The identification of the circular forms of SV40 DNA in whole infected cell preparations

VIROLOGY

62, 395-407

(1973)

The Identification

of the Circular Infected

Forms

Cell

E. H. HARLEY Department

of Biochemical Pathology, University Street,

of SV40

DNA

in Whole

Preparations AND

University London,

J. S. WHITE College Hospital WClE 6J.J

Medical

School,

Accepted December S1, 1472

The supercoiled and nicked circular forms of HV40 virus DNA, in a crude preparation from whole infected cells, have been resolved both from one another and from linear DNA by a simple one-st,age process. This has been achieved using polyecrylamide gel elec’trophoresis, a technique hitherto little applied to the analysis of DNA. The separations are assisted by the phenomenon that high molecular weight linear DNA, even of considerable size heterogeneit,y, is concentrated into a narrow band on gel electrophoresis. A factor which greatly assists resolution of the circular DNA species is the differential effect of varying acrylamide concentration in the gel on the respective electrophoretic mobility of linear as compared with circular forms of DNA. It is proposed that, this approach should serve as a simple and versatile means of identifging circular DNA species in simple whole rell DNA extracts. INTRODUCTION

When DNA is extracted from cell cultures infected with oncogenic DNA viruses, such as SV40 or polyoma, a mixture of viral and cell DNA is obtained and any further analysis requires the separation of viral from cell DNA. Several procedures have been developed to achieve such a separation. Careful extraction by the Hirt procedure results in a fairly pure virus DNA preparation but involves a loss of 20 % of the viral DNA int,o the cell DNA fraction (Hirt, 1967). Column chromatography using methylated albuminkieselguhr is effective in separat#ing polyoma DNA from infected cells, but only after heat denaturation of all forms of DSA other than supercoiled DXA (Sheinin, 1966). One of the most generally useful techniques is isopycnic centrifugation in caesium chloride. This will separate DI\‘A species if they have significantly different base ratios (Vinograd and Hearst, 1962)) but the het,erogeneity of the cell DNA fragments with rcspcct to base ratio often precludes adequate scparat,ion. The use of the intcrcalat-

ing dye cthidium bromide (Waring, 1966) has more recently enabled good preparations of the supercoiled form of circular DNA to be achieved (Radloff et al. 1967), since constraints imposed by the supercoiling result in decreased binding of the dye. Supercoiled DNA will then band in a different position to other forms of DNA on isopycnic centrifugation. Rate zonal centrifugation in alkaline solutions is a useful procedure for scparat,ing viral D?r’A (Vinograd et aE., 196;i), but results in dcnaturation of all except the supercoiled DXA. Although some of thesr t’echniques produce excellent separations of supercoiled DNA, they &her result in denaturation of other forms of DNA, such as nicked circular or linear Drc’A, present in the infected cell, or they are demanding on time and apparatus. Thus they are better suited to USCas prrparative techniques. The most generally used analytical technique for resolving circular viral Dlu’,4 species is rate zonal srdimmtation in neutral densit’y gradients (Crawford, 1963; Dulbecco and Vogt, 1963; Weil and

395 Copyright All rights

0 1973 by Academic Press, of reproduction in any form

Inc. reserved.

Vinograd, 1963). However, this also has some disadvantages: the viral DNA must Ploducfion of cells and virus. C:V-1 monkey first be purified from cell DNA, n-hose kidney cells (American Type eulturo collecheterogeneity would otherwise confuse scdi- tion, CCL 70) were grown in IO-02 rolling mentation profiles, care must be taken to bottles. Confluent monolayers were infected avoid concentration-dependent dist’urbances with the large plaque strain of SV40 virus, in sedimentation rate (Hearst and Vinograd, obtained from Dr. S. Kit, at an input 1961; Studier, 1965), and the differences in multiplicity of 10 PFU/cell in the prcsencc of sedimentation coefficient between nicked 10 &i/ml [methgl-3H]thymidine. Eight days circular and linear viral DNA are not after infect,&, the cells and free virions \vc’rc, sufficient for adequate resolut’ion. first harvested by freezing the medium, and Gel electrophoresis has proved to be an shaking the bottle as the medium thalvcd, excellent, technique for producing fine and then pelleted by centrifugation at analytical separations of some nucleic acid 40,000 rpm for 1.5 hr in the S X 50 ml fixedelectrophoresis in angle rotor of an MSE superspeed 65 ultraspecies. In particular, polyacrylamide gels is now a standard centrifuge. For some experiments, virus leas method for RNA analysis (Richards et al., purified from this pelleted material by 1965; Loening, 1967; Peacock and Dingman, isopycnic banding in a preformed linear 1967). Hayward and Smith (1972) have 20-60% CsCl gradient, centrifuged for 20 achieved good separations of single-stranded hr at 39,000 rpm and 4’ in t’he 3 X 5 ml DNA species in agarose, and agarose-acrylaswing-out bucket rotor of the MSE supermide gels, but they, and Loening (1967), speed 50 ultracentrifuge. Control monohave observed that double-stranded DNA layers were grown and labelled under species separate poorly on gel electrophoreidentical conditions, but without virus sis. In consequence, apart from some degree infection. of success in the separation of satellite The CCL2 strain of HeLa cells were DNA (Zeiger et al., 1971), and of small grown in 10 oe rolling bottles as described by DNA fragments of specific size (Williamson Harley and Rees (1972). 1970; Danna and Nathans, 1971), this Media. CV-1 cells were grown in Eagle’s technique has found little applicat’ion to the Basal medium containing Hank’s salts, study of double-stranded DNA. However, supplemented with 10% foetal bovine, resolution of the circular forms of Polyoma serum. Extra amino acids and vitamins were DNA has been achieved by Thorne (1967), added to bring the final concentration to using agar gel electrophoresis, and by Harley twice normal. HeLa cells were grown in et al. (1972), using polyacrylamide gel Eagle’s Minimal Essential Medium suppleelectrophoresis. mcnted with 10% calf serum, nonessential The purpose of the present investigation amino acids, and tryptose phosphate broth has been to determine whether, and under (0x0). Both media contained 100 units of what conditions, the circular forms of SV40 penicillin and 100 pg of st,reptomycin per DNA can be identified in simple extracts of milliliter of medium. &Iedia concentrates whole infected cells, using polyacrylamide were obtained from Flow Laborat,orirs, gel elcctrophoresis. The results have shown Irvine, Scotland. that the supercoiled and nicked circular Chemicals. All chemicals used in the forms of SV40 DNA can br separated both extract’ion and analysis of DNA were BDH from each other and from linear DNA by “Analar” grade reagents unlrss otherwise this method. Furthermore, by varying the specified. [Methyl-3H]t,hymidim (5 Ci/ concentration of acrylamide in hhe gels, mmolc) and (2-14C]thymidine (50 mCi/ differential mobility characterist,ics arc mmole) n-cre purchased from the Radiobrought to light which will provide addichemical Centre, Amersham, /*England. T. tional criteria for the identification of the Ethidium bromide was a kind gift from ur. and N,S’circular SV40 DNA species in whole cell Watson Fuller. Acrylamide, methyltnebisacrylamide were recrystallized extracts.

GEL ELECTROPHORESIS

as described by Loening (1967). Electrophoretically purified pancreatic deoxyribonuclease I, and pancreatic ribonuclease were purchased from Sigma. Scintillation fluid consisted of 8% naphthalene (Fisons) and 0.4 % BBOT (Ciba) dissolved in a 6.5:3.5 mixture of toluene and 2-methoxyethanol. Extraction of DNA. DNA was extracted from infected and uninfected cells by lysis in a solution of 1% SDS in 1 N sodium perchlorate (pH 7.5) at a concentration of 1 to 2 X 10’ cells/ml. After deproteination by four sequential chloroform extractions, DNA was precipitated by the addition of an equal volume of 95% ethanol, the mixture being left to stand for at least 1 hr at -15”. The DNA was centrifuged down at 9000 g for 10 min and redissolved in sterile SSC. Samples were stored at -70”. To extract DNA from purified virus, CsCl was first removed by dialysis against 0.01 M Tris buffer pH 7.0. The virus suspension was then mixed with an equal volume of 2 % SDS in 2 N sodium perchlorate, gently mixed, then allowed to stand for 10 min. The extraction was then continued as described above. Viral DNA was stored at -70”. Centrifugation. Isopycnic centrifugation was performed in caesium chloride gradients containing 330 pg/ml ethidium bromide. 3H-Labelled DNA was dissolved in 0.01 M Tris pH 7.9 and mixed with CsCl solution to give a final volume of 5 ml and density of 1.5981 g ml-‘. The solution was centrifuged for 48 hr at 40,000 rpm and 20” in the 8 X 14 ml fixed-angle rotor of an MSE superspeed 65 ultracentrifuge. The tube was pierced and 15.set fractions were collected achieving const’ant flow with an LKB varioperpex pump. Aliquots were removed, 5 ~1 for density measurement by refractometry in an Abbe 60 refractometer, and 3 ~1 for radioactivity determination. The regions of the gradient containing the separat’ed DNA species were bulked as illustrated in Fig. 3, and the ethidium bromide was removed by a 72-hr dialysis against, repeated changes of SSC. Rate zonal sedimentation in neutral density gradients was performed as described previously (Harley et al., 1972).

OF SV40 DNA

391

Polyacrylamide gel electrophoresis. Electrophoresis was performed as described by Loening (1967). Monomer solutions were prepared by dilution from a stock 12% w/v acrylamide solution containing 0.6 % w/v N , N’-methylene bisacrylamide; 2.0 % was found to be the lowest acrylamide concentration that would form a gel, provided that polymerization was carried out below 4”. Electrophoresis was performed between 20 and 24” for 4 hr at 12.5 V cm-’ on cylindrical 0.6 X 10 cm gels. A maximum of 5 pg DNA was layered on each gel. Gels were fractionated on a Mickle Laboratories Gel Slicer, and l-mm slices were transferred to plastic scintillation vials. Radioactivity detewninafion. Radioactivity was eluted from gel slices by an 18-hr incubation in 0.2 ml of 1 N HCl followed by at least 18 hr further incubation after the addition of 0.2 ml 2 N ammonium hydroxide solution. The aqueous sample was mixed with 15 ml scintillation fluid, and radioactivity was measured in an Intertechnique ABAC SL40 liquid scintillation spectrometer programmed to compute disintegrations per minute, under variable quench conditions, in a mixed isotope situation. The efficiency of 3H counting is 25 % and that of 14Cis 50 %. Deoxyribrmuclease digestion. Quantities, 1.1 pg, of some 3H-labelled DNA preparations were incubat’ed \vith various concentrations of DNase I for 5 min at 24” in 10 mM NaCl, 1.5 mM MgCL , 10 mM Tris. HCI, pH 7.4 in a total volume of 100 ~1. The reaction was terminated by the addition of 10 ~1 of 10 mM EDTA + 2% w/v SDS; 0.5 tig W-labelled HeLa DNA was then added to each sample. To facilitate layering onto the gels, sufficient glycerol was added tjo give a final concentration of I5 %. RESULTS

Monolayers of CV-1 cells were infected with SV40 virus and labelled with 3H-thymidine as described above. Uninfected monolayers were treated similarly. The DNA extracted from these monolayers was analyzed both by polyacrylamide gel electrophoresis and by rate zonal centrifugation in neutral glycerol density gradient,s. The re-

398

HARLEY

.4NII WHITI,:

b

Slice

No.

FIG. 1. Comparative analysis of 3H-labelled DNA extracted from SV40 infected and uninfected CV-1 cells by polyacrylamide gel electrophoresis and rate tonal centrifugation. Electrophoresis was performed on 2.0y0 gels as described in Materials and Methods, and centrifugation in 14.5 ml of neutral linear glycerol density gradients at 75,000 g for 15 hr at 15”. Electropherograms show DNA profiles from (a) uninfected CV-1 cells and (b) SV40-infected CV-1 cells; (c) and (d) are the corresponding density gradient profiles. The direction of electrophoresis is from left to right, and the direction of sedimentation is from right to left. @---a, 3H disintegrations per minute (dpm). sults, a comparison between the two analytical methods, are presented in Fig. 1. Figures la and c show that the DNA extracted from uninfected cells sediments as a broad band between about 10 and 30 S (Fig. lc), but is concentrated into a single sharp peak on gel electrophoresis (Fig. la). In this experiment, equal quantities of labelled DNA were not layered onto the gel and gradient,

Fraction FIG.

No.

1 c-d.

but when this is done, a full quantitative rccovery of radioactivity is achieved. Quite different electrophoretlc and sedimentation patterns (Figs. lb and d, respectively) are shown by DNA extracted from infected monolayers. In Fig. ld, peaks are discernible but poorly resolved on rate zonal sedimentation, but three peaks are clearly resolved on gel electrophoresis (Fig. lb). The peak occupying an intermediate position in Fig. lb, corresponding in position to the single peak in Fig. la, may represent the host cell linear DNA similarly concentrated into a narrow band. One of the features of most practical significance in Fig. 1 is the way in which the broadly sedimcnting DNA from cont’rol cells is concentrated quantitatively into a single narrow band on gel clrctrophoresis. 14C-Labelled HcLa DNA ITas, therefore, used in some subsequent experiments as a general marker for high molecular

GEL ELECTROPHORESIS

399

OF SV40 DNA

b

.Ij 4

60

Slice FIG. 2. ElectroDhoresis of DNA extracted (c) 3.0% gels. O-0, 3H dpm.

20

80

40

60

20

40

No.

from SV40-infected CV-1 cells on: (a) 2.07’$, (b) 2.50/,,

weight linear DNA species on polyacrylamide gels. It seems likely that the additional peaks of label in Fig. lb represent SV40 DNA, although the possibility remains at this stage that they only represent a modified form of cell DNA. Treatment with pancreatic RNase produces no change in the profiles. A further preparation of DKA from SV40 infected cells, labelled and extracted in a similar way to the above, was analyzed on a range of gels of varying concentration in order to determine the conditions producing optimal separations of the various components. Fig. 2 illustrates the results of electrophoresis on three different concentrations of gel. It is apparent that very different separations are achieved depending on the gel concentration chosen. On 2.0 and 3.0% gels, peaks of label are clearly resolved, in contrast with the poor separation on the gel of intermediate concentrat,ion (2.5 %). At 2.0 %, the secondary peak is running in advance of the main band, and behind on the 3.0% gel. On the 2.5% gel this peak is not resolvrd, but there is a shoulder on the slow running side of the main band, which is absent from the

main bands on the other two gels. In other words, increasing the gel concentration results in a differential effect on the mobility of these various 3H-labelled DNA species. Having established that separations could be achieved on certain concentrations of polyacrylamide gel, it was necessary to identify t,he various species observed. One way to achieve this is to prepare the various species of DlYA known to be synthesized in SV40-infected cells using standard methods, and to subject the separated species to electrophoresis with a known marker DNA. Supercoiled DNA was therefore separated from linear DNA (and nicked circular DNA) by isopycnic centrifugation in CsCl-ethidium bromide. Figure 3 illustrates t’he results of such a separation using the whole infected cell DNA preparation which gave the profiles in Fig. 2. The heavier band corresponds to supcrcoiled DNA and the lighter band corresponds to linear together with nickedcircular DNA (Radloff et al., 1967). DNA was collected from each band as indicated in the figure. To prepare nicked circular DNA the supercoiled preparation was stored at 4” for a few weeks; under these condit,ions a signifi-

HARLEY

AND WHITE

-690 T z 650 E m 610 ,570 ,530

Fraction

z. .L_ e B

No.

FIG. 3. Caesium chIoride/ethidium bromide centrifugation of DNA extracted from SV40-infected CV-1 cells. Centrifugation was for 48 hr at 120,000 g and 20”; 0.27-ml fractions were cut, from which 3-~1 aliquots were taken for radioactivity determination. 5+1 aliquots were removed from alternate fractions for density determination (a.... 0). The square brackets indicate fractions of the gradient bulked for subsequent analyses. a----•,8H dpm.

cant proportion of supercoiled DNA nicks to give rise to the nicked circular form (Crawford and Black, 1964). A second cycle of CsCl-ethidium bromide centrifugation thus enabled pure nicked circular DNA to be collected from the lighter band of the gradient. These separate fractions were then subjected to electrophoresis on 2.0 and 3.0% gels, after mixing each with marker 14C-labelled linear DNA from HeLa cells. The results are illustrated in Fig. 4. The profiles in Fig. 4a and b show that supercoiled DNA migrates faster than the linear [14C]DNA marker on a 2.0 % gel, but slower than the marker on a 3.0 % gel. The linear [3H] DNA, as might be expected, migrates very close to the linear [14C]DNA marker in each case (Figs. 4c and d). The nicked circular DNA gives rise to a peak of label (Fig. 4e and f) migrating more slowly than any of the other labelled peaks at both gel concentrations. The electrophoretic mobility of these species relative to the linear [“C]DNA marker are summarized as Rf

values at each gel concentration in Table I. On the basis of these values, a slo~~ly migrating peak in Figs. 4c and d can now be identified as nicked circular DNA, a species which one would expect to find contaminating this linear DNA preparation. The identification of the “H-labelled linear and nicked circular DNA species in the above argument is to some extent dependent on the assumption that supercoiled DNA undergoes single strand nicking on storage at 4”. To provide additional evidence in support of these conclusions, some further analyses were undertaken. Figure 5a illustrates the results of electrophoresis on a 2.0% gel of DNA extracted from material resulting from a purification procedure intended to remove a proportion of the linear host cell DKA. This was prepared from the same batch of infected cells from which the DNA in Fig. 1 was derived. With much of the host cell linear DNA removed by this procedure the profile should, therefore, show a reduction in size of the 3H peak corresponding to linear DNA. Linear HeLa [14C]DNA was added as a marker. Comparison with Fig. lb (illustrating the corresponding whole cell preparations) shows that the central peak of R, = 1.1 is indeed now much reduced in size. Figure 5b shows the same preparation reanalyzed on a 2.0 % gel after 4 weeks’ storage at 4’ in SSC. The fast migrating peak is much reduced in size, and the slow migrating peak is now the predominant feature of the 3H profile, a picture compatible with a slow conversion on storage of supercoiled to nicked circular DNA. However, the intermediate 3H peak seems also relatively larger than in Fig. 5a. This figure demonstrated the nature of the changes undergone by a preparation of SV40 DNA when stored at 4”. To confirm therefore, that the slowest migrating peak on both 2.0 and 3.0% gels represents nicked circular SV40 DNA, the supercoiled DNA preparation from the CsCIethidium bromide gradient’ was treated with a range of concentrations of pancreatic DNase in order deliberately to introduce single-strand nicks (Vinograd et al., 1965). The digests were performed as described in the Materials and Methods section and the

GEL

ELECTROPHORESIS

401

OF SV40 DNA

b

I , 7

.5

-4

-3

-2

I

i d

.

d

-5

-4

-3

-2

--I

an

FIG. 4. Electrophoresis of DNA fractions prepared from L3HI thymidine labelled SV40 infected CV-1 cells by CsCl/ethidium bromide centrifugation as shown in Fig. 3. Electropherograms show heavy band DNA analyzed on (a) 2.0%, and (b) 3.0% gels, light band DNA analyzed on (c) 2.075, and (d) 3.0% gels, and light band DNA from a second cycle of CsCl/ethidium bromide centrifugation of stored heavy linear HeLa DNA was added to each band DNA, analyzed on (e) 2.0’7& and (f) 3.0% gels. l*C-labelled preparation before electrophoresis. a--@, 3H dpm; Ok.e.0, W dpm.

402

HARLEY

ANI) WHITE

n!-Y? Slice No. FIG.

products of digestion were analyzed on 2.0% gels. The results arc illustrated in Fig. 6; a control digestion wit&out DNase in the incubation mixture gave a profile identical to that in Fig. 4a. As DNase concentration is increased, thcrc is a progressive shift of “Hlabelled material from the faster to the slower migrating peak, until at high DKaw concentration only a small 3H peak remains in the intermediate position corresponding to the 14Clinear DNA marker. This confirms that the slower moving 3H peak represents the nicked circular form of SV40 DNA, and suggests that the small 3H peak in the intermediate position which remains at higher DNase concentration represents linear DNA, since this will bc produced from the circular forms when two single-strand nicks on adjacent strands are close enough together. This peak is not an artifact caused by faulty spillovcr corrections for i4C. A feature requiring further clarification is

4

I

e-f.

TABLE

1

ELECTROPHORBTIC MOBILITIES OF 3H-L~~~~~~~ DNA SPECIIZSFROM SV40 INFWTED CV-1 CELLS RKLATIVE TO 14C LINIZAR DNA INTERNAL MARKERS 1

I?, 3H:‘4C(f SEM) DI\IA

1-p 2.0% Acrylamide

Supercoiled DNA Linear DNA Nicked circular DNA -___

I

1.19 f 0.01 1.00 f 0.01 0.91 xk 0.01

3.0% Acrylamide

l---

0.78 f 1.01 f 0.50 f

0.03 0.01 0.04 -

the variation in the relat,ivc positions of the circular and linear DNA species observed in Figs. 2 and 4. In order to do this the three 3H DNA preparations, supercoiled, linear, and nicked circular, which gave the results illustrated in Fig. 4 were analyzed on a range

GEL

ELECTROPHORESIS

OF SV4O DNA

403

and nicked circular DNA both from each other and from linear forms of DNA, in a one-stage process. In addition, the method is rapid, simple, and requires inexpensive apparatus. Since selectively denaturing conditions such as heat or alkali are not required, all forms of DXA are separated in the nativo state. There is no requirement for intercalating dyes, with the problem of t’heir subsequent removal. One of the main reasons for the success of this method is the phenomenon of the linear, heterogeneous host cell DNA being concentrated into a narrow band on the gel. Quantitative recovery of counts from either 14Clabelled HcLa DNA or 3H-labelled CV-1 DXA was found in this region, and no contamination of other regions of the gel occurs. It is not known why linear DNA preparations of considerable size heterogeneity Slice No. should behave in this way on electrophoresis FIG. 5. Electrophoresis of $H-labelled DNA exin polyacrylamide gels. Danna and Nathans tracted from purified SV40 virus and mixed with (1971) have shown separations on polyW-labelled HeLa DNA. (a) Electrophoresis acrylamidc gels of (presumably) linear shortly after purification. (b) electrophoresis after double-stranded DNA species produced by storage for 4 weeks in SSC at 4”. 0. . . . 0 ,14C dpm; the action of restriction cndonuclcase on a--@, 3H dpm. SV40 DNA. This result would seem at first sight paradoxical to the results reported here; of gel concentrations between 2.0 and 3.2% however, the molecular weight (MW) of the acrylamide. Mobility determinations for restriction endonuclease derived DNA fracthese various SV40 DNA species were then tions are all less than about 7 X 105, conderived from duplicate analyses on gels in siderably smaller than the heterogenous this concentration range. Results were nor3H linear CV-1 DXA or the linear HeLa malized using a 14C HeLa DNA standard [‘*CID NA. It may be, therefore, that linear curve and the observed mobility of the 14C DXA of low MW can separate out on polylinear marker DNA run on the same gel. acrylamide gels in a manner related to Absolute mobility is plotted against gel conMW, but that there is a minimum value centration in Fig. 7a and h$ (relative to l4C for the mobility at any fixed gel concenlinear HeLa DNA) is plotted against gel trat’ion which is approached asymptotically concentration in Fig. 7b. as the MW increases, i.e., over a cerThese graphs characterize the way in tain hIW value, any size of linear DNA which the electrophoretic mobility of each will migrate at virtually the same velocity. of these species varies with gel concentration, The slightly greater mobility of linear [3H]and suggests that analysis of DNA preparaDXA relative t’o linear [l(C]DNA (irretions across a range of gel concentrations can spective of gel concentration) observed in provide data of value in the identification of many of the figures in this paper sugcircular DNA species. gests that the size of the former is sufficiently small for its mobility to depart from DISCUSSION the limiting value discussed above. This sugPolyacrylamide gel electrophoresis has gestion is supported by the results of rate several distinct advantages over other tech(Fig. l), which shows niques for separating SV40 DNA. The chief zonal centrifugation of these is a clear separation of supercoiled that the DNA remaining at the time of harb

4”

404

HARLEY

AND WHITE

FIG. 6. Electrophoresis of supercoiled 3Klabelled SV40 DNA, partially digested with pancreatic DNase I at 4 different concentrations of enzyme: (a) 0.001 pg/ml, (b) 0.01 pg/ml, (c) 0.1 pg/ml, (d) (d) 1.0 pg/ml. The digests were performed as in Mat.erials and Methods, and 14CHeLa DNA was added to the samples after the termination of digestion. O--O, 3H dpm; 0. . . 0, ‘$C dpm.

vest of the infected cells is reproducibly of much smaller size t,han DNA extracted from uninfected cells. There is also some evidence in Figs. 1,2, and 4 of a skewed distribution of the 3H counts under the linear peak to give a faster migrating tail, a feature not observed for the linear [14C]DNA peak. This could represent short fragments of degraded host cell linear DNA in infected cells, harvested after cycles of virus replication had resulted in considerable cytopathic effect. There is no evidence for a separation of linear SV40 DNA from host cell linear DNA by this technique, nor would it necessarily be expected, even assuming linear SV40 DNA to be present’. It

may be noted that there is no evidence in Kg. 6d of any DKA of low MW running faster than the 14C-labelled linear DNA marker. One might) expect that fragments of varying size, resulting from the DNase degradation, would migrate in a heterogeneous band ahead of the main peak of linear DNA. Such a result is achieved n-hen linear DNA is subjected to sonication (unpublished observations), but this does not occur on DNase degradation, presumably because the enzyme degrades the DNA direct1.y to a size well below that which all linear DNA migrates as a single peak. The decrease in electrophoretic mobility

GEL ELECTROPHORESIS

20

2.2 Gel

24 Concentration

2.6

2.8

3.0

3-2

3.4

%*crylam!de

40.5

OF SV40 DNA

2.2

2.0 Gel

2’4

Conoentratlan

2.6

2.8

3.0

30

%Acrvlamide

FIG. 7. The relationship between electrophoretic mobility and gel concentration for supercoiled, nicked circular, and linear DNA. (left) Absolute electrophoretic mobility (ordinate) plotted against gel concentration (abscissa). Mobility values for 3H-labelled DNA species were normalized using a standard curve for r4C-labelled HeLa linear DNA. The latter was derived from a total of 52 observations of the electrophoretic mobility of linear [WIDNA across this range of gel concentrations and is expressed as a plot of the second-order polynomial equation describing these mobility values, with an F value of 260, between gel concentrations of 2.0 and 3.47,. (right) Rf values relating electrophoretie mobility of 3H-labelled DNA species to linear [WIDNA, plotted against gel concentration. RI values were derived by dividing the absolute electrophoretic mobility of each PHIDNA species by that of the linear [W]DNA at each gel concentration. e----O, supercoiled DNA; O-.-.-,-. 0, nicked circular DNA; +---f, linear 3H-labelled DNA; l . . .a, linear [WIDNA standard curve.

with increasing gel concentration, graphically represented in Fig. 7, is more pronounced for the circular than for the linear DNA species. Reference to this figure facilitates the choice of a suitable gel concentration in which to produce desired separations; for example, supercoiled SV40 DP\rA is well separated on either 2.0 or 3.2 % gels, whereas nicked circular DNA is best separated from the other two species on a 3.0% gel. Xo attempt has been made in Fig. 7 to draw curves through the data points, with the exception of those describing the mobility of linear [14C]DNA. Many replicate analyses would be required for accuracy, and the tendency is avoided to derive mathematical relationships which are likely to prove misleading without much more thorough knowledge of the

theory of gel electrophoresis of nucleic acids. However, Fig. 7 clearly emphasizes that the electrophoretic mobility of DNA varies with gel concentration in a manner which is dependent on the structure of the DNA. The main application of the present work should relate to the identification of supercoiled and nicked circular SV40 DNA in whole cell preparations. The clear separations should enable the circular DXA species to be detected at an early stage in the infective cycle, and will show the presence of nicked circular molecules which other techniques are unable to separate adequately from linear DNA. This should be valuable in the study of the intracellular replication cycle of SV40 virus. Since the circular forms of SV40 DNA are

406

HARLEY

AN11 WHITE

easily separated by polyacrylamide gel elect,rophoresis, it may be assumed that many other supcrcoiled and open circular DNA molecules may prove equally easy to wparate, for example, those of other DKA viruses, plasmids, kinetoplasts, and mitochondria. The mobilit’y characteristics of other circular DNA species may not, how ever, be identical to those of SV40, since there is no season to suppose that the sizeindependent mobility of high molecular weight linear DNA is also characteristic of t,he circular forms of DKA. In consequrnce, it would be necessary t’o calibrate the system under investigation by a gel concrntration range analysis of the purified circular species and graphical representation of the rcwlts as in Fig. 7. Work is in progress to extend this study to compare the mobility across a ra,nge of gel concentrations of various classes of nucleic acids, including DNA and RNA in both single- and double-stranded forms, DXA-RNA hybrids, and linear as opposed to circular forms (Harley et al., 1973). ACKNOWLEIXMENTS We acknowledge the capable technical assistance of Miss C. I. Johnson, and thank Professor K. R. Rees and Dr. V. Defendi for helpful and critical review of the manuscript. This work was supported by a grant from the Cancer Research Campaign. One of the authors (E. H. H.) holds a Beit Memorial Research Fellowship. REFERENCES L. V. (1963). The physical characteristics of polyoma virus. II. The nucleic arid. Virology 19, 279-282. CRAWFORD, L. V., and BLACK, P. H. (1964). The nucleic acid of simian virus 40. Virology 24, 38% 392. &NNA, K., and NATHANS, 1>. (1971). Sperific cleavage of simian virus 40 DNil by restriction endonuclease of Hemophilus ir$uetlzae. Proc. CRAWFORD,

Nat. Acad. Sci. U.S. 68,2913-2917.

R., and VOGT, M. (1963). Evidence for a ring structure of polyoma virus DNA. Proc.

DULBECCO,

Nat. Acad. Sci. U.S. SO, 236-243.

E. H., and REES, K. It. (1972). Mitochondrial RNA in mycoplasma infected HeLa cells. Biochim. Biophys. Acta 259,228-238. HARLEY, E. H., WHITE, J. S., and RUBERY, E. D. (1972). The resolution of polyoma DNA by HARLEY,

polyac~rylamide gel electrophoresis. 22, 41-15. H.\RLJ~:Y,

E.

II.,

WHITI,:,

J.

S.

:tnd

PBHS /,
K.

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