2 hr, again washed with 2 x SSC, incubated with 10 pg/ml RNase at 37” for a period of 40 min, again washed with 2 x SSC, air-dried, mounted on Whatman 3M paper and subjected to autoradiography (Noscreen film, type NS-5T, Kodak) for 1 week. DNA:DNA hybridization was carried out at 68” in 1.0 M NaCl buffered with 0.14 M Na-phosphate, pH 6.8. Before annealing both viral and cellular DNAs were degraded to oligonucleotides of 300 bases in length by boiling in 0.3 M NaOH for 20 min. DNA extracted from nuclei of transformed cells was degraded to oligonucleotides with the average size of 150 bases in length by boiling in 0.3 M NaOH for 30 min. DNA from nuclei digested with DNase I had the same average length, as judged by sedimentation in the alkaline sucrose gradient (data not shown). Samples taken after different times of incubation at 68” were diluted lofold in 0.14 M Na-phosphate, pH 6.8, and stored at 4’ until assayed by chromatography on hydroxylapatite (Gallimore et al., 1974; Sharp et al., 1974). Calculations. The derivation of equa-
VIRAL
349
SEQUENCES
tions and the methods of calculation have been described in detail elsewhere (Sharp et al., 1974b). The time required for 50% of the “‘P-labeled probe of DNA to reanneal in the presence of calf thymus DNA (t, ,2p) and in the presence of transformed cell DNA (t,J was calculated from Equation 1: tll:! (or tl12p) =
t
l/f+ - 1
[II
where hh is the fraction of [,“P]DNA remaining single stranded at time t. The quantity of viral DNA per diploid quantity of transformed cell DNA was calculated as follows: the average value of tl,2p/tl,a was calculated and multiplied by the quantity of ““P-labeled viral DNA per milliliter of hybridization solution. Knowing the concentration of transformed cell DNA initially added to this mixture, the number of copies of each fragment of Ad5 DNA per diploid quantity of cell DNA (3.9 x 10’” daltons) (Sober, 1968) can be readily calculated. Theoretical curves for the renaturation kinetics of [“‘P]DNA in conditions where transformed cell DNA contain sequences homologous to only a fraction of probe were derived from Equation 2: l/f5 =
1 X (r + l)t/tllrp
1-x + 1 + t/t,,zp + 1
121
where X is the fraction of the “‘P-labeled probe assumed to be present in the transformed cells and r is the molar ratio of these viral sequences in cell DNA to those of the ““P-labeled viral DNA probe (Sharp et al., 1974b). RESULTS
Adenovirus 5 DNA sequences in transformed cells. We have digested Ad5 DNA with restriction endonuclease HpaI to obtain specific fragments of the viral genome (Fig. 1). With these fragments we have assayed for the presence of Ad5 DNA sequences in five lines of transformed cells. Four of the lines (FKAdl, FKAd5, FKAd6, and DFKl) are independent viral transformants of rat embryo cells. The remaining one (FKAugustDNA3) is a line of rat
350
3 -ID3 : * EC3 j ! Ad 5 3 8; _
FROLOVA
4
b x
I
I
AND
=1
Ecdl-J
FIG. 1. Eco RI and HpaI cleavage sites on adenovirus 5 DNA. The positions of the two Eco RI and the six HpaI cleavage sites on Ad5 DNA are shown (1 and 2, respectively), and HpaI+EcoRI cleavage sites are shown (3), the solid horizontal line depicting the viral genome. These data are taken from Flint et al. (1976).
embryo cells transformed by Ad5 DNA. Before using specific HpaI fragments of Ad5 DNA in an analysis of the viral DNA sequences present in DNA from transformed cells, it was necessary to demonstrate that all fragments were capable of annealing to viral DNA sequences. The conditions in which ““P-labeled probe DNA was annealed in the presence of transformed cell DNA were closely reproduced in reconstruction experiment in which the hybridization mixtures contained a large amount of unrelated, control cell DNA and a small amount of viral DNA. The data obtained were treated as described in Materials and Methods to give the values shown in Table 1. The rates of reannealing of all “P-labeled fragments were accelerated in the presence of unlabeled Ad5 DNA by an amount close to that expected from the quantity of viral DNA added. Thus we concluded that the HpaI fragments of Ad5 DNA reannealed at rates proportional to their initial molar concentrations in the reaction mixture. The effect of transformed cell DNA on the reassociation kinetics of the “‘P-labeled fragments can be used to determine the concentration of each fragment in DNA extracted from transformed cells. We measured the rates of reannealing of “2P-labeled specific fragments of Ad5 DNA in the presence of DNA extracted from Ad5-transformed cells, and calf thymus DNA. Figure 2 shows the kinetics observed with the ““P-labeled HpaI fragments of Ad5
ZALMANZON
DNA in the presence of DNA extracted from FKAdl and FKAd5 lines of Ad5transformed cells. The rates of reannealing of A + B, D, F, and G fragments in the presence of DNA extracted from transformed cells and calf thymus DNA are the same. However, DNA extracted from transformed cells increases the rate of reannealing of E and C fragments compared with the control DNA. From the map positions of the fragments (Fig. 1) it is clear that these lines of transformed cells do not contain any viral sequences that lie to the right of HpaI fragment C. The DNA of ‘“P-labeled HpaI fragment E reanneals in the presence of transformed cell DNA with apparent second-order kinetics, but the renaturation rate of HpaI fragment C deviates from second order in the presence of DNA extracted from FKAdl and FKAd5 cells. TABLE
1
RENATUHATION OF Ad5 DNA ~~~_____. Fragment
OF .‘?-LABELED HpaI FRAGMENTS IN THE PRESENCE OF UNLARELED ADENOVIRUS 5 DNA” .~.___ Adenovirus 5 DNA concentra(0 g/t, 2) tion (&ml x 10 ‘) ~-___
A+B c D E F G
_______~. 23.6 + 0.24 8.83 + 0.43 5.37 +_ 0.15 2.71 + 0.24 3.13 -c 0.11 2.05 + 0.25
Calculated from Added to hyobserved rate of bridization reannealing reaction 2.57 2.40 2.72 3.01 3.09 2.92
2.65 2.65 2.65 2.65 2.65 2.65
” ?-labeled HpaI fragment (specific activity 3.0-6.0 x 10’ cpm/pg) were mixed with the quantity of unlabeled Ad5 DNA shown in column 4, and hybridization reactions were performed as described in legend to Fig. 2. Six samples were withdrawn at various times during the renaturation reaction and the fraction of [‘“P]DNA remaining single-stranded was detected by chromatography on hydroxylapatite. The ratio of the time required for half of the r”PlDNA of restriction endonuclease fragments of Ad5 DNA to reanneal in the absence and presence of unlabeled Ad5 DNA was determined for each time point using Equation 1. These ratios were used to calculate average values of ( tl,Lp/tlr2) and their standard deviations (column 2). From the increase in the rate of hybridization of the “P-labeled probe DNAs in the presence of unlabeled, viral DNA the amount of adenovirus 5 DNA in each reaction mixture was calculated.
TRANSCRIPTION
OF
VIRAL
SEQUENCES
351
The increases in rates of renaturation of HpaI fragments of Ad5 DNA in the presence of DNA extracted from FKAd6 cells compared with calf thymus DNA and the number of copies of different fragments per diploid quantity of FKAd6 DNA are shown 0.4 014 in Table 2. Thus, just as transformed cell lines de!lpo I-G 46 scribed previously contain the segment of viral DNA that maps at the extreme left3 hand end of the Ad5 genome (Flint et al., 2 1976), so the three cell lines which were investigated carry only this partial copy of 1 a viral sequences. 5,5 :c I,5 2,o The results of an experiment in which tit, the kinetics of renaturation of specific HpaI IP fragments of Ad5 DNA were measured in FIG. 2. Kinetics of reassociation of ““P-labeled the presence of DNA extracted from DFKl HpaI fragments of Ad5 DNA in the presence of calf and FKAugustDNAS cells and in the presthymus DNA and DNA extracted from the Ad&transformed cell lines, FKAdl and FKAd5. “P-labeled fragence of calf thymus DNA are shown in Fig. ments of Ad5 DNA were prepared as described under 3 and Table 2. Clearly DNA extracted from Materials and Methods. The concentrations of calf these cells increases the rate of reannealing thymus DNA and FKAdl and FKAd5 DNAs were 1.0 of all labeled HpaI fragments, so the cells and 1.5 mg/ml, when the probes were HpaI fragments carry not only the left end of Ad5 genome A-F and HpaI fragment G, respectively. The concenbut almost all regions of it. The magnitude trations of the “P-labeled probe DNAs were HpaI of the increases in the renaturation rates fragments A + B, 6.80 x 10 ’ pg/ml; HpaI fragment produced by cell DNA varied from fragC, 6.39 x 10 ’ pg/ml; HpaI fragment D, 5.99 x 10-j ment to fragment and from cell line to cell pg/ml; HpaI fragment E, 7.05 x 10 ’ pg/ml; HpaI line. For example, DFKl cells contain the fragment F, 5.80 X 1O-J pg/ml; HpaI fragment G, 6.40 x 10 ’ Fg/ml. Before hybridization cellular and viral highest amount of viral DNA per diploid DNAs were degraded by boiling in 0.3 M NaOH for 20 quantity of cell DNA: 1.4, 2.0, 4.5, 4.1, 3.1 min, to lengths of about 300 nucleotides. Hybridization copies of HpaI fragments A + B, C, D, E, reactions contained l.OMNaCI, 0.14MNa-phosphate, F, and G, respectively. Although, unlike pH 6.8, and 0.4% sodium dodecyl sulfate, and were other known Ad5-transformed cell lines, incubated at 68”, samples being withdrawn at intervals DFKl cells seem to contain a full set of for analysis by chromatography on hydroxylapatite. viral genes, they never produce infectious W, Renaturation of [ r’P]DNA in the presence of calf virus, so it is possible that more detailed thymus DNA, 0, renaturation of [‘jLP]DNA in the investigation will detect some deletions in presence of FKAd5 DNA at concentrations listed viral DNA sequences present in DFKl above for each probe; 0, renaturation of [“PlDNA in the presence of FKAdl DNA. The curves shown in cells. panels l-6 are theoretical ones, calculated from EquaViral RNA sequences in Ad5-transtion 2 (see text). formed cells. Viral nuclear RNA sequences in the three lines of Ad5-transformed cells The curves in Fig. 2 (panel 2) are theoretical (FKAd5, DFKl, and FKAugustDNAS) curves calculated on the premise that dipwere assayed on nitrocellulose filters by loid transformants contain only a part of hybridization of [:‘“P]RNA extracted from the total sequences of HpaI fragment C. the nuclei of transformed cells to specific Because of the good fit between the experendonuclease restriction fragments of unimental data and the theoretical curves we labeled Ad5 DNA. Such assay allows the conclude that FKAdl and FKAd5 cells condetection of fragments which are completain sequences homologous to about 10%~ mentary to the nuclear RNA sequences of and 50% of the HpaI fragment C, respectransformed cells. For hybridization expertively. iments we have used specific fragments
352
FROLOVA
AND
ZALMANZON
TABLE
RENATURATIONOF HpaI Cell line
2
FRAGMENTSOF Ad5DNA INTHE~RESENCEOF DNAs LINESOFRATCELLSTRANSFORMEDBYADENOVIRUS~" Fragment
2. FKAd5
3. FKAd6
4. DFKl
5. FKAugustDNA3
A+B C D E F G A+B C D E F G A+B C D E F G A+B C D E F G A+B C D E F G
____~
Equivalent/diploid DNA assuming Complete ment
1. FKAdl
EXTRACTEDFROMFIVE
1.04 1.41 1.13 1.97 0.93 0.98 1.04 3.34 1.15 2.25 1.13 0.91 2.09 2.26 0.95 3.64 1.09 1.32 8.24 4.85 5.23 2.35 2.66 2.31 2.52 3.98 2.80 3.07 1.65 1.76
* zk + f f f f f f f f f -c f f f + f f +f -c + f f -t -+ f -c f
0.07 0.15 0.14 0.34 0.12 0.20 0.31 0.18 0.14 0.31 0.28 0.29 1.18 0.86 0.13 1.01 0.47 0.12 2.42 1.64 1.45 0.79 0.43 0.16 0.35 0.73 0.58 0.17 0.04 0.08
0.0 0.2 0.0 2.7 0.0 0.0 0.0 1.3 0.0 3.8 0.0 0.0 0.2 0.7 0.0 7.7 0.0 1.0 1.4 2.0 4.5 4.0 4.1 3.1 0.3 1.6 1.9 6.2 1.6 1.8
seg-
quantity of ceH sequences homologous to Percentage of the fragment 2.7(10%)
h
3.8(50?;)’
n Hybridization reactions were performed as described in legend to Fig. 2. The specific activity of the “Plabeled probe DNA ranged from 3.0 to 6.0 x 10’ cpm/pg. Six samples were withdrawn at various times during the renaturation reaction and the fraction of [,“PlDNA remaining single-stranded was determined by chromatography on hydroxylapatite. For all probe DNAs, the data were treated as follows; the ratio of the time required for half of the ‘“P-labeled probe DNA to reanneal in the presence of calf thymus DNA (tromp) and in the presence of transformed cell DNA (tlir) was determined at each time point using Equation 1. These ratios were used to calculate average values of (t,/p/t, L) and their standard deviations (column 3). From the increase in the rate of reannealing of the “‘P-labeled probe DNAs in the presence of transformed cell DNA, the number of equivalents of the sequences of each of the probe DNAs in a diploid quantity of rat DNA (3.9 X IO’” daltons) was calculated assumming that the transformed cells contained all of the sequences of the probe DNA. ‘These values are derived from the theoretical curves, calculated as described in the text, shown in the panels of Fig. 2.
which were obtained after simultaneous digestion of unlabeled Ad5 DNA with HpaI and Eco RI restriction endonucleases. After treatment of Ad5 DNA with these two en-
zymes we were able to separate in 1% agarose slab gels HpaI-A and two main fragments which Eco RI produced from the HpaI-B fragment (X and Eco RI-C).
OF VIRAL
TRANSCRIPTION tlpa I- 0
43 3 2 1 l!!!i4
0.q
0,e
1:1,,
/~p;p$fy
353
SE:QUF,NCES
different viral DNA sequences before and after mild DNAse I digestion of isolated nuclei of transformed cells. Specific fragments of Ad5 DNA generated by cleavage with restriction endonuclease HpaI have been used to assay for the presence of the Ad5 DNA sequences in different DNA preparations from transformed cells. We measured the rate of reannealing of “‘P-labeled HpaI fragments in
II 0,:
C.8
O,?
0.8
c.5 l,o 1.5 2,0
FIG. 3. Kinetics of reassociation of ‘“P-labeled HpI fragments of Ad5 DNA in the presence of calf thymus DNA and DNA extracted from the Ad5-transformed cell lines, DFKI and FKAugustDNA3. Hybridization reactions were performed as described in the legend to Fig. 2. W, Renaturation of [ “PlDNA in the presence of calf thymus DNA; 0, renaturation of [“I’JDNA in the presence of DFKl DNA at concentrations listed at the legend to Fig. 2 for each probe; 0, renaturation of [‘“PJDNA in the presence of FKAugustDNA3 DNA. The curves shown in panels l-6 are theoretical ones, calculated from Equation 2.
Figure 4 depicts the results of such an experiment. Nuclear RNAs extracted from any of cell lines are complementary to the HpaI fragments E and C. Nuclear RNA from FKAd5 cells is complementary to only these DNA fragments, e.g., both viral DNA sequences detected by methods described above. After hybridization of “‘Plabeled nuclear RNA from DFKl and FKAugustDNA3 cells to the fragments of At15 DNA radioactive spots were localized at the positions of all fragments which were transferred to the filter (Fig. 4, b and c). Thus, nuclear RNAs from the cells which carry not only the left-hand end of Ad5 DNA but practically all regions of Ad5 genome hybridize to the fragments which represent not only the left-hand end of Ad5 DNA. Sensitivity of viral DNA sequences to DNAse I digestion of nuclei of transformed cells. In order to study chromatin conformation of viral sequences in transformed cells we measured the concentrations of
D
Eco RI-C
E F a
b 4. Hybridization
C
d
of “P-labeled nuclear RNA extracted from transformed cells to Ad5 DNA fragments generated by restriction endonucleases HpoI and Eco RI. Ad5 DNA was digested with &a1 and Eco RI enzymes and electrophoresed through a 1% agarose gel as described under Materials and Methods. The DNA bands were photographed under ultraviolet light in the presence of 0.5 pg/ml ethidium bromide. The fragments were transferred to nitrocellulose strips, and then hybridized to 1-3 x 10’ cpm of “I’labeled nuclear RNA from transformed cells. Radioactivity was detected by autoradiography. Autoradiograms of the nitrocellulose strip after hybridization to labeled FKAd5 RNA (a); DFKI RNA (b); and FKAugustDNAY (cl. (d) Photograph of the ethidium bromide fluorescence of Ad5 HpaI+Eco RI fragments separated in a 1% agarose gel (HpaI-G and Y fragments have run off the gel). FIG.
354
FHOLOVA
AND
the presence of DNA extracted from native nuclei of transformed cells and compared it with the rate of reannealing of the same fragments in the presence of equivalent amounts of DNA extracted from the nuclei of transformed cells which were digested with DNAse I. The renaturation rates of the “2P-labeled viral DNA fragments in the presence of calf thymus DNA were used as controls. Such assays permit the detection of a preferential sensitivity or resistance of specific portions of the integrated adenovirus DNA to DNase I. The results of renaturation experiments are shown in Table 3 and in Figs. 5-7. Figure 5 depicts reannealing of ““P-labeled HpaI fragments of Ad5 DNA in the presence of different DNA preparations from FKAd5 cells. There is an evident difference between the rates of reassociation of HpaI fragments C and E in the presence of two preparations of DNA extracted from transformed cells. Thus, the sequences of viral DNA present in transformed cells which are complementary to the C and E fragments are sensitive to DNase I digestion. Because the rate of renaturation of labeled HpaI fragments C in the presence of DNA extracted from DNase I digested nuclei of FKAd5 cells and calf thymus DNA are almost equal, we conclude that practically all sequences of HpaI fragment C present in FKAd5 nuclei are completely sensitive to DNase I. The sequences which are complementary to the HpaI fragment E are present in DNA extracted from DNase I digested nuclei in detectable amounts. This means that some of these sequences in the FKAd5 cell DNA are stable to DNase I treatment. The number of copies of DNase I stable sequences of HpaI fragment E per diploid quantity of FKAd5 DNA was calculated (Table 3). DNA extracted from the DNase I digested nuclei of FKAugustDNA3 cells increases the rate of renaturation of HpaI-E fragment only. The sequences of the other Ad5 DNA fragments present in this cell line are completely sensitive to DNase I digestion of nuclei (Fig. 6). The results of renaturation experiments with DNA extracted from DNase I treated and untreated nuclei of DFKl cells are
ZALMANZON
shown in Fig. 7. The sequences complementary to all HpaI fragments of Ad5 DNA were detected in DNA extracted from digested nuclei of DFKl cells. There were no differences in the rates of reannealing of HpaI fragments F and G in the presence of DNA extracted from native and digested nuclei of DFKl cells, so these fragments were stable to DNase I digestion. The partial sensitivity of the sequences complementary to the HpaI fragments A + B, C, D, and E was detected. The number of copies of HpaI fragments which were stable to DNase I digestion of nuclei per diploid quantity of cell DNA in this line presented in Table 3. The sequences of Hpa I fragment E present in transformed cells were partially sensitive to DNase I treatment of nuclei from all cell lines studied. DISCUSSION
In experiments reported here we have studied viral genomes, their transcription and chromatin conformation in several lines of Ad5-transformed rat embryo cells. The HpaI restriction endonuclease fragments of Ad5 DNA have been used as probes of different regions of Ad5 genome to define viral DNA sequences present in transformed cell DNA. The results are summarized in Fig. 8 which shows both regions of the Ad5 genome found in FKAdl, FKAd5, FKAd6, DFKl, and FKAugustDNA3 cells and the frequency per diploid quantity of DNA at which they occur. Three lines of Ad5-transformed cells (FKAdl, FKAd5, and FKAd6) are similar to the known Ad2- and Ad5transformed cell lines. All of them carry only a part of the viral genome and those viral DNA sequences present map in the left-hand end of the Ad5 DNA. DFKl and FKAugustDNA3 cells are reminiscent of some Ad2-transformed cell lines which contain sequences from almost all regions of viral genome but in different numbers of copies. All Ad5-transformed cell lines contain sequences homologous to the HpaI fragments E and C, but they vary in the content of other Ad5 DNA fragments. The FKAdl cells contain the minimal fraction of viral genome known (6%). Although the
TRANSCRIPTION
OF
FRAGMENTS WITH DNase
OF Ad5 I NUCLEI
VIRAL
TABLE RENATUKATION I)IGESTEII
OF ?LAREI,ED AND UNIIIGESTED
3 DNA
IN THF: PRMENCF, OF DNAs EXTRACTED OF THREE LINES OF RAT CF.I,LS TRANSFORMED
ADENOVIR~JS
DNA
1. 1)NA from digested with clei of FKAd5 cells
DNase
DNA from undigested with nuclei of FKAd5 cells
2. DNA from digested with clei of DFKI cell cells
I nu-
DNase
DNase
DNA from undigested with nuclei of DFKI cells
I
I nu-
DNAse
I
FKOM BY
5”
Fragment
preparation
355
SEQUENCES
Equivalent/diploid quantity of cell DNA, assuming sequences homologous to complete fragment
A+B C D E F G A+B C D E F G
0.0 0.2 0.0 1.3 0.0 0.0 0.0 1.2 0.0 4.1 0.0 0.0
A+B C D E F G A+B C D E F G
0.4 0.7 1.2 2.0 3.7 3.0 1.2 2.2 4.1 3.8 3.8 2.7
f
3. DNA from digested clei of FKAugust
with DNase DNA3 cells
DNA from undigested FKAugustDNA3 cells
I nu-
nuclei
0 Hybridization reactions were labeled probe DNA ranged from the renaturation reaction and the raphy on hydroxylapatite. For all
of
A+B C D E F G A+B C D E F G
1.13 0.96 0.92 3.03 1.09 0.93 3.60 6.32 4.53 4.88 1.68 1.62
f 0.25 f 0.18 f 0.24 t 0.64 F 0.18 f 0.11 it 0.54 f 0.78 +- 0.18 rf; 1.03 + 0.09 AC 0.15
0.0 0.0 0.0 3.5 0.0 0.0 0.2 1.3 2.1 6.7 1.1 2.0
performed as described in the legend to Fig. 5. The specific activity of the ‘“P3.0 to 6.0 x 10’ cpm/pg. Six samples were withdrawn at various times during fraction of [ “P]DNA remaining single-stranded was detected by chromatogprobe DNAs, the data were treated as described in the legend to Table 2.
minimal fragment found earlier in group C adenovirus transformed cells comprised left-hand 12% of viral DNA (Flint et al., 1976) our results are consistent with those of Graham et al. (1974) who were able to transform cells with the 7% of the left-hand
adenovirus DNA fragment. So any viral functions required for the maintenance of the transformed cell phenotype are encoded by about 6-7s of the left end of Ad5 DNA. The nature of the observed differences in the content of various viral sequences in
356
FROLOVA
I:. 64
0.8
c.4
0.8
05I
AND
: :I 10 I '5, 20,
!tt, ip
5. Kinetics of reassociation of “Plabeled Hpd fragments of Ad5 DNA in the presence of calf thymus DNA and DNA extracted from digested and undigested with DNase I nuclei of FKAd5 cells. ,“I’labeled fragments of Ad5 DNA were prepared as described in Materials and Methods. The concentration of calf thymus DNA and DNAs extracted from FKAd5 digested and undigested nuclei was 1.5 m&ml. The concentrations of the “P-labeled probe DNAs were HpuI fragments A+B, 4.08 x 10 ’ &ml; H,nnI fragment C, 4.36 X IO ’ pg/ml: HpnI fragment D, 5.05 X 10 ’ &ml; HpnI fragment E. 6.10 x 10 ’ &ml; Hpnl fragment F, 5.80 x 10 ’ pg/ml; HpnI fragment G, 6.53 x IO ’ pg/ml. Nuclei of FKAdS cells were digested with 20 &ml DNase I until 20’:: of DNA became acid soluble. DNA from undigested nuclei and DNA which was stable to 1)Nase I digestion were extracted and used in hybridization experiments. Before hybridization viral DNA and DNA extracted from the undigested with DNase I nuclei were degraded by boiling in 0.3 M NaOH for :JO min. to lengths of about 150 nucleotides. Hybridization reactions contained 1 .O M NaCl, 0.14 M Na-phosphate, pH 6.8, and 0.40 sodium dodecyl sulfate, and were incubated at 68”. samples being withdrawn at intervals for analysis hv chromatography on hydroxylapatite. n . Kenaturation of [“I’]DNA in the presence of calf thymus DNA; 0, renaturation of [“I’]DNA in the presence of DNA extracted from digested with DNase I nuclei of FKAd5 cells; Q, renaturation of [ “I’JDNA in the presence of DNA extracted from undigested with 11Nase 1 nuclei of FKAd5 cells. The curves shown in panels 1-6 are theoretical ones, calculated from Equation 2. FIG.
different But we forming FKAd6 infection of Ad5
transformed cell lines is not clear. can suggest the role of the transviral DNA-FKAdl, FKAd5, and cell lines which were obtained after of the cells with the same stock have more similarities in the pat-
ZALMANZON
tern of HpaI fragments with each other than with the lines obtained with another stock of virus (DFKl) or viral DNA (FKAugustDNAS). Nuclear RNA transcripts which were complementary to the Hpa-E and HpaI-C fragments of Ad5 DNA were found in the nuclei of all cell lines. Although the maintenance of transformation in adenovirus transformed cells is though to be coded by 6-7s of the Ad5 genome and mapped at the extreme Ieft end of Ad5 genome, in some transformed cell lines (DFKI, FKAugustDNA3) not only is the extreme left end of the genome transcribed, but nuclear RNA from these cell lines is complementary to all viral DNA fragments corresponding to the viral DNA sequences of the cell line detected by the renaturation experiments. In order to study the chromatin conformation of the integrated viral sequences in
FIG. 6. Kinetics of reassociation of “I’-labeled HpaI fragments of Ad5 DNA in the presence of calf thymus DNA and DNA extracted from digested (2O’i; of acid-soluble material) and undigested with DNase I nuclei of FKAugustDNA3 cells. Hybridization reactions were performed as described in the legend to Fig. 5. n , Renaturation of [‘“P]DNA in the presence of calf thymus DNA; 0, renaturation of [,‘“P]DNA m the presence of DNA extracted from digested with DNase I nuclei of FKAugustDNAY cells; 0, renaturation of r”P]DNA in the presence of DNA extracted from undigested with DNase I nuclei of FKAugustDNA3 cells. The curves shown in panels l-6 are theoretical ones, calculated from Equation 2.
TRANSCRIPTION
OF
t !. i3 FIG. 7. Kinetics of reassociation of ‘“‘P-labeled HpuI fragments of Ad5 DNA in the presence of calf thyrnus DNA and DNA extracted from digested (20% of acid-soluble material) and undigested with DNase I nuclei of DFKI cells. Hybridization reactions were performed as described in the legend to Fig. 5. n , Kenaturation of [“PjDNA in the presence of calf thyrnus DNA; 0, renaturation of [ “I’]DNA in the presence of DNA extracted from digested with DNase I nuclei of DFKl cells; 0, renaturation of [-“P]DNA in the presence of DNA extracted from undigested with DNase I nuclei of DFKl cells. The curves shown in panels 1-6 are theoretical ones, calculated from Equation 2.
VIRAL
SEQUENCES
transformed cells we have analyzed sensitivity of different regions of viral genome to DNAse I. By comparison of the reassociation rates of restriction endonuclease fragments of Ad5 DNA in the presence of DNA extracted from native nuclei of transformed cells, nuclei digested with DNAse I and calf thymus DNA it was possible to determine which regions of Ad5 genome present in transformed cells were preferentially sensitive to DNAse I. The results are shown in Fig. 9. All the cell lines studied have different patterns of DNAse I sensitivity and their own peculiarities, but partial digestion of sequences which are complementary to HpaI-E fragment is common to all these cell lines. For the integrated adenoviral genes it was shown earlier that transcriptional activity correlates with the DNAse I sensitivity of the DNA sequence (it means that transcribed DNA sequences are more sensitive to DNAse I than nontranscribed) (Flint and Weintraub, 1977; Frolova et al., 1978). From this point of view simple explanations of the partial sensitivity of the left region of Ad5 DNA to DNAse I are that not all copies of this fragment are active in transcription, or that only part of the sequences of this region are transcribed. The latter explanation is more probable because of the observation that the renaturation A
FKAdl
2.1 -A
FKAd5
-
357
R
;
c
16
19
38
02
FK August DNA3
62 ----
16
18
03
FIG. 8. Viral DNA sequences in rat cells transformed by adenovirus 5. The number of copes of various segments of Ad5 DNA that are present in diploid quantities of DNA extracted from each of the transformed cell lines is shown. The figures given for DFKI, FKAugustDNA3, and FKAdG cells are taken directly from Table 1 and are calculated assuming that the total sequence of each of the fragments is present in the cells. The number of copies of &a1 fragment C present in each diploid quantity of DNA extracted from FKAdl and FKAd5 cells was calculat.ed on the assumption that only part of the sequences of the fragment are present in the cells (102 and 50’; for FKAdl and FKAd5 cells, respectively).
358
FROLOVA
eaulv./diplold
AND
auont
c:rlDNAii”r,rl E
C
sb
DFK 1
E
C
G
A.0
F
0
FKAuguslDNA3
E
C
G
A+B
F
D Hpaf Ad5 DNA fragments
FIG. 9. Sensitivity of different regions of viral genome present in Ad5transformed cells to DNase I digestion of nuclei. (a) Sensitivity of viral sequences present in FKAd5 cells to DNase I digestion of nuclei. (b) Sensitivity of viral sequences present in DFKl cells to DNase I digestion of nuclei. (c) Sensitivity of viral sequences present in FKAugustDNA3 cells to DNase I digestion. a, The number of copies of various segments of Ad5 DNA that are sensitive to DNase 1 digestion; 0, the number of copies of various segments of Ad5 DNA that are stable to DNase I digestion per diploid quantities of cell DNA.
rate of fragment E in the presence of DNA extracted from nuclei digested with DNAse I deviates from second order kinetics. This suggests that only a part of viral sequences complementary to the E fragment, are present in this cell DNA preparation. This conclusion is supported by recent detailed analysis in which it was established that only half of the left end integrated adenovirus sequences in Ad5transformed hamster cells are in a chromatin conformation that permits transcription to occur (Flint and Weintraub, 1977). We have found that sequences corresponding to the HpaI fragment F in DFKl cells were not sensitive to DNAse I, although nuclear RNA which was complementary to this fragment have been observed in DFKl cells. It may well be that this phenomenon is to be accounted for by
ZALMANZON
the fact that the cell cultures used for this work were asynchronous. In the case when DNA sequences corresponding to HpaI fragment F are transcribed a very short period before cell division, a very small part of cells in population have these sequences in the chromatin conformation which is sensitive to DNAse I. Our data indicate the partial sensitivity of the right regions of Ad5 genome present in DFKl cells to DNAse I digestion. The renaturation rate of HpaI fragment D in the presence of DNA extracted from the nuclei digested with DNAse I does not deviate from the second order kinetic. So, it is likely that not all copies of a particular sequence present in transformed cells are involved in transcription. These possible differences in the sensitivity of copies can suggest the dependence of activity of viral genes on the distribution of them over the host genome. Detailed studies similar to those described above can provide new information about transcriptional activity of viral genes in transformed cells and its possible relationship to the arrangement of viral DNA sequences integrated in host cell DNA. The approach used for study of chromatin conformation of viral genes seems to be valid permiting one to solve the problem of the involvement of the different copies of the same gene in the process of transcription. REFERENCES BOTCHAN, M., TOPP, M., and SAMBROOK, J. (1976). The arrangement of simian virus 40 sequences in the DNA of transformed cells. Cell 9, 269-287. DF.NHARDT, D. T. (1966). A membrane filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23, 641-646. FLINT, S. J., SAMRROOK, J., WILLIAMS, J. F., and SHARP, I’. A. (1976). Viral nucleic acid sequences in transformed cells. IV. A study of the sequences of adenovirus 5 DNA and RNA in four lines of adenovirus 5-transformed rodent cells using specific fragments of the viral genome. Virology 72,456-470. FLINT, S. J., and SHARP, I’. A. (1976). Adenovirus transcription. V. Quantitation of viral RNA sequences in adenovirus 2.infected and transformed cells. J. Mol. Bid. 106, 749-771. FLINT, S. J., and WEINTRAUB, H. (1977). An altered subunit configuration associated with the actively transcribed DNA of integrated adenovirus genes.
TRANSCRIPTION
OF
Cell 12, 783-794. FHOI,OVA, I3. I., ZAI,MANZON, E. S., LUCANIDIN, E. M., and GEOHGIEV, G. P. (1978). Studies on the transcription of viral genome in adenovirus 5-transformed cells. Nut. Acid. Res. 5, l-11. GALLIMORE, P. H., SHARP, P. A., and SAMBROOK, J. (1974). Viral DNA in transformed cells. II. A study of the sequences of adenovirus 2 DNA in nine lines of transformed rat cells using specific fragments of the viral genome. J. Mol. Biol. 89, 49-72. GAREI., A., and AXEL, R. (1976). DNase I preferentially digests active chromatin. Proc. Nut. Acad. Sci. USA 73, 3966-3970. GELB, L. D., KOHNE, D. IX., and MARTINE, M. A. (1971). Quantitation of simian virus 40 sequences in African Green Monkey, mouse and virus-transformed cells. J. Mol. Biol. 57, 129-145. GRAHAM, F. L., AHRAHAMS, I’. .J., MUI,DER, C.. HEIJNEKER. H. L.. WARNAAR, S. O., DE VRIES. F. A. J., FIERS. W., and VAN DER En. A. J. (1974). Studies on in c’itro transformation by DNA and DNA fragments of human adenoviruses and simian virus 40. Cold Spring Harbor Symp. Quant. Riol. 39,
638-650. GREEN. M., and PINA. M. (1963). Biochemical studies on adenovirus multiplication. IV. Isolation, purification and chemical analysis of adenovirus. Virology 20, 199-207. LEWY, B. W., and DIXON, G. H. (1977). Renaturation kinetics of cDNA complementary to cytoplasmic polyadenylated RNA from rainbow trout testis. Accessibility of transcribed genes to pancreatic DNase. Nucl. Acid. Res. 4, 883-898. LONBERG-HOLM, K., and PHILIPSON, L. (1969). Early events in virus-cell infection in an adenovirus system. J. Viral. 4, 323-338. I’ETTEKSSON, U.. and QAMHKOOK, J. (1973). The amount of viral DNA in the genome of cells trans-
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SEQUENCES
359
formed by adenovirus type 2. J. Mol. Bid. 73, 125-130. SAMBROOK, J., BOTCHAN, M., GAI,I,IMORE, H. P., OZANNE, H., PETTERSSON, U., WILLIAMS, J., and SHARP, 1’. A. (1974). Viral DNA sequences in cells transformed by simian virus 40, adenovirus type 2 and adenovirus type 5. Cold Spring Harbor Svmp. Qunnt. Biol. 39, 615-632. SCHERRER, K. (1969). Isolation and sucrose gradient analysis of RNA In “Fundamental Techniques in Virology” (K. Habel and N. I’. Zalzman, eds.), pp. 413-432. Academic Press, New York. SHARP, P. A., SUGDEN, B., and SAMBROOK, .J. (1974a). Detection of two restriction endonuclease activities in H. pwainfluenrae using analytical agarose-ethidium bromide gel electrophoresis. Biochemistry 12, 3055-3063. SHARP, I’. A., PETTERSSON, U., and SAMHROOK, d. (19741)). Viral DNA in transformed cells. I. A Study of the sequences of adenovirus 2 DNA in a line of transformed cells using specific fragments of the viral genome. J. Mol. Bid. 86, 709-726. SoHER, H. led.) (1968). “Handbook of Biochemistry”, pp. 14-58. Cleveland Rubber Co., Cleveland, Ohio. QOUTHE~N, E. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. ,J. Mol. Bid. 98, 503-518. WEINTRAUB, H., and GROUI)I~‘F:, M. (1976). Active and inactive conformation of chromatin subunits. Science 193, 848-8.56. YOSHIMOKY. Ii. N. (1971). Ph.D. thesis. 1Jniversity of California, San Francisco, Calif. ZAI.MANZON, K. S., FROLOVA, E. I.. SAVINA, A. A., RICHTER, B., TURETSKAYA, R. I,.. and BOBHOVA, N. R. (1978). Isolation and characterization of seven lines of rat embryo cells transformed by adenovirus type five and its DNA. Moleculnmqvn biologicl, USSR, in press.
89, 347-359
Transcription
(1978)
of Viral
Sequences
in Cells Type
E. I. FROLOVA Institute
of Molecular
Biology,
Academy
by Adenovirus
5 E. S. ZALMANZON
AND
Accepted
Transformed
of Sciences May
of the
USSR,
Moscou~,
USSR
8, 1978
‘I’he kinetics of renaturation of ‘“P-labeled restriction endonuclease fragments of adenovirus 5 DNA were measured in the presence of calf thymus DNA and DNA extracted from five lines of adenovirus 5-transformed cells. The number of copies of different fragments per diploid quantity of cell DNA was calculated. All five transformed cell lines contained sequences homologous to the HpaI fragment E, but varied in the other adenovirus 5 DNA sequences. Thus, the sequences which lie on the left-hand end of adenovirus 5 DNA must specify any viral functions required for the maintenance of the transformed cell phenotype. Transcription of viral sequences in three lines of adenovirus 5-transformed cells was studied by hybridization of [ “I’]RNA from transformed cell nuclei with restriction endonuclease fragments of adenovirus 5 DNA which were attached to a nitrocellulose filter. Almost all viral DNA sequences present in transformed cells were transcribed into nuclear RNA. Preferential digestion of transcribed viral sequences with DNase I was analyzed using kinetics of renaturation of “P-labeled specific fragments of adenovirus 5 DNA in the presence of DNA extracted from DNase I treated nuclei of transformed cells. Only part of the sequences of HpaI fragment E comprising the left 47’ of the viral genome was sensitive to DNase I digestion in all lines of transformed cells which were studied. All viral sequences prcscnt in the DFKl ccl1 line were partially sensitive to DNase I digestion. INTRODUCTION
Knowledge of the structure and transcription of the integrated viral genomes in transformed cells is necessary for understanding of the mechanisms of cell transformation and regulation of gene expression in eukaryotic cells. Viral genomes in transformed cell DNA usually are investigated using restriction fragments of viral DNA by measuring the increase in their reassociation rates by t,ransformed DNA (Gelb et al., 1971). Using this technique it has been shown that no line of rat and hamster cells transformed by group C adenoviruses examined contains a complete copy of the viral genome and the extent of viral sequences and the number of copies present per diploid quantity of transformed cell DNA varies in different cell lines (Gallimore et al., 1974; Sambrook et al., 1974; Sharp et al., 1974b; Flint et al., 1976). The segment of viral DNA comprising the left hand 12% of the viral DNA was
common to all adenovirus transformed cells. Studies on the transcription of adenovirus genes in transformed cells demonstrated that only a portion of the viral sequences which represent the early adenoviral genes are transcribed (Flint et al., 1976). From this type of analysis it is not clear whether all the copies of a certain sequence are active in transcription, however, with a few exceptions, the number of RNA transcripts of a given sequence closely parallels the number of copies of viral DNA, from cell line to cell line (Flint and Sharp, 1976). In this paper we described the experiments which have been performed using five lines of Ad5transformed cells. Four of them (DFKl, FKAdl, FKAd5, and FKAd6) were independently isolat,ed after infection of rat primer embryonic fibroblasts by Ad5 and one was obtained after transformation of rat embryo cells by Ad5 DNA. The biological characteristics of these lines (growth properties, sensitivity to the repetitive Ad5 0042~6822/78/0892-0347$02.00/O Copyright 0 19% hy Academic
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348
FHOLOVA
AND
ZALMANZON
were isolated, digested with DNase I (Worthington) as described by Weintraub and Groudine (1976) and DNA from native and DNase I digested nuclei was isolated (Weintraub and Groudine, 1976). Transformed cell DNA was isolated as described by Gallimore et al. (1974). RNA. [““PIRNA was extracted from isolated nuclei of Ad5-transformed cells and purified by the method of Scherrer (1969). Restriction endonucleases. Endonuclease Eco RI was isolated from Escherichia coli RY-13 (Yoshimory, 1971). HpaI was prepared from Hemophilusparainfluenzae according to Sharp et al. (1974a). Preparation of specific fragments of Ad 5 DNA. HpaI restriction fragments of Ad5 DNA were separated by electrophoresis on 0.7 x 30 cm cylindrical gels of 0.7% agarose (Flint et al., 1976) and prepared as described by Gallimore et al. (1974). Combined restriction of Ad5 DNA by HpaI and Eco RI was made in incubation mixture containing 10 mM Tris-HCl, pH 7.4, 10 mM MgC12,6 n&f KCl, 1 mMdithiothreito1, and 5 mM NaCI. Resulting fragments were sepMATERIALS AND METHODS arated by electrophoresis on a 0.4 x 12 x Cells. Lines of rat embryo cells trans- 15 cm slab gel containing 1% agarose. formed by Ad5 were grown in glass roller Radioactive labeling of DNA. HpaI rebottles (New Brunswick) in Eagle’s mini- striction fragments of Ad5 DNA were lamal essential medium (MEM), supple- beled to specific activities of l-8 x 10’ mented with 10% calf serum. KB cells for cpm/pg using four deoxy[n-“‘Plnucleotide cultivation of Ad5 virus were grown in sus- triphosphates (250 Ci/mmol, Amersham) pension cultures in MEM supplemented and DNA-polymerase I (Fraction VII, with 5% calf serum. Boehringer Mannheim Biochemicals) (Bot‘QP-labeled cells were prepared in the than et al., 1976). The reaction was termifollowing manner: cells were washed three nated by the addition of sodium dodecyl times during 1 hr and one time during 12 hr sulfate (SDS) and EDTA to final concenwith phosphate-free MEM, containing 25 trations of 0.5% and 0.01 M, respectively. mM Hepes and 1% dialyzed calf serum and The radioactive Ad5 DNA HpaI fragments then incubated for 4 hr in the same medium were freed from proteins by two extractions with phenol and separated from unincorwith 500 &i per ml of H:,‘“PO, (carrier-free, porated deoxynucleotide triphosphates by Amersham). Virus. Ad5 was propagated in suspension passagethrough a column (0.5 x 20 cm) of G-50 Sephadex equilibrated with 0.01 M culture of KB cells at an input multiplicity of IO plaque-forming units (PFU) per cell. Tris-HCl, pH 7.7, 0.001 M EDTA. Further Virus was purified by a modification of the separation was obtained by chromatogramethod of Green and Pina (1963) (Lonberg- phy on hydroxylapatite. Eluates from hyHolm and Philipson, 1969). droxylapatite column (in 0.4 M Na-phosphate, pH 6.8) were then used for reassociaViral DNA. Ad5 DNA was extracted from purified virions as described (Petters- tion kinetic experiments. Blotting. A gel containing DNA fragson and Sambrook, 1973). Cell DNA. Nuclei from transformed cells ments was immersed in 0.2 M NaOH and infection, T-antigen content, oncogenicity for newborn rats, etc.) were described in more detail elsewhere (Zalmanzon et al., 1978). In the present work, we examined viral DNA sequences in these cell lines by reassociation kinetics and determined the number of copies of different fragments of Ad5 DNA per diploid quantity of cell DNA. Transcribed viral sequences in these cell lines were investigated by analysis of labeled nuclear RNA transcripts. As it has been shown for globin gene (Weintraub and Groudine, 1976), ovalbumin gene (Garel and Axel, 1976) and total structural genes (Lewy and Dixon, 1977) the actively transcribed sequences have an altered chromatin conformation that reflected by preferential digestion of these DNA sequences by DNAse I. We used mild DNAse I digestion of transformed cell nuclei to study chromatin conformation, possibly reflecting transcriptional activity, of different regions of the integrated viral genomes in four lines of Ad5-transformed cells.
TRANSCRIPTION
OF
0.6 A4 NaCl for 45 min at room temperature and neutralized in 1.0 M Tris-HCl, pH 7.4, and 0.6 M NaCl for a further period of 45 min (Botchan et al., 1976). The DNA was then transferred onto a sheet of nitrocellulose (B6, Schleicher and Schull) using the method described by Southern (1975). The filter containing the DNA was washed with 2 x SSC, allowed to dry in air for 1-2 hr and heated in vacua at 80” for 4 hr. HLybridization conditions. DNA:RNA filter hybridization was carried out at 65” for 16 hr in 6 X SSC, 0.5% SDS, 0.001 M EDTA. Before hybridization a strip of nitrocellulose filter (4 mm x 11 cm) with HpaI + Eco RI Ad5 restriction fragments was soaked in a solution containing 0.02% FicoIl, 0.02% bovine serum albumine, and 0.02% polyvinylpyrrolidone dissolved in 2 x SSC (Denhardt, 1966) for a period of 4-5 hr at 65”. The filter was then incubated under the above conditions with 10 x 10” cpm of [“‘P]RNA from the nuclei of transformed cells in a volume 0.2 ml. Following hybridization the nitrocellulose filter was washed with 2 x SSC, incubated in 7 M urea, 2 x SSC, 0.5% SDS at 42” for a period of 2 hr, again washed with 2 x SSC, incubated with 10 pg/ml RNase at 37” for a period of 40 min, again washed with 2 x SSC, air-dried, mounted on Whatman 3M paper and subjected to autoradiography (Noscreen film, type NS-5T, Kodak) for 1 week. DNA:DNA hybridization was carried out at 68” in 1.0 M NaCl buffered with 0.14 M Na-phosphate, pH 6.8. Before annealing both viral and cellular DNAs were degraded to oligonucleotides of 300 bases in length by boiling in 0.3 M NaOH for 20 min. DNA extracted from nuclei of transformed cells was degraded to oligonucleotides with the average size of 150 bases in length by boiling in 0.3 M NaOH for 30 min. DNA from nuclei digested with DNase I had the same average length, as judged by sedimentation in the alkaline sucrose gradient (data not shown). Samples taken after different times of incubation at 68” were diluted lofold in 0.14 M Na-phosphate, pH 6.8, and stored at 4’ until assayed by chromatography on hydroxylapatite (Gallimore et al., 1974; Sharp et al., 1974). Calculations. The derivation of equa-
VIRAL
349
SEQUENCES
tions and the methods of calculation have been described in detail elsewhere (Sharp et al., 1974b). The time required for 50% of the “‘P-labeled probe of DNA to reanneal in the presence of calf thymus DNA (t, ,2p) and in the presence of transformed cell DNA (t,J was calculated from Equation 1: tll:! (or tl12p) =
t
l/f+ - 1
[II
where hh is the fraction of [,“P]DNA remaining single stranded at time t. The quantity of viral DNA per diploid quantity of transformed cell DNA was calculated as follows: the average value of tl,2p/tl,a was calculated and multiplied by the quantity of ““P-labeled viral DNA per milliliter of hybridization solution. Knowing the concentration of transformed cell DNA initially added to this mixture, the number of copies of each fragment of Ad5 DNA per diploid quantity of cell DNA (3.9 x 10’” daltons) (Sober, 1968) can be readily calculated. Theoretical curves for the renaturation kinetics of [“‘P]DNA in conditions where transformed cell DNA contain sequences homologous to only a fraction of probe were derived from Equation 2: l/f5 =
1 X (r + l)t/tllrp
1-x + 1 + t/t,,zp + 1
121
where X is the fraction of the “‘P-labeled probe assumed to be present in the transformed cells and r is the molar ratio of these viral sequences in cell DNA to those of the ““P-labeled viral DNA probe (Sharp et al., 1974b). RESULTS
Adenovirus 5 DNA sequences in transformed cells. We have digested Ad5 DNA with restriction endonuclease HpaI to obtain specific fragments of the viral genome (Fig. 1). With these fragments we have assayed for the presence of Ad5 DNA sequences in five lines of transformed cells. Four of the lines (FKAdl, FKAd5, FKAd6, and DFKl) are independent viral transformants of rat embryo cells. The remaining one (FKAugustDNA3) is a line of rat
350
3 -ID3 : * EC3 j ! Ad 5 3 8; _
FROLOVA
4
b x
I
I
AND
=1
Ecdl-J
FIG. 1. Eco RI and HpaI cleavage sites on adenovirus 5 DNA. The positions of the two Eco RI and the six HpaI cleavage sites on Ad5 DNA are shown (1 and 2, respectively), and HpaI+EcoRI cleavage sites are shown (3), the solid horizontal line depicting the viral genome. These data are taken from Flint et al. (1976).
embryo cells transformed by Ad5 DNA. Before using specific HpaI fragments of Ad5 DNA in an analysis of the viral DNA sequences present in DNA from transformed cells, it was necessary to demonstrate that all fragments were capable of annealing to viral DNA sequences. The conditions in which ““P-labeled probe DNA was annealed in the presence of transformed cell DNA were closely reproduced in reconstruction experiment in which the hybridization mixtures contained a large amount of unrelated, control cell DNA and a small amount of viral DNA. The data obtained were treated as described in Materials and Methods to give the values shown in Table 1. The rates of reannealing of all “P-labeled fragments were accelerated in the presence of unlabeled Ad5 DNA by an amount close to that expected from the quantity of viral DNA added. Thus we concluded that the HpaI fragments of Ad5 DNA reannealed at rates proportional to their initial molar concentrations in the reaction mixture. The effect of transformed cell DNA on the reassociation kinetics of the “‘P-labeled fragments can be used to determine the concentration of each fragment in DNA extracted from transformed cells. We measured the rates of reannealing of “2P-labeled specific fragments of Ad5 DNA in the presence of DNA extracted from Ad5-transformed cells, and calf thymus DNA. Figure 2 shows the kinetics observed with the ““P-labeled HpaI fragments of Ad5
ZALMANZON
DNA in the presence of DNA extracted from FKAdl and FKAd5 lines of Ad5transformed cells. The rates of reannealing of A + B, D, F, and G fragments in the presence of DNA extracted from transformed cells and calf thymus DNA are the same. However, DNA extracted from transformed cells increases the rate of reannealing of E and C fragments compared with the control DNA. From the map positions of the fragments (Fig. 1) it is clear that these lines of transformed cells do not contain any viral sequences that lie to the right of HpaI fragment C. The DNA of ‘“P-labeled HpaI fragment E reanneals in the presence of transformed cell DNA with apparent second-order kinetics, but the renaturation rate of HpaI fragment C deviates from second order in the presence of DNA extracted from FKAdl and FKAd5 cells. TABLE
1
RENATUHATION OF Ad5 DNA ~~~_____. Fragment
OF .‘?-LABELED HpaI FRAGMENTS IN THE PRESENCE OF UNLARELED ADENOVIRUS 5 DNA” .~.___ Adenovirus 5 DNA concentra(0 g/t, 2) tion (&ml x 10 ‘) ~-___
A+B c D E F G
_______~. 23.6 + 0.24 8.83 + 0.43 5.37 +_ 0.15 2.71 + 0.24 3.13 -c 0.11 2.05 + 0.25
Calculated from Added to hyobserved rate of bridization reannealing reaction 2.57 2.40 2.72 3.01 3.09 2.92
2.65 2.65 2.65 2.65 2.65 2.65
” ?-labeled HpaI fragment (specific activity 3.0-6.0 x 10’ cpm/pg) were mixed with the quantity of unlabeled Ad5 DNA shown in column 4, and hybridization reactions were performed as described in legend to Fig. 2. Six samples were withdrawn at various times during the renaturation reaction and the fraction of [‘“P]DNA remaining single-stranded was detected by chromatography on hydroxylapatite. The ratio of the time required for half of the r”PlDNA of restriction endonuclease fragments of Ad5 DNA to reanneal in the absence and presence of unlabeled Ad5 DNA was determined for each time point using Equation 1. These ratios were used to calculate average values of ( tl,Lp/tlr2) and their standard deviations (column 2). From the increase in the rate of hybridization of the “P-labeled probe DNAs in the presence of unlabeled, viral DNA the amount of adenovirus 5 DNA in each reaction mixture was calculated.
TRANSCRIPTION
OF
VIRAL
SEQUENCES
351
The increases in rates of renaturation of HpaI fragments of Ad5 DNA in the presence of DNA extracted from FKAd6 cells compared with calf thymus DNA and the number of copies of different fragments per diploid quantity of FKAd6 DNA are shown 0.4 014 in Table 2. Thus, just as transformed cell lines de!lpo I-G 46 scribed previously contain the segment of viral DNA that maps at the extreme left3 hand end of the Ad5 genome (Flint et al., 2 1976), so the three cell lines which were investigated carry only this partial copy of 1 a viral sequences. 5,5 :c I,5 2,o The results of an experiment in which tit, the kinetics of renaturation of specific HpaI IP fragments of Ad5 DNA were measured in FIG. 2. Kinetics of reassociation of ““P-labeled the presence of DNA extracted from DFKl HpaI fragments of Ad5 DNA in the presence of calf and FKAugustDNAS cells and in the presthymus DNA and DNA extracted from the Ad&transformed cell lines, FKAdl and FKAd5. “P-labeled fragence of calf thymus DNA are shown in Fig. ments of Ad5 DNA were prepared as described under 3 and Table 2. Clearly DNA extracted from Materials and Methods. The concentrations of calf these cells increases the rate of reannealing thymus DNA and FKAdl and FKAd5 DNAs were 1.0 of all labeled HpaI fragments, so the cells and 1.5 mg/ml, when the probes were HpaI fragments carry not only the left end of Ad5 genome A-F and HpaI fragment G, respectively. The concenbut almost all regions of it. The magnitude trations of the “P-labeled probe DNAs were HpaI of the increases in the renaturation rates fragments A + B, 6.80 x 10 ’ pg/ml; HpaI fragment produced by cell DNA varied from fragC, 6.39 x 10 ’ pg/ml; HpaI fragment D, 5.99 x 10-j ment to fragment and from cell line to cell pg/ml; HpaI fragment E, 7.05 x 10 ’ pg/ml; HpaI line. For example, DFKl cells contain the fragment F, 5.80 X 1O-J pg/ml; HpaI fragment G, 6.40 x 10 ’ Fg/ml. Before hybridization cellular and viral highest amount of viral DNA per diploid DNAs were degraded by boiling in 0.3 M NaOH for 20 quantity of cell DNA: 1.4, 2.0, 4.5, 4.1, 3.1 min, to lengths of about 300 nucleotides. Hybridization copies of HpaI fragments A + B, C, D, E, reactions contained l.OMNaCI, 0.14MNa-phosphate, F, and G, respectively. Although, unlike pH 6.8, and 0.4% sodium dodecyl sulfate, and were other known Ad5-transformed cell lines, incubated at 68”, samples being withdrawn at intervals DFKl cells seem to contain a full set of for analysis by chromatography on hydroxylapatite. viral genes, they never produce infectious W, Renaturation of [ r’P]DNA in the presence of calf virus, so it is possible that more detailed thymus DNA, 0, renaturation of [‘jLP]DNA in the investigation will detect some deletions in presence of FKAd5 DNA at concentrations listed viral DNA sequences present in DFKl above for each probe; 0, renaturation of [“PlDNA in the presence of FKAdl DNA. The curves shown in cells. panels l-6 are theoretical ones, calculated from EquaViral RNA sequences in Ad5-transtion 2 (see text). formed cells. Viral nuclear RNA sequences in the three lines of Ad5-transformed cells The curves in Fig. 2 (panel 2) are theoretical (FKAd5, DFKl, and FKAugustDNAS) curves calculated on the premise that dipwere assayed on nitrocellulose filters by loid transformants contain only a part of hybridization of [:‘“P]RNA extracted from the total sequences of HpaI fragment C. the nuclei of transformed cells to specific Because of the good fit between the experendonuclease restriction fragments of unimental data and the theoretical curves we labeled Ad5 DNA. Such assay allows the conclude that FKAdl and FKAd5 cells condetection of fragments which are completain sequences homologous to about 10%~ mentary to the nuclear RNA sequences of and 50% of the HpaI fragment C, respectransformed cells. For hybridization expertively. iments we have used specific fragments
352
FROLOVA
AND
ZALMANZON
TABLE
RENATURATIONOF HpaI Cell line
2
FRAGMENTSOF Ad5DNA INTHE~RESENCEOF DNAs LINESOFRATCELLSTRANSFORMEDBYADENOVIRUS~" Fragment
2. FKAd5
3. FKAd6
4. DFKl
5. FKAugustDNA3
A+B C D E F G A+B C D E F G A+B C D E F G A+B C D E F G A+B C D E F G
____~
Equivalent/diploid DNA assuming Complete ment
1. FKAdl
EXTRACTEDFROMFIVE
1.04 1.41 1.13 1.97 0.93 0.98 1.04 3.34 1.15 2.25 1.13 0.91 2.09 2.26 0.95 3.64 1.09 1.32 8.24 4.85 5.23 2.35 2.66 2.31 2.52 3.98 2.80 3.07 1.65 1.76
* zk + f f f f f f f f f -c f f f + f f +f -c + f f -t -+ f -c f
0.07 0.15 0.14 0.34 0.12 0.20 0.31 0.18 0.14 0.31 0.28 0.29 1.18 0.86 0.13 1.01 0.47 0.12 2.42 1.64 1.45 0.79 0.43 0.16 0.35 0.73 0.58 0.17 0.04 0.08
0.0 0.2 0.0 2.7 0.0 0.0 0.0 1.3 0.0 3.8 0.0 0.0 0.2 0.7 0.0 7.7 0.0 1.0 1.4 2.0 4.5 4.0 4.1 3.1 0.3 1.6 1.9 6.2 1.6 1.8
seg-
quantity of ceH sequences homologous to Percentage of the fragment 2.7(10%)
h
3.8(50?;)’
n Hybridization reactions were performed as described in legend to Fig. 2. The specific activity of the “Plabeled probe DNA ranged from 3.0 to 6.0 x 10’ cpm/pg. Six samples were withdrawn at various times during the renaturation reaction and the fraction of [,“PlDNA remaining single-stranded was determined by chromatography on hydroxylapatite. For all probe DNAs, the data were treated as follows; the ratio of the time required for half of the ‘“P-labeled probe DNA to reanneal in the presence of calf thymus DNA (tromp) and in the presence of transformed cell DNA (tlir) was determined at each time point using Equation 1. These ratios were used to calculate average values of (t,/p/t, L) and their standard deviations (column 3). From the increase in the rate of reannealing of the “‘P-labeled probe DNAs in the presence of transformed cell DNA, the number of equivalents of the sequences of each of the probe DNAs in a diploid quantity of rat DNA (3.9 X IO’” daltons) was calculated assumming that the transformed cells contained all of the sequences of the probe DNA. ‘These values are derived from the theoretical curves, calculated as described in the text, shown in the panels of Fig. 2.
which were obtained after simultaneous digestion of unlabeled Ad5 DNA with HpaI and Eco RI restriction endonucleases. After treatment of Ad5 DNA with these two en-
zymes we were able to separate in 1% agarose slab gels HpaI-A and two main fragments which Eco RI produced from the HpaI-B fragment (X and Eco RI-C).
OF VIRAL
TRANSCRIPTION tlpa I- 0
43 3 2 1 l!!!i4
0.q
0,e
1:1,,
/~p;p$fy
353
SE:QUF,NCES
different viral DNA sequences before and after mild DNAse I digestion of isolated nuclei of transformed cells. Specific fragments of Ad5 DNA generated by cleavage with restriction endonuclease HpaI have been used to assay for the presence of the Ad5 DNA sequences in different DNA preparations from transformed cells. We measured the rate of reannealing of “‘P-labeled HpaI fragments in
II 0,:
C.8
O,?
0.8
c.5 l,o 1.5 2,0
FIG. 3. Kinetics of reassociation of ‘“P-labeled HpI fragments of Ad5 DNA in the presence of calf thymus DNA and DNA extracted from the Ad5-transformed cell lines, DFKI and FKAugustDNA3. Hybridization reactions were performed as described in the legend to Fig. 2. W, Renaturation of [ “PlDNA in the presence of calf thymus DNA; 0, renaturation of [“I’JDNA in the presence of DFKl DNA at concentrations listed at the legend to Fig. 2 for each probe; 0, renaturation of [‘“PJDNA in the presence of FKAugustDNA3 DNA. The curves shown in panels l-6 are theoretical ones, calculated from Equation 2.
Figure 4 depicts the results of such an experiment. Nuclear RNAs extracted from any of cell lines are complementary to the HpaI fragments E and C. Nuclear RNA from FKAd5 cells is complementary to only these DNA fragments, e.g., both viral DNA sequences detected by methods described above. After hybridization of “‘Plabeled nuclear RNA from DFKl and FKAugustDNA3 cells to the fragments of At15 DNA radioactive spots were localized at the positions of all fragments which were transferred to the filter (Fig. 4, b and c). Thus, nuclear RNAs from the cells which carry not only the left-hand end of Ad5 DNA but practically all regions of Ad5 genome hybridize to the fragments which represent not only the left-hand end of Ad5 DNA. Sensitivity of viral DNA sequences to DNAse I digestion of nuclei of transformed cells. In order to study chromatin conformation of viral sequences in transformed cells we measured the concentrations of
D
Eco RI-C
E F a
b 4. Hybridization
C
d
of “P-labeled nuclear RNA extracted from transformed cells to Ad5 DNA fragments generated by restriction endonucleases HpoI and Eco RI. Ad5 DNA was digested with &a1 and Eco RI enzymes and electrophoresed through a 1% agarose gel as described under Materials and Methods. The DNA bands were photographed under ultraviolet light in the presence of 0.5 pg/ml ethidium bromide. The fragments were transferred to nitrocellulose strips, and then hybridized to 1-3 x 10’ cpm of “I’labeled nuclear RNA from transformed cells. Radioactivity was detected by autoradiography. Autoradiograms of the nitrocellulose strip after hybridization to labeled FKAd5 RNA (a); DFKI RNA (b); and FKAugustDNAY (cl. (d) Photograph of the ethidium bromide fluorescence of Ad5 HpaI+Eco RI fragments separated in a 1% agarose gel (HpaI-G and Y fragments have run off the gel). FIG.
354
FHOLOVA
AND
the presence of DNA extracted from native nuclei of transformed cells and compared it with the rate of reannealing of the same fragments in the presence of equivalent amounts of DNA extracted from the nuclei of transformed cells which were digested with DNAse I. The renaturation rates of the “2P-labeled viral DNA fragments in the presence of calf thymus DNA were used as controls. Such assays permit the detection of a preferential sensitivity or resistance of specific portions of the integrated adenovirus DNA to DNase I. The results of renaturation experiments are shown in Table 3 and in Figs. 5-7. Figure 5 depicts reannealing of ““P-labeled HpaI fragments of Ad5 DNA in the presence of different DNA preparations from FKAd5 cells. There is an evident difference between the rates of reassociation of HpaI fragments C and E in the presence of two preparations of DNA extracted from transformed cells. Thus, the sequences of viral DNA present in transformed cells which are complementary to the C and E fragments are sensitive to DNase I digestion. Because the rate of renaturation of labeled HpaI fragments C in the presence of DNA extracted from DNase I digested nuclei of FKAd5 cells and calf thymus DNA are almost equal, we conclude that practically all sequences of HpaI fragment C present in FKAd5 nuclei are completely sensitive to DNase I. The sequences which are complementary to the HpaI fragment E are present in DNA extracted from DNase I digested nuclei in detectable amounts. This means that some of these sequences in the FKAd5 cell DNA are stable to DNase I treatment. The number of copies of DNase I stable sequences of HpaI fragment E per diploid quantity of FKAd5 DNA was calculated (Table 3). DNA extracted from the DNase I digested nuclei of FKAugustDNA3 cells increases the rate of renaturation of HpaI-E fragment only. The sequences of the other Ad5 DNA fragments present in this cell line are completely sensitive to DNase I digestion of nuclei (Fig. 6). The results of renaturation experiments with DNA extracted from DNase I treated and untreated nuclei of DFKl cells are
ZALMANZON
shown in Fig. 7. The sequences complementary to all HpaI fragments of Ad5 DNA were detected in DNA extracted from digested nuclei of DFKl cells. There were no differences in the rates of reannealing of HpaI fragments F and G in the presence of DNA extracted from native and digested nuclei of DFKl cells, so these fragments were stable to DNase I digestion. The partial sensitivity of the sequences complementary to the HpaI fragments A + B, C, D, and E was detected. The number of copies of HpaI fragments which were stable to DNase I digestion of nuclei per diploid quantity of cell DNA in this line presented in Table 3. The sequences of Hpa I fragment E present in transformed cells were partially sensitive to DNase I treatment of nuclei from all cell lines studied. DISCUSSION
In experiments reported here we have studied viral genomes, their transcription and chromatin conformation in several lines of Ad5-transformed rat embryo cells. The HpaI restriction endonuclease fragments of Ad5 DNA have been used as probes of different regions of Ad5 genome to define viral DNA sequences present in transformed cell DNA. The results are summarized in Fig. 8 which shows both regions of the Ad5 genome found in FKAdl, FKAd5, FKAd6, DFKl, and FKAugustDNA3 cells and the frequency per diploid quantity of DNA at which they occur. Three lines of Ad5-transformed cells (FKAdl, FKAd5, and FKAd6) are similar to the known Ad2- and Ad5transformed cell lines. All of them carry only a part of the viral genome and those viral DNA sequences present map in the left-hand end of the Ad5 DNA. DFKl and FKAugustDNA3 cells are reminiscent of some Ad2-transformed cell lines which contain sequences from almost all regions of viral genome but in different numbers of copies. All Ad5-transformed cell lines contain sequences homologous to the HpaI fragments E and C, but they vary in the content of other Ad5 DNA fragments. The FKAdl cells contain the minimal fraction of viral genome known (6%). Although the
TRANSCRIPTION
OF
FRAGMENTS WITH DNase
OF Ad5 I NUCLEI
VIRAL
TABLE RENATUKATION I)IGESTEII
OF ?LAREI,ED AND UNIIIGESTED
3 DNA
IN THF: PRMENCF, OF DNAs EXTRACTED OF THREE LINES OF RAT CF.I,LS TRANSFORMED
ADENOVIR~JS
DNA
1. 1)NA from digested with clei of FKAd5 cells
DNase
DNA from undigested with nuclei of FKAd5 cells
2. DNA from digested with clei of DFKI cell cells
I nu-
DNase
DNase
DNA from undigested with nuclei of DFKI cells
I
I nu-
DNAse
I
FKOM BY
5”
Fragment
preparation
355
SEQUENCES
Equivalent/diploid quantity of cell DNA, assuming sequences homologous to complete fragment
A+B C D E F G A+B C D E F G
0.0 0.2 0.0 1.3 0.0 0.0 0.0 1.2 0.0 4.1 0.0 0.0
A+B C D E F G A+B C D E F G
0.4 0.7 1.2 2.0 3.7 3.0 1.2 2.2 4.1 3.8 3.8 2.7
f
3. DNA from digested clei of FKAugust
with DNase DNA3 cells
DNA from undigested FKAugustDNA3 cells
I nu-
nuclei
0 Hybridization reactions were labeled probe DNA ranged from the renaturation reaction and the raphy on hydroxylapatite. For all
of
A+B C D E F G A+B C D E F G
1.13 0.96 0.92 3.03 1.09 0.93 3.60 6.32 4.53 4.88 1.68 1.62
f 0.25 f 0.18 f 0.24 t 0.64 F 0.18 f 0.11 it 0.54 f 0.78 +- 0.18 rf; 1.03 + 0.09 AC 0.15
0.0 0.0 0.0 3.5 0.0 0.0 0.2 1.3 2.1 6.7 1.1 2.0
performed as described in the legend to Fig. 5. The specific activity of the ‘“P3.0 to 6.0 x 10’ cpm/pg. Six samples were withdrawn at various times during fraction of [ “P]DNA remaining single-stranded was detected by chromatogprobe DNAs, the data were treated as described in the legend to Table 2.
minimal fragment found earlier in group C adenovirus transformed cells comprised left-hand 12% of viral DNA (Flint et al., 1976) our results are consistent with those of Graham et al. (1974) who were able to transform cells with the 7% of the left-hand
adenovirus DNA fragment. So any viral functions required for the maintenance of the transformed cell phenotype are encoded by about 6-7s of the left end of Ad5 DNA. The nature of the observed differences in the content of various viral sequences in
356
FROLOVA
I:. 64
0.8
c.4
0.8
05I
AND
: :I 10 I '5, 20,
!tt, ip
5. Kinetics of reassociation of “Plabeled Hpd fragments of Ad5 DNA in the presence of calf thymus DNA and DNA extracted from digested and undigested with DNase I nuclei of FKAd5 cells. ,“I’labeled fragments of Ad5 DNA were prepared as described in Materials and Methods. The concentration of calf thymus DNA and DNAs extracted from FKAd5 digested and undigested nuclei was 1.5 m&ml. The concentrations of the “P-labeled probe DNAs were HpuI fragments A+B, 4.08 x 10 ’ &ml; H,nnI fragment C, 4.36 X IO ’ pg/ml: HpnI fragment D, 5.05 X 10 ’ &ml; HpnI fragment E. 6.10 x 10 ’ &ml; Hpnl fragment F, 5.80 x 10 ’ pg/ml; HpnI fragment G, 6.53 x IO ’ pg/ml. Nuclei of FKAdS cells were digested with 20 &ml DNase I until 20’:: of DNA became acid soluble. DNA from undigested nuclei and DNA which was stable to 1)Nase I digestion were extracted and used in hybridization experiments. Before hybridization viral DNA and DNA extracted from the undigested with DNase I nuclei were degraded by boiling in 0.3 M NaOH for :JO min. to lengths of about 150 nucleotides. Hybridization reactions contained 1 .O M NaCl, 0.14 M Na-phosphate, pH 6.8, and 0.40 sodium dodecyl sulfate, and were incubated at 68”. samples being withdrawn at intervals for analysis hv chromatography on hydroxylapatite. n . Kenaturation of [“I’]DNA in the presence of calf thymus DNA; 0, renaturation of [“I’]DNA in the presence of DNA extracted from digested with DNase I nuclei of FKAd5 cells; Q, renaturation of [ “I’JDNA in the presence of DNA extracted from undigested with 11Nase 1 nuclei of FKAd5 cells. The curves shown in panels 1-6 are theoretical ones, calculated from Equation 2. FIG.
different But we forming FKAd6 infection of Ad5
transformed cell lines is not clear. can suggest the role of the transviral DNA-FKAdl, FKAd5, and cell lines which were obtained after of the cells with the same stock have more similarities in the pat-
ZALMANZON
tern of HpaI fragments with each other than with the lines obtained with another stock of virus (DFKl) or viral DNA (FKAugustDNAS). Nuclear RNA transcripts which were complementary to the Hpa-E and HpaI-C fragments of Ad5 DNA were found in the nuclei of all cell lines. Although the maintenance of transformation in adenovirus transformed cells is though to be coded by 6-7s of the Ad5 genome and mapped at the extreme Ieft end of Ad5 genome, in some transformed cell lines (DFKI, FKAugustDNA3) not only is the extreme left end of the genome transcribed, but nuclear RNA from these cell lines is complementary to all viral DNA fragments corresponding to the viral DNA sequences of the cell line detected by the renaturation experiments. In order to study the chromatin conformation of the integrated viral sequences in
FIG. 6. Kinetics of reassociation of “I’-labeled HpaI fragments of Ad5 DNA in the presence of calf thymus DNA and DNA extracted from digested (2O’i; of acid-soluble material) and undigested with DNase I nuclei of FKAugustDNA3 cells. Hybridization reactions were performed as described in the legend to Fig. 5. n , Renaturation of [‘“P]DNA in the presence of calf thymus DNA; 0, renaturation of [,‘“P]DNA m the presence of DNA extracted from digested with DNase I nuclei of FKAugustDNAY cells; 0, renaturation of r”P]DNA in the presence of DNA extracted from undigested with DNase I nuclei of FKAugustDNA3 cells. The curves shown in panels l-6 are theoretical ones, calculated from Equation 2.
TRANSCRIPTION
OF
t !. i3 FIG. 7. Kinetics of reassociation of ‘“‘P-labeled HpuI fragments of Ad5 DNA in the presence of calf thyrnus DNA and DNA extracted from digested (20% of acid-soluble material) and undigested with DNase I nuclei of DFKI cells. Hybridization reactions were performed as described in the legend to Fig. 5. n , Kenaturation of [“PjDNA in the presence of calf thyrnus DNA; 0, renaturation of [ “I’]DNA in the presence of DNA extracted from digested with DNase I nuclei of DFKl cells; 0, renaturation of [-“P]DNA in the presence of DNA extracted from undigested with DNase I nuclei of DFKl cells. The curves shown in panels 1-6 are theoretical ones, calculated from Equation 2.
VIRAL
SEQUENCES
transformed cells we have analyzed sensitivity of different regions of viral genome to DNAse I. By comparison of the reassociation rates of restriction endonuclease fragments of Ad5 DNA in the presence of DNA extracted from native nuclei of transformed cells, nuclei digested with DNAse I and calf thymus DNA it was possible to determine which regions of Ad5 genome present in transformed cells were preferentially sensitive to DNAse I. The results are shown in Fig. 9. All the cell lines studied have different patterns of DNAse I sensitivity and their own peculiarities, but partial digestion of sequences which are complementary to HpaI-E fragment is common to all these cell lines. For the integrated adenoviral genes it was shown earlier that transcriptional activity correlates with the DNAse I sensitivity of the DNA sequence (it means that transcribed DNA sequences are more sensitive to DNAse I than nontranscribed) (Flint and Weintraub, 1977; Frolova et al., 1978). From this point of view simple explanations of the partial sensitivity of the left region of Ad5 DNA to DNAse I are that not all copies of this fragment are active in transcription, or that only part of the sequences of this region are transcribed. The latter explanation is more probable because of the observation that the renaturation A
FKAdl
2.1 -A
FKAd5
-
357
R
;
c
16
19
38
02
FK August DNA3
62 ----
16
18
03
FIG. 8. Viral DNA sequences in rat cells transformed by adenovirus 5. The number of copes of various segments of Ad5 DNA that are present in diploid quantities of DNA extracted from each of the transformed cell lines is shown. The figures given for DFKI, FKAugustDNA3, and FKAdG cells are taken directly from Table 1 and are calculated assuming that the total sequence of each of the fragments is present in the cells. The number of copies of &a1 fragment C present in each diploid quantity of DNA extracted from FKAdl and FKAd5 cells was calculat.ed on the assumption that only part of the sequences of the fragment are present in the cells (102 and 50’; for FKAdl and FKAd5 cells, respectively).
358
FROLOVA
eaulv./diplold
AND
auont
c:rlDNAii”r,rl E
C
sb
DFK 1
E
C
G
A.0
F
0
FKAuguslDNA3
E
C
G
A+B
F
D Hpaf Ad5 DNA fragments
FIG. 9. Sensitivity of different regions of viral genome present in Ad5transformed cells to DNase I digestion of nuclei. (a) Sensitivity of viral sequences present in FKAd5 cells to DNase I digestion of nuclei. (b) Sensitivity of viral sequences present in DFKl cells to DNase I digestion of nuclei. (c) Sensitivity of viral sequences present in FKAugustDNA3 cells to DNase I digestion. a, The number of copies of various segments of Ad5 DNA that are sensitive to DNase 1 digestion; 0, the number of copies of various segments of Ad5 DNA that are stable to DNase I digestion per diploid quantities of cell DNA.
rate of fragment E in the presence of DNA extracted from nuclei digested with DNAse I deviates from second order kinetics. This suggests that only a part of viral sequences complementary to the E fragment, are present in this cell DNA preparation. This conclusion is supported by recent detailed analysis in which it was established that only half of the left end integrated adenovirus sequences in Ad5transformed hamster cells are in a chromatin conformation that permits transcription to occur (Flint and Weintraub, 1977). We have found that sequences corresponding to the HpaI fragment F in DFKl cells were not sensitive to DNAse I, although nuclear RNA which was complementary to this fragment have been observed in DFKl cells. It may well be that this phenomenon is to be accounted for by
ZALMANZON
the fact that the cell cultures used for this work were asynchronous. In the case when DNA sequences corresponding to HpaI fragment F are transcribed a very short period before cell division, a very small part of cells in population have these sequences in the chromatin conformation which is sensitive to DNAse I. Our data indicate the partial sensitivity of the right regions of Ad5 genome present in DFKl cells to DNAse I digestion. The renaturation rate of HpaI fragment D in the presence of DNA extracted from the nuclei digested with DNAse I does not deviate from the second order kinetic. So, it is likely that not all copies of a particular sequence present in transformed cells are involved in transcription. These possible differences in the sensitivity of copies can suggest the dependence of activity of viral genes on the distribution of them over the host genome. Detailed studies similar to those described above can provide new information about transcriptional activity of viral genes in transformed cells and its possible relationship to the arrangement of viral DNA sequences integrated in host cell DNA. The approach used for study of chromatin conformation of viral genes seems to be valid permiting one to solve the problem of the involvement of the different copies of the same gene in the process of transcription. REFERENCES BOTCHAN, M., TOPP, M., and SAMBROOK, J. (1976). The arrangement of simian virus 40 sequences in the DNA of transformed cells. Cell 9, 269-287. DF.NHARDT, D. T. (1966). A membrane filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23, 641-646. FLINT, S. J., SAMRROOK, J., WILLIAMS, J. F., and SHARP, I’. A. (1976). Viral nucleic acid sequences in transformed cells. IV. A study of the sequences of adenovirus 5 DNA and RNA in four lines of adenovirus 5-transformed rodent cells using specific fragments of the viral genome. Virology 72,456-470. FLINT, S. J., and SHARP, I’. A. (1976). Adenovirus transcription. V. Quantitation of viral RNA sequences in adenovirus 2.infected and transformed cells. J. Mol. Bid. 106, 749-771. FLINT, S. J., and WEINTRAUB, H. (1977). An altered subunit configuration associated with the actively transcribed DNA of integrated adenovirus genes.
TRANSCRIPTION
OF
Cell 12, 783-794. FHOI,OVA, I3. I., ZAI,MANZON, E. S., LUCANIDIN, E. M., and GEOHGIEV, G. P. (1978). Studies on the transcription of viral genome in adenovirus 5-transformed cells. Nut. Acid. Res. 5, l-11. GALLIMORE, P. H., SHARP, P. A., and SAMBROOK, J. (1974). Viral DNA in transformed cells. II. A study of the sequences of adenovirus 2 DNA in nine lines of transformed rat cells using specific fragments of the viral genome. J. Mol. Biol. 89, 49-72. GAREI., A., and AXEL, R. (1976). DNase I preferentially digests active chromatin. Proc. Nut. Acad. Sci. USA 73, 3966-3970. GELB, L. D., KOHNE, D. IX., and MARTINE, M. A. (1971). Quantitation of simian virus 40 sequences in African Green Monkey, mouse and virus-transformed cells. J. Mol. Biol. 57, 129-145. GRAHAM, F. L., AHRAHAMS, I’. .J., MUI,DER, C.. HEIJNEKER. H. L.. WARNAAR, S. O., DE VRIES. F. A. J., FIERS. W., and VAN DER En. A. J. (1974). Studies on in c’itro transformation by DNA and DNA fragments of human adenoviruses and simian virus 40. Cold Spring Harbor Symp. Quant. Riol. 39,
638-650. GREEN. M., and PINA. M. (1963). Biochemical studies on adenovirus multiplication. IV. Isolation, purification and chemical analysis of adenovirus. Virology 20, 199-207. LEWY, B. W., and DIXON, G. H. (1977). Renaturation kinetics of cDNA complementary to cytoplasmic polyadenylated RNA from rainbow trout testis. Accessibility of transcribed genes to pancreatic DNase. Nucl. Acid. Res. 4, 883-898. LONBERG-HOLM, K., and PHILIPSON, L. (1969). Early events in virus-cell infection in an adenovirus system. J. Viral. 4, 323-338. I’ETTEKSSON, U.. and QAMHKOOK, J. (1973). The amount of viral DNA in the genome of cells trans-
VIRAL
SEQUENCES
359
formed by adenovirus type 2. J. Mol. Bid. 73, 125-130. SAMBROOK, J., BOTCHAN, M., GAI,I,IMORE, H. P., OZANNE, H., PETTERSSON, U., WILLIAMS, J., and SHARP, 1’. A. (1974). Viral DNA sequences in cells transformed by simian virus 40, adenovirus type 2 and adenovirus type 5. Cold Spring Harbor Svmp. Qunnt. Biol. 39, 615-632. SCHERRER, K. (1969). Isolation and sucrose gradient analysis of RNA In “Fundamental Techniques in Virology” (K. Habel and N. I’. Zalzman, eds.), pp. 413-432. Academic Press, New York. SHARP, P. A., SUGDEN, B., and SAMBROOK, .J. (1974a). Detection of two restriction endonuclease activities in H. pwainfluenrae using analytical agarose-ethidium bromide gel electrophoresis. Biochemistry 12, 3055-3063. SHARP, I’. A., PETTERSSON, U., and SAMHROOK, d. (19741)). Viral DNA in transformed cells. I. A Study of the sequences of adenovirus 2 DNA in a line of transformed cells using specific fragments of the viral genome. J. Mol. Bid. 86, 709-726. SoHER, H. led.) (1968). “Handbook of Biochemistry”, pp. 14-58. Cleveland Rubber Co., Cleveland, Ohio. QOUTHE~N, E. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. ,J. Mol. Bid. 98, 503-518. WEINTRAUB, H., and GROUI)I~‘F:, M. (1976). Active and inactive conformation of chromatin subunits. Science 193, 848-8.56. YOSHIMOKY. Ii. N. (1971). Ph.D. thesis. 1Jniversity of California, San Francisco, Calif. ZAI.MANZON, K. S., FROLOVA, E. I.. SAVINA, A. A., RICHTER, B., TURETSKAYA, R. I,.. and BOBHOVA, N. R. (1978). Isolation and characterization of seven lines of rat embryo cells transformed by adenovirus type five and its DNA. Moleculnmqvn biologicl, USSR, in press.