JOURNAL OF VIROLOGY, Aug. 1978, p. 307-312
Vol. 27, No. 2
0022-538X/78/0027-0307$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Presence of Free Viral DNA in Simian Virus 40-Transformed Nonproducer Cells L. DAYA-GROSJEAN AND R. MONIER*
Institut de Recherches Scientifiques sur le Cancer, 94800 Villejuif, France Received for publication 11 April 1978
Extracts from several simian virus 40 (SV40)-transformed nonproducer cells prepared by the hot-phenol procedure normally used to extract cellular RNA. These extracts contained SV40 infectious units. Part of the infectious units were identified as SV40 form I DNA molecules. The results of reconstruction experiments suggest that SV40 form I DNA is extractable by the hot-phenol procedure because of its fast renaturation rate. The significance of the presence of free viral DNA in nonproducer transformed cells is discussed. were
Simian virus 40 (SV40)-transformed cells usually contain a variable amount of virus-specific genetic information integrated into the cell genome (2, 10). In their classical study on the integrated state of the SV40 genome in transformed nonpermissive mouse cells, Sambrook et al. (11) failed to detect the simultaneous presence of free viral DNA molecules. During the course of a recent study on the transfection of permissive monkey cells with cellular DNA preparations extracted from nonproducer transformed cells, we considered it necessary to reexamine the matter. Although most of the transformed cells at our disposal apparently did not contain infectious free viral DNA molecules that could be extracted by the conventional Hirt procedure (5), we observed that the use of an unorthodox technique, namely extracting the cells in the presence of phenol at 600C (12), enabled us to detect free viral DNA in most of the transformed cell lines that we examined. Part of the infectious units have been identified as SV40 form I DNA. The significance of these observations with respect to the mechanism of extraction and to the state of the viral genome in the transformed cells is discussed.
rat cells are lung epithelioid cells transformed in vitro with SV40 form I DNA (Lasne et al., manuscript in preparation). With the exception of the CHK-SVLP clone 5.1 cells, which are known to produce significant amounts of viral DNA and virions (7), none of the transformed cells produced virions at the passages used in this study, as shown by titration of cell extracts on CV-1 cells. All the nonproducer clones have been tested for their ability to produce SV40 virions after fusion with permissive CV-1 cells. Infectious SV40 was rescuable from all of them. Monkey CV-1 cells were used for virus or viral DNA infection. All cells were grown in Eagle minimal medium, supplemented with 10% tryptose broth and 10% calf serum (SORGA). Preparation of viral DNA. CV-1 cells were infected at a multiplicity of infection of 0.001 PFU/cell with SV40 strain LP (14). Viral DNA was selectively extracted by the Hirt technique and purified by equilibrium sedimentation in cesium chloride-ethidium bromide gradients according to published procedures
(5,8).
Preparation of linear SV40 DNA. Form I SV40 DNA was treated with restriction endonuclease EcoRI in 0.1 M Tris-hydrochloride (pH 7.5)-10 mM MgCl2 for 1 h at 37°C. Linear DNA was separated by electrophoresis on 1.4% agarose gels run in 40 mM Trishydrochloride (pH 7.8)-20 mM sodium acetate-2 mM MATERIALS AND METHODS EDTA buffer and further purified by sedimentation in Cells. TSV-5 clone 2 Syrian hamster cells were a cesium chloride-ethidium bromide gradient as dederived from a tumor induced by SV40 injection, as scribed before. Extraction of transformed cells in the presence described by Tournier et al. (15). CHK-SVLP clone 5.1 Chinese hamster cells are a subclone of Chinese of phenol at 60°C. This extraction was carried out as hamster kidney cells transformed in vitro as described described by Girard (4). In brief, approximately 109 by Lavialle et al. (7). MKSV clone liD mouse cells cells were lysed with 0.5% sodium dodecyl sulfate in are a subclone of mouse kidney cells transformed in 100 ml of 10 mM EDTA-10 mM sodium acetate (pH vitro (16). V5 F4 clones 1 and 4 rat cells are lines of 5.1). The lysate was extracted with an equal volume of newborn rat kidney cells transformed in vitro with buffer-saturated phenol at 60°C for 3 min with vigorSV40 form I DNA by Lasne et al. (C. Lasne, L. Daya- ous shaking. The aqueous phase and the interphase Grosjean, P. Nardeux, I. Chourouinkov, and R. Mon- were reextracted twice with phenol for 2 min at 60°C. ier, manuscript in preparation). Vii Fl clones 1 and 3 The final aqueous phase was precipitated at -20°C 307
308
J. VIROL.
DAYA-GROSJEAN AND MONIER
with 2 volumes of 95% ethanol. For direct titration, the precipitate was redissolved in lx SSC (0.15 M NaCl-0.015 M sodium citrate), treated with RNase (see below), deproteinized with phenol, and dialyzed against lx SSC. Cs2SO4 equilibrium sedimentation. The material extracted with phenol at 600C was redissolved in 1 mM Tris-hydrochloride (pH 7.4)-l mM EDTA and dialyzed against the same buffer. The solution was mixed with an equal volume of saturated Cs2SO4, and sedimentation was performed in a Spinco SW50 rotor at 37,000 rpm for 60 h at 150C. The refractive index of each fraction was read, and fractions corresponding to DNA or RNA densities (1.47 g/cm3 and 1.65 g/cm3, respectively) were pooled and dialyzed against lx ssc.
Radioactive labeling of viral DNA during a productive infection. Confluent CV-1 cells were infected at a multiplicity of infection of 25 PFU/cell. [3H]thymidine (specific activity, 25 Ci/mmol; 10 ,zCi/ml) was added 24 h after infection. Radioactively labeled viral DNA was extracted at the indicated times either by the Hirt procedure (5) or with phenol at 600C as described above. Analysis of [H]DNA extracted from productively infected cells. This analysis was perfonned by sedimentation on 5 to 20% sucrose density gradients prepared in 0.7 M NaCl-0.3 N NaOH-1 mM EDTA in a Spinco SW50 rotor at 50,000 rpm for 110 min at 40C. Fractions of 0.2 ml were collected and counted in toluene-PPO (2,5-diphenyloxazole)-POPOP [1,4-bis(5-phenyloxazolyl)benzene] scintillation liquid in a Packard counter. Enzymatic treatments of the material extracted from transformed cells. Pancreatic RNase (Calbiochem, grade A, 5x crystallized) (10 mg/ml in water) was preincubated at 900C for 10 min. The material extracted with phenol at 600C from transformed cells was dialyzed against SSC (pH 7.0). Pancreatic RNase was added at a final concentration of 100 ,ug/ml, and the mixture was incubated for 1 h at 370C. Digestion with DNase I (Worthington, RNase free) was carried out in 10 mM Tris-hydrochloride (pH 8.0)-60 mM MgCl2-10 mM EDTA at 370C for 1 h at a final enzyme concentration of 100 ,g/ml. At the end of the incubation periods, the digested material was deproteinized with phenol and ethanol precipitated before further analysis. Plaque assay of material extracted from transformed cells. Hot-phenol extracts were dissolved in lx SSC and dialyzed against the same buffer before assay. Plaque titration was carried out on confluent 60-mm CV-1 plates (106 cells per plate) by adding 0.2 ml of extract per plate containing 1 mg of DEAEdextran per ml according to the procedure of Kit et al. (6). The activity of the samples titrated is either expressed as PFU per milliliter or converted to viral DNA molecules per cell extracted. The conversion was done on the basis of a control titration in which known quantities of purified SV40 DNA were titrated (as described above) in the presence of phenol extracts of 109 untransformed cells. The average specific infectivity obtained for form I SV40 DNA was 105 PFU/jLg. Identification of infectious material in trans-
formed cell extracts. (i) SV40 was identified in plaques obtained after titration on CV-1 cells by infection of CV-1 cover-slip cultures, which were then examined for the presence of SV40 T antigen. After fixation, the cover-slip cultures were washed with phosphate-buffered saline and treated with hamster anti-SV40 tumor serum for 60 min at 370C. The cover slips were then rinsed three times with phosphatebuffered saline and treated for 30 min at 370C with an anti-hamster fluorescent serum (Pasteur Institute). After further washing in phosphate-buffered saline, the cover-slip cultures were mounted in buffered glycerine and examined for fluorescent nuclei. (ii) SV40 form I DNA was identified in transformed cell extracts by equilibrium sedimentation in cesium chloride-ethidium bromide gradients (5, 8). Fractions from the gradients were analyzed, after dialysis against lx SSC, by transfection on CV-1 clone 1 cells. The cultures were inoculated for 30 min at 370C in the presence of 1 mg of DEAE-dextran per ml and incubated for 12 days in fresh medium at 370C. The cells were then disrupted by three alternate cycles of quick freeze and thawing, the cell debris was removed by centrifugation, and the supernatant fluid was tested, for each fraction, for infectious SV40 by plaque assay.
RESULTS
Extraction of SV40 infectious units from Syrian hamster TSV-5 clone 2 by the hotphenol procedure. When extracts from Syrian hamster TSV-5 clone 2 cells were tested for infectivity on CV-1 cells, plaques with characteristic SV40-like morphology were regularly detected. They were identified as SV40 plaques by showing that upon reinfection with the material present in a few of them, CV-1 cells became positive for the specific SV40 nuclear T antigen as shown by indirect immunofluorescence. The number of PFU recovered from 109 cells varied from experiment to experiment (Table 1). Identification of the infectious units as SV40 DNA. To identify the chemical nature of the SV40 infectious units present in hot-phenol extracts from TSV-5 clone 2, a number of tests were performed, the results of which are described in Table 2. After addition of NaCl to 1 M final concentration at 00C, an abundant precipitate, containing most of the high-molecular-weight RNA present in the extract, was collected by sedimentation. All of the infectivity in the original extract remained in the supematant, i.e., behaved as DNA or low-molecular-weight RNAs (Table 2). When the infectious material was banded in Cs2SO4, all of the infectivity was recovered at the density of DNA and none in the RNA-contaiing fractions (Table 2). Finally, the infectious material was sensitive to DNase and insensitive to RNase (Table 2). RNase treatment actually produced an increase in the infectivity of the extract. The initial ex-
VOL. 27, 1978
FREE VIRAL DNA IN SV40-TRANSFORMED CELLS
309
TABLE 1. SV4O infectivity ofhot-phenol extracts from transformed cells DNA Cell line molecules/ no. celLs cell' 128 70 131 13 138 2 7 x 10-3 TSV-5 clone 55 227 35 526 45 536 20
TSV-5 clone 2 cells were analyzed by equilibrium sedimentation in cesium chloride-ethidium bromide gradients, which allows the separation PasagePFU1(?SV40 of circular supercoiled DNA molecules from linear or relaxed-circle DNA owing to their difference in density. The gradients were collected, and each fraction was tested for infectivity as described in Material and Methods. Infectious SV40 was recovered partly in the fractions banding at the density of form I DNA and partly in the form m DNA fractions (Fig. 1). We can therefore conclude that at least a portion of the 24 12 infectivity in the phenol extracts is due to DNA 29 0 molecules that are circular supercoils. 30 0 MKSV clone ll/D 5 x 10-3 Efficiency of the hot-phenol method in 35 50 50 37 extracting SV40 form I DNA. To further confirm that the hot-phenol extraction procedure is 2 able to selectively extract SV40 form I DNA 2 x 10-2 V5 F4 clone 1 38° from cells, reconstruction experiments were performed. Known amounts of SV40 form I or form 11 200 2 x 10-2 V5 F4 clone4 III DNA, expressed in terms of PFU, were added to CV-1 cells. The mixtures were then extracted either with the conventional Hirt procedure or 0 32 0 21 0 Vll Fl clone 3 with the hot-phenol procedure, and the re0 0 321 covered SV40 DNA was titrated on CV-1 cells (Table 3). The hot-phenol procedure proved to 0 0 321 Vll Fl clone 3 be quite efficient in selectively extracting small amounts of form I DNA, whereas it extracted CHK-SVLP clone 43 10 1 form HI DNA with a very low efficiency. On the 5.1 other hand, the Hirt procedure was equally ef'As calculated from the highest value of PFU in ficient in extracting both DNA forms. previous column. SV40-infected CV-1 cells, labeled with [3H]were also extracted with the conventhymidine, TABLE 2. Characterization of infectious unit in hotphenol extracts of TSV-5 clone 2 Determination PFU/mn1 High-salt precipitation (1 M NaCI) Initial extract .......................... 1.5 Precipitate ............................ 0.0
Supernatant ...........................
1.0
Equilibrium sedimentation in Cs2SO4 RNA fraction .......................... DNA fraction ..........................
0.0 3.0
Sensitivity to nucleases Initial extract ..........................
3.0 Extract after RNase treatment ...... .... 9.0 Extract after DNase treatment ...... .... 0.0 In all instances, the titrated solution was adjusted to a constant volume, and plaque titration was performed as described in the text.
tracts always contained a large amount of RNA, which either could compete with DNA for entry into the cells or be cytotoxic. From these tests, we conclude that the SV40 infectious units are DNA molecules. To further characterize these molecules, extracts from
Fraction No
FIG. 1. A phenol extract of TSV-5 clone 2 cells was analyzed on a cesium chloride-ethidium bromide gradient. Fractions from the gradient were transfected on CV-I cells, and, after 12 days ofincubation, lysates for each fraction were titrated by plaque assay. A part of the actiuvity was recovered in the region banding at the density of form I DNA, the remainder in the region ofform III DNA fractions.
310
DAYA-GROSJEAN AND MONIER
J. VIROL.
TABLE 4. Alkaline sucrose gradient analysis of tional Hirt procedure and with the hot-phenol procedure, and the quantities of DNA extracted CHithymidine-labeled DNA extracted from infected CV-I cells by the Hirt and hot-phenol methodse were compared by analysis on alkaline sucrose cpm of labeled DNA peak by procedure": gradients. Three radioactive peaks were de- Time tected in both types of extracts: a minor 16 to after Hot phenol Hirt 18S peak, which contains cellular DNA and infection SV40 single-strand circles and linears; a major 18S 53S
Vol. 27, No. 2
0022-538X/78/0027-0307$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Presence of Free Viral DNA in Simian Virus 40-Transformed Nonproducer Cells L. DAYA-GROSJEAN AND R. MONIER*
Institut de Recherches Scientifiques sur le Cancer, 94800 Villejuif, France Received for publication 11 April 1978
Extracts from several simian virus 40 (SV40)-transformed nonproducer cells prepared by the hot-phenol procedure normally used to extract cellular RNA. These extracts contained SV40 infectious units. Part of the infectious units were identified as SV40 form I DNA molecules. The results of reconstruction experiments suggest that SV40 form I DNA is extractable by the hot-phenol procedure because of its fast renaturation rate. The significance of the presence of free viral DNA in nonproducer transformed cells is discussed. were
Simian virus 40 (SV40)-transformed cells usually contain a variable amount of virus-specific genetic information integrated into the cell genome (2, 10). In their classical study on the integrated state of the SV40 genome in transformed nonpermissive mouse cells, Sambrook et al. (11) failed to detect the simultaneous presence of free viral DNA molecules. During the course of a recent study on the transfection of permissive monkey cells with cellular DNA preparations extracted from nonproducer transformed cells, we considered it necessary to reexamine the matter. Although most of the transformed cells at our disposal apparently did not contain infectious free viral DNA molecules that could be extracted by the conventional Hirt procedure (5), we observed that the use of an unorthodox technique, namely extracting the cells in the presence of phenol at 600C (12), enabled us to detect free viral DNA in most of the transformed cell lines that we examined. Part of the infectious units have been identified as SV40 form I DNA. The significance of these observations with respect to the mechanism of extraction and to the state of the viral genome in the transformed cells is discussed.
rat cells are lung epithelioid cells transformed in vitro with SV40 form I DNA (Lasne et al., manuscript in preparation). With the exception of the CHK-SVLP clone 5.1 cells, which are known to produce significant amounts of viral DNA and virions (7), none of the transformed cells produced virions at the passages used in this study, as shown by titration of cell extracts on CV-1 cells. All the nonproducer clones have been tested for their ability to produce SV40 virions after fusion with permissive CV-1 cells. Infectious SV40 was rescuable from all of them. Monkey CV-1 cells were used for virus or viral DNA infection. All cells were grown in Eagle minimal medium, supplemented with 10% tryptose broth and 10% calf serum (SORGA). Preparation of viral DNA. CV-1 cells were infected at a multiplicity of infection of 0.001 PFU/cell with SV40 strain LP (14). Viral DNA was selectively extracted by the Hirt technique and purified by equilibrium sedimentation in cesium chloride-ethidium bromide gradients according to published procedures
(5,8).
Preparation of linear SV40 DNA. Form I SV40 DNA was treated with restriction endonuclease EcoRI in 0.1 M Tris-hydrochloride (pH 7.5)-10 mM MgCl2 for 1 h at 37°C. Linear DNA was separated by electrophoresis on 1.4% agarose gels run in 40 mM Trishydrochloride (pH 7.8)-20 mM sodium acetate-2 mM MATERIALS AND METHODS EDTA buffer and further purified by sedimentation in Cells. TSV-5 clone 2 Syrian hamster cells were a cesium chloride-ethidium bromide gradient as dederived from a tumor induced by SV40 injection, as scribed before. Extraction of transformed cells in the presence described by Tournier et al. (15). CHK-SVLP clone 5.1 Chinese hamster cells are a subclone of Chinese of phenol at 60°C. This extraction was carried out as hamster kidney cells transformed in vitro as described described by Girard (4). In brief, approximately 109 by Lavialle et al. (7). MKSV clone liD mouse cells cells were lysed with 0.5% sodium dodecyl sulfate in are a subclone of mouse kidney cells transformed in 100 ml of 10 mM EDTA-10 mM sodium acetate (pH vitro (16). V5 F4 clones 1 and 4 rat cells are lines of 5.1). The lysate was extracted with an equal volume of newborn rat kidney cells transformed in vitro with buffer-saturated phenol at 60°C for 3 min with vigorSV40 form I DNA by Lasne et al. (C. Lasne, L. Daya- ous shaking. The aqueous phase and the interphase Grosjean, P. Nardeux, I. Chourouinkov, and R. Mon- were reextracted twice with phenol for 2 min at 60°C. ier, manuscript in preparation). Vii Fl clones 1 and 3 The final aqueous phase was precipitated at -20°C 307
308
J. VIROL.
DAYA-GROSJEAN AND MONIER
with 2 volumes of 95% ethanol. For direct titration, the precipitate was redissolved in lx SSC (0.15 M NaCl-0.015 M sodium citrate), treated with RNase (see below), deproteinized with phenol, and dialyzed against lx SSC. Cs2SO4 equilibrium sedimentation. The material extracted with phenol at 600C was redissolved in 1 mM Tris-hydrochloride (pH 7.4)-l mM EDTA and dialyzed against the same buffer. The solution was mixed with an equal volume of saturated Cs2SO4, and sedimentation was performed in a Spinco SW50 rotor at 37,000 rpm for 60 h at 150C. The refractive index of each fraction was read, and fractions corresponding to DNA or RNA densities (1.47 g/cm3 and 1.65 g/cm3, respectively) were pooled and dialyzed against lx ssc.
Radioactive labeling of viral DNA during a productive infection. Confluent CV-1 cells were infected at a multiplicity of infection of 25 PFU/cell. [3H]thymidine (specific activity, 25 Ci/mmol; 10 ,zCi/ml) was added 24 h after infection. Radioactively labeled viral DNA was extracted at the indicated times either by the Hirt procedure (5) or with phenol at 600C as described above. Analysis of [H]DNA extracted from productively infected cells. This analysis was perfonned by sedimentation on 5 to 20% sucrose density gradients prepared in 0.7 M NaCl-0.3 N NaOH-1 mM EDTA in a Spinco SW50 rotor at 50,000 rpm for 110 min at 40C. Fractions of 0.2 ml were collected and counted in toluene-PPO (2,5-diphenyloxazole)-POPOP [1,4-bis(5-phenyloxazolyl)benzene] scintillation liquid in a Packard counter. Enzymatic treatments of the material extracted from transformed cells. Pancreatic RNase (Calbiochem, grade A, 5x crystallized) (10 mg/ml in water) was preincubated at 900C for 10 min. The material extracted with phenol at 600C from transformed cells was dialyzed against SSC (pH 7.0). Pancreatic RNase was added at a final concentration of 100 ,ug/ml, and the mixture was incubated for 1 h at 370C. Digestion with DNase I (Worthington, RNase free) was carried out in 10 mM Tris-hydrochloride (pH 8.0)-60 mM MgCl2-10 mM EDTA at 370C for 1 h at a final enzyme concentration of 100 ,g/ml. At the end of the incubation periods, the digested material was deproteinized with phenol and ethanol precipitated before further analysis. Plaque assay of material extracted from transformed cells. Hot-phenol extracts were dissolved in lx SSC and dialyzed against the same buffer before assay. Plaque titration was carried out on confluent 60-mm CV-1 plates (106 cells per plate) by adding 0.2 ml of extract per plate containing 1 mg of DEAEdextran per ml according to the procedure of Kit et al. (6). The activity of the samples titrated is either expressed as PFU per milliliter or converted to viral DNA molecules per cell extracted. The conversion was done on the basis of a control titration in which known quantities of purified SV40 DNA were titrated (as described above) in the presence of phenol extracts of 109 untransformed cells. The average specific infectivity obtained for form I SV40 DNA was 105 PFU/jLg. Identification of infectious material in trans-
formed cell extracts. (i) SV40 was identified in plaques obtained after titration on CV-1 cells by infection of CV-1 cover-slip cultures, which were then examined for the presence of SV40 T antigen. After fixation, the cover-slip cultures were washed with phosphate-buffered saline and treated with hamster anti-SV40 tumor serum for 60 min at 370C. The cover slips were then rinsed three times with phosphatebuffered saline and treated for 30 min at 370C with an anti-hamster fluorescent serum (Pasteur Institute). After further washing in phosphate-buffered saline, the cover-slip cultures were mounted in buffered glycerine and examined for fluorescent nuclei. (ii) SV40 form I DNA was identified in transformed cell extracts by equilibrium sedimentation in cesium chloride-ethidium bromide gradients (5, 8). Fractions from the gradients were analyzed, after dialysis against lx SSC, by transfection on CV-1 clone 1 cells. The cultures were inoculated for 30 min at 370C in the presence of 1 mg of DEAE-dextran per ml and incubated for 12 days in fresh medium at 370C. The cells were then disrupted by three alternate cycles of quick freeze and thawing, the cell debris was removed by centrifugation, and the supernatant fluid was tested, for each fraction, for infectious SV40 by plaque assay.
RESULTS
Extraction of SV40 infectious units from Syrian hamster TSV-5 clone 2 by the hotphenol procedure. When extracts from Syrian hamster TSV-5 clone 2 cells were tested for infectivity on CV-1 cells, plaques with characteristic SV40-like morphology were regularly detected. They were identified as SV40 plaques by showing that upon reinfection with the material present in a few of them, CV-1 cells became positive for the specific SV40 nuclear T antigen as shown by indirect immunofluorescence. The number of PFU recovered from 109 cells varied from experiment to experiment (Table 1). Identification of the infectious units as SV40 DNA. To identify the chemical nature of the SV40 infectious units present in hot-phenol extracts from TSV-5 clone 2, a number of tests were performed, the results of which are described in Table 2. After addition of NaCl to 1 M final concentration at 00C, an abundant precipitate, containing most of the high-molecular-weight RNA present in the extract, was collected by sedimentation. All of the infectivity in the original extract remained in the supematant, i.e., behaved as DNA or low-molecular-weight RNAs (Table 2). When the infectious material was banded in Cs2SO4, all of the infectivity was recovered at the density of DNA and none in the RNA-contaiing fractions (Table 2). Finally, the infectious material was sensitive to DNase and insensitive to RNase (Table 2). RNase treatment actually produced an increase in the infectivity of the extract. The initial ex-
VOL. 27, 1978
FREE VIRAL DNA IN SV40-TRANSFORMED CELLS
309
TABLE 1. SV4O infectivity ofhot-phenol extracts from transformed cells DNA Cell line molecules/ no. celLs cell' 128 70 131 13 138 2 7 x 10-3 TSV-5 clone 55 227 35 526 45 536 20
TSV-5 clone 2 cells were analyzed by equilibrium sedimentation in cesium chloride-ethidium bromide gradients, which allows the separation PasagePFU1(?SV40 of circular supercoiled DNA molecules from linear or relaxed-circle DNA owing to their difference in density. The gradients were collected, and each fraction was tested for infectivity as described in Material and Methods. Infectious SV40 was recovered partly in the fractions banding at the density of form I DNA and partly in the form m DNA fractions (Fig. 1). We can therefore conclude that at least a portion of the 24 12 infectivity in the phenol extracts is due to DNA 29 0 molecules that are circular supercoils. 30 0 MKSV clone ll/D 5 x 10-3 Efficiency of the hot-phenol method in 35 50 50 37 extracting SV40 form I DNA. To further confirm that the hot-phenol extraction procedure is 2 able to selectively extract SV40 form I DNA 2 x 10-2 V5 F4 clone 1 38° from cells, reconstruction experiments were performed. Known amounts of SV40 form I or form 11 200 2 x 10-2 V5 F4 clone4 III DNA, expressed in terms of PFU, were added to CV-1 cells. The mixtures were then extracted either with the conventional Hirt procedure or 0 32 0 21 0 Vll Fl clone 3 with the hot-phenol procedure, and the re0 0 321 covered SV40 DNA was titrated on CV-1 cells (Table 3). The hot-phenol procedure proved to 0 0 321 Vll Fl clone 3 be quite efficient in selectively extracting small amounts of form I DNA, whereas it extracted CHK-SVLP clone 43 10 1 form HI DNA with a very low efficiency. On the 5.1 other hand, the Hirt procedure was equally ef'As calculated from the highest value of PFU in ficient in extracting both DNA forms. previous column. SV40-infected CV-1 cells, labeled with [3H]were also extracted with the conventhymidine, TABLE 2. Characterization of infectious unit in hotphenol extracts of TSV-5 clone 2 Determination PFU/mn1 High-salt precipitation (1 M NaCI) Initial extract .......................... 1.5 Precipitate ............................ 0.0
Supernatant ...........................
1.0
Equilibrium sedimentation in Cs2SO4 RNA fraction .......................... DNA fraction ..........................
0.0 3.0
Sensitivity to nucleases Initial extract ..........................
3.0 Extract after RNase treatment ...... .... 9.0 Extract after DNase treatment ...... .... 0.0 In all instances, the titrated solution was adjusted to a constant volume, and plaque titration was performed as described in the text.
tracts always contained a large amount of RNA, which either could compete with DNA for entry into the cells or be cytotoxic. From these tests, we conclude that the SV40 infectious units are DNA molecules. To further characterize these molecules, extracts from
Fraction No
FIG. 1. A phenol extract of TSV-5 clone 2 cells was analyzed on a cesium chloride-ethidium bromide gradient. Fractions from the gradient were transfected on CV-I cells, and, after 12 days ofincubation, lysates for each fraction were titrated by plaque assay. A part of the actiuvity was recovered in the region banding at the density of form I DNA, the remainder in the region ofform III DNA fractions.
310
DAYA-GROSJEAN AND MONIER
J. VIROL.
TABLE 4. Alkaline sucrose gradient analysis of tional Hirt procedure and with the hot-phenol procedure, and the quantities of DNA extracted CHithymidine-labeled DNA extracted from infected CV-I cells by the Hirt and hot-phenol methodse were compared by analysis on alkaline sucrose cpm of labeled DNA peak by procedure": gradients. Three radioactive peaks were de- Time tected in both types of extracts: a minor 16 to after Hot phenol Hirt 18S peak, which contains cellular DNA and infection SV40 single-strand circles and linears; a major 18S 53S
