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The physical characteristics of polyoma virus
VIROLOGY
22,
149-152
(1964)
The
Physical
Characteristics
of
Polyoma
Virus
IV. The Size of the DNA L. V. CRAWFORD’ Institute
of Virology,
University
Accepted
October
of Glasgow,
Scotland
Y, 1963
The DNA of polyoma virus has been examined by sedimentation velocity and equilibrium density gradient band-width measurement. Two velocity components were present in the DNA preparations with sedimentation coefficients of 21 S and 15.5 S. It is suggested that the 21 S component represents the intact DNA of the virus, a circular molecule with a molecular weight of 3.5 million, and that the 15.5 S component results from the opening of the 21 S ring. Two forms of the slow component were observed, differing by 0.01 g/ml in density.
purification of the virus and separation of “full” and “empty” particles by equilibrium density gradient centrifugation in buffered RbCl have also been described (Crawford et al., 1962). The nucleic acid preparations used in this work were derived solely from “full” particles to avoid any possible confusion caused by material derived from “empty” particles. DNA extraction. Two methods of DNA extraction were used. Details of the detergent method employing sodium dodecyl sulphate, modified from that described by Watson and Littlefield (1960), have already been given (Crawford, 1963). Phenol extraction by the method of DiMayorca et al., 1959, as modified by Weil (1961)) was also used. Where highly purified suspensions were being extracted a small amount of gelatin was added (100 erg/ml). After three extractions with phenol the aqueous phase was dialyzed to remove residual phenol and the DNA was sedimented (80,000 g for 6 hours). The DNA pellet was allowed to resuspend itself in a suitable buffer at 4” without agitation. Analytical centrijugation. A Spinco model E analytical centrifuge with ultraviolet absorption optics was used for these experiments. Sedimentation coefficients were measured using a 12-mm cell with 4” filled-Epon cen-
INTRODUCTION
In a previous pap& a preliminary description of the properties of the DNA of polyoma virus was presented (Crawford, 1963). In that paper it was not possible to determine the molecular weight of the DNA unequivocally owing to the presence of two components in the preparation and to uncertainties in the relation between sedimentation coefficients and DNA molecular weights. This paper reports the results of an examination of polyoma DNA by sedimentation velocity determination and equilibrium density gradient centrifugation. MATERIALS
AND
METHODS
I/‘irus strain. The strain of polyoma virus used arose during the propagation of the Toronto strain (McCulloch et al., 1959) and differed from it in that it formed small plaques in the standard mouse embryo plaque assay. Virus production and purification. The method used for virus production was based on the extraction of virus from the debris of infected mouse embryo cells with receptordestroying enzyme (Crawford, 1962). The 1 Member perimental
of the Medical Virus Research
Research Unit,.
Council
Exi49
150
CRAWFORD TABLE
1
SEDIMENTATION COEFFICIENTS AND MOLECULAR WEIGHTS OF POLYOMA DNA Sedimentation coefficient
Molecular
weight -
-
21 s 15.5
Equation’
ing the bottom of the tube, drops were collected and the optical density at 260 rnp of each sample was determined after suitable dilution. The samplescorresponding to each component were pooled and the DNA was sedimented (80,000 g for 6 hours). RESULTS
s
1 2
equations used are: Eq. 1. S?o,w = 0.063 MO.31 (Doty ef al., 1958) Eq. 2. &o,w = 0.080 k”‘.35 (Burgi and Hershey, Since the 21 S component is circular, weight cannot be calculated from its coefficient by the above equations.
2.9 3.4
x x
106 106
a The
1963) its molecular
sedimentation
terpiece, at 20” and 42,040 rpm. The DNA concentration was between 5 and 40 pg/ml in 0.15 M NaCl containing either 0.02 M phosphate buffer or 0.015 M citrate buffer. Equilibrium density gradient centrifugation in CsCl (Meselson et al., ,1957) was used to determine band-width molecular weights. The DNA sample in CsCl (density 1.71 g/ml) containing Tris buffer (0.01 M, pH 8.5) was spun in a 12-mm cell with 4” filledEpon centerpiece. The solution was introduced slowly into the cell with a glasspipette through an enlarged loading port. After 3-4 days at 31,410 rpm (21 S component) or 35,600 rpm (15.5 S component) no further change in band profile could be detected. Ultraviolet absorption photographs were taken and scanned with a Joyce Loebl microdensitometer to determine the shapes and positions of the bands or boundaries. The calculated band-width molecular weights have been converted from the cesium salt of DNA to the sodium salt by multiplying by 331/441. Sucrose density gradient centrifugation. Linear gradients of 20% to 3% sucrose in 0.15 M NaCl, 0.015 M citrate buffer, pH 6.8, were prepared with the mixing device described bg Britten and Roberts (1960). The sample of DNA was layered on top of the gradient forming an inverse gradient of DNA and a continuation of the sucrose gradient from 3% to 0 %. The gradients were spun at 30,000 rpm in an SW 39 rotor, Spinco model L, for 9 hours. After punctur-
Sedimentation Velocity Samples of polyoma DNA extracted by either the phenol or detergent methods show two sedimentation velocity components, as previously described (Crawford, 1963) and shown in Table 1. The two components did not appear to be in equilibrium with each other since each could be obtained free of the other by sucrosedensity gradient centrifugation. The difference in sedimcntation coefficient between the two components is probably due to a difference in shape, as suggestedby Dulbecco and Vogt (1963), the fast component being circular. As shown by Freifelder and Davison (1963) heating T7 phage DNA in the presence of formaldehyde results in a progressive increase in sedimentation coefficient as t’he double helical structure collapses. This rise is followed by a sharp fall as the two strands separate. Similar experiments under identical conditions with the 21 S component of polyoma virus DNA gave different results. The sedimentation coefficient increased after exposure to elevated temperatures, but this rise was not followed by a fall. Even when heated in the presence of 12% HCHO to 70” for 10 minutes, more than 15” above the temperature at which strand separation would be expected to occur, the sedimentation coefficient of the main boundary remained high, 48 S. Apparently the double helical structure of the DNA collapsed, but the strands remained intertwined with each other owing to the circularity of the structure. Equilibrium Density Gradient Centrifugation. When the 21 S component of polyoma virus DNA, isolated by sucrose density gradient centrifugation, was centrifuged in CsCl, the band obtained was symmetrical and approximately Gaussian. The band profile and a graph of the square of band width against DNA concentration are shown in
PHYSICAL
CHARACTERISTICS
OF
POLYOMA
VIRUS.
151
IV
lecular weight by Burgi and Hershey (1963) has removed one of the main difficulties in the interpretation of sedimentation studies. Previously the values obtained from the equations of Doty et al. (1958) and Rubenstein et al. (1961) were widely different in the region considered here. The molecular weight now obtained for the 15.5 S component is 2.9 million (Doty et al., 1958) or 3.4 million (Burgi and Hershey, 1963). The molecular weight of the 21 S component is probably the same since the more compact shape of this component, shown by Dulbecco and Vogt (1963) to be circular, would result in more rapid sedimentation. The relation between sedimentation coefficient and molecular weight for circular molecules of this type may be given by SZOW= 0.105 MO.35, a
I
0 T
.a FIG. 1. Equilibrium density gradient of 21 S component. Approximately 2 rg of the 21 S component of polyoma virus DNA extracted with phenol was spun at 31,410 rpm as described under Materials and Methods. The microdensitometer trace is shown inset on the graph of the square of band width against DNA concentration derived from it.
Fig. 1. The method of presentation is taken from Thomas and Berns (1961). The molecular weight calculated for the 21 S component by this method is close to 2.5 X lo6 both for phenol-extracted and detergentextracted DNA. A similar experiment with the 15.5 S component produced the profile shown in Fig. 2. The profile is symmetrical but not Gaussian. This could be due to the presence of two types of molecule of similar shape but different density, forming overlapping bands. It is not possible to estimate molecular weight from band width in this case. The difference in density between the two types of slow component was estimated to be 0.01 g/ml. DISCUSSION
Recently a reassessment of the relation between sedimentation coefficient and mo-
L
.6 c /cMAX .4
.o
.2
‘c \
FIG. 2. Equilibrium density gradient of 15.5 S component. Approximately 3 rg of the 15.5 S component of polyoma virus DNA extracted with phenol was spun at 35,666 rpm as described under Materials and Methods. The microdensitometer trace is shown inset on the graph of the square of band width against DNA concentration derived from it.
152
CRAWFORD
modification of the equation of Burgi and Hershey (1963). The equation is based on studies of polyoma DNA and papilloma DNA (Crawford, in preparation) and does not appear to apply to molecules larger than papilloma DNA. The molecular weight of the 21 S cornponent of polyoma virus DNA, estimated from band-width measurements in equilibrium density gradients, was 2.5 million, as compared to the figure of 3.4 million above. The reason for this difference is not known. Other DNA’s have been found to give bandwidth molecular weights lower than those derived from other methods (Thomas and Berns, 1961). The band profile of the 15.5 S component was such as to suggest that two types of slow component exist, similar in size but differing in density. This density difference could result from the presence of material other than DNA in one type of slow component. If this were so it may be calculated that a protein with a molecular weight of about lo5 attached to the lighter type would be sufficient to account for the difference in density between the two types. This speculation may be of interest in connection with the nature of the linkage which holds the two ends of the linear molecule in the form of a ring. It is conceivable that some protein concerned with the replication of the DNA might remain attached to the molecule, linking the two ends to form a ring. The value proposed for the molecular weight of polyoma DTU’A, 3.4 million, is lower than the value reported for the DNA content of the virus particle, 6 to 8.5 million (Crawford et al., 1962). This would seem to suggest that the virus particle contains two molecules of DNA. A much more likely explanation would be that the estimate of the DNA content of the particle is too high. The value reported depended on particle counting and systematic errors, including the loss of particles onto glassware during dilution, are difficult to detect or avoid in this method. In view of this uncertainty it seemsmore reasonable to suggest that polyoma virus contains a single DNA molecule, with a molecular weight of approximately 3.4 million.
REFERENCES BRITTEN,
R. J.,
and
ROBERTS,
resolutiondensity gradient
R. B. (1960). sedimentation
High anal-
ysis. BURGI,
Science 131, 32-33. E., and HERSHEY, A. D. (1963). Sedimentation rate as a measure of molecular weight of DNA. Biophys. J. 3,309-321. CRdWFORD, L. V. (1962). The adsorption of polyoma virus. virology 18, 177-181. CRAWFORD, L. V. (1963). The physical characteristics of polyoma virus. II. The nucleic acid. Virology 19, 279-282. CRAWFORD, L. V., CRAWFORD, E. M., and W.~TSON, D. H. (1962). The physical characteristics of polyoma virus. I. Two types of particle. ViTiroZogy 18, 170-176. DIMAYORCA, G. A., EDDY, B. E., STEWART, S. E., HUNTER, W. S., FRIEND, C., and BENDICH, A. (1959). Isolation of infectious deoxyribonucleic acid from SE polyoma-infected tissue cultures. Proc. Natl. Acad. Sci. U.S. 45, 1805-1808. DOTY, P., MCGILL? B. B., and RICE, S. (1958).
The propertiesof sonicfragmentsof deoxyribonucleic acid. Proc. Natl. Acad. Sci. U.S. 44, 432-438. DULBECCO, R., and VOGT, M. (1963). Evidence for a ring structure of polyoma virus Dh’A. Proc. Natl. Acad. Sci. U.S. 50, 236-243. FREIFELDER, D., and DAVISON, P. F. (1963). Physicochemical studies on the reaction between formaldehyde and DNA. Biophys. J. 3, 49-63. MCCULLOCH, E. A., HOWATSON, A. F., SIMINOVITCH, L., AXELRAD, A. A., and HIM, A. W. (1959). A cytopathogenic agent from mammary tumour in a C3H mouse that produces tumours in Swiss mice and hamsters. Nature 183, 15351536. MESELSON, M., STAHL, F., and VINOGRAD, J. (1957). Equilibrium sedimentation of macromolecules in density gradients. Proc. Natl. Acad. Sci. U.S. 43, 581-588. RUBENSTEIN, I., THOMAS, C. A., JR., and HERSHEY, A. D. (1961). The molecular weights of T2 bacteriophage DNA and its first and second breakage products. Proc. Natl. Acad. Sci. lid’. 47, 1113-1122. THOMAS, C. A., JR., and BERNS, K. I. (1961). The physical characterization of DNA molecules released from T2 and T4 bacteriophage. J. Mol. Biol. 3, 277-288. WATSON, J. D., and LITTLEFIELD, J. W. (1960). Some properties of DNA from Shope papilloma virus. J. Mol. Biol. 2, 161-165. WEIL, R. (1961). A quantitative assay for a subviral infective agent related to polyoma virus. Virology 14, 46-53.
22,
149-152
(1964)
The
Physical
Characteristics
of
Polyoma
Virus
IV. The Size of the DNA L. V. CRAWFORD’ Institute
of Virology,
University
Accepted
October
of Glasgow,
Scotland
Y, 1963
The DNA of polyoma virus has been examined by sedimentation velocity and equilibrium density gradient band-width measurement. Two velocity components were present in the DNA preparations with sedimentation coefficients of 21 S and 15.5 S. It is suggested that the 21 S component represents the intact DNA of the virus, a circular molecule with a molecular weight of 3.5 million, and that the 15.5 S component results from the opening of the 21 S ring. Two forms of the slow component were observed, differing by 0.01 g/ml in density.
purification of the virus and separation of “full” and “empty” particles by equilibrium density gradient centrifugation in buffered RbCl have also been described (Crawford et al., 1962). The nucleic acid preparations used in this work were derived solely from “full” particles to avoid any possible confusion caused by material derived from “empty” particles. DNA extraction. Two methods of DNA extraction were used. Details of the detergent method employing sodium dodecyl sulphate, modified from that described by Watson and Littlefield (1960), have already been given (Crawford, 1963). Phenol extraction by the method of DiMayorca et al., 1959, as modified by Weil (1961)) was also used. Where highly purified suspensions were being extracted a small amount of gelatin was added (100 erg/ml). After three extractions with phenol the aqueous phase was dialyzed to remove residual phenol and the DNA was sedimented (80,000 g for 6 hours). The DNA pellet was allowed to resuspend itself in a suitable buffer at 4” without agitation. Analytical centrijugation. A Spinco model E analytical centrifuge with ultraviolet absorption optics was used for these experiments. Sedimentation coefficients were measured using a 12-mm cell with 4” filled-Epon cen-
INTRODUCTION
In a previous pap& a preliminary description of the properties of the DNA of polyoma virus was presented (Crawford, 1963). In that paper it was not possible to determine the molecular weight of the DNA unequivocally owing to the presence of two components in the preparation and to uncertainties in the relation between sedimentation coefficients and DNA molecular weights. This paper reports the results of an examination of polyoma DNA by sedimentation velocity determination and equilibrium density gradient centrifugation. MATERIALS
AND
METHODS
I/‘irus strain. The strain of polyoma virus used arose during the propagation of the Toronto strain (McCulloch et al., 1959) and differed from it in that it formed small plaques in the standard mouse embryo plaque assay. Virus production and purification. The method used for virus production was based on the extraction of virus from the debris of infected mouse embryo cells with receptordestroying enzyme (Crawford, 1962). The 1 Member perimental
of the Medical Virus Research
Research Unit,.
Council
Exi49
150
CRAWFORD TABLE
1
SEDIMENTATION COEFFICIENTS AND MOLECULAR WEIGHTS OF POLYOMA DNA Sedimentation coefficient
Molecular
weight -
-
21 s 15.5
Equation’
ing the bottom of the tube, drops were collected and the optical density at 260 rnp of each sample was determined after suitable dilution. The samplescorresponding to each component were pooled and the DNA was sedimented (80,000 g for 6 hours). RESULTS
s
1 2
equations used are: Eq. 1. S?o,w = 0.063 MO.31 (Doty ef al., 1958) Eq. 2. &o,w = 0.080 k”‘.35 (Burgi and Hershey, Since the 21 S component is circular, weight cannot be calculated from its coefficient by the above equations.
2.9 3.4
x x
106 106
a The
1963) its molecular
sedimentation
terpiece, at 20” and 42,040 rpm. The DNA concentration was between 5 and 40 pg/ml in 0.15 M NaCl containing either 0.02 M phosphate buffer or 0.015 M citrate buffer. Equilibrium density gradient centrifugation in CsCl (Meselson et al., ,1957) was used to determine band-width molecular weights. The DNA sample in CsCl (density 1.71 g/ml) containing Tris buffer (0.01 M, pH 8.5) was spun in a 12-mm cell with 4” filledEpon centerpiece. The solution was introduced slowly into the cell with a glasspipette through an enlarged loading port. After 3-4 days at 31,410 rpm (21 S component) or 35,600 rpm (15.5 S component) no further change in band profile could be detected. Ultraviolet absorption photographs were taken and scanned with a Joyce Loebl microdensitometer to determine the shapes and positions of the bands or boundaries. The calculated band-width molecular weights have been converted from the cesium salt of DNA to the sodium salt by multiplying by 331/441. Sucrose density gradient centrifugation. Linear gradients of 20% to 3% sucrose in 0.15 M NaCl, 0.015 M citrate buffer, pH 6.8, were prepared with the mixing device described bg Britten and Roberts (1960). The sample of DNA was layered on top of the gradient forming an inverse gradient of DNA and a continuation of the sucrose gradient from 3% to 0 %. The gradients were spun at 30,000 rpm in an SW 39 rotor, Spinco model L, for 9 hours. After punctur-
Sedimentation Velocity Samples of polyoma DNA extracted by either the phenol or detergent methods show two sedimentation velocity components, as previously described (Crawford, 1963) and shown in Table 1. The two components did not appear to be in equilibrium with each other since each could be obtained free of the other by sucrosedensity gradient centrifugation. The difference in sedimcntation coefficient between the two components is probably due to a difference in shape, as suggestedby Dulbecco and Vogt (1963), the fast component being circular. As shown by Freifelder and Davison (1963) heating T7 phage DNA in the presence of formaldehyde results in a progressive increase in sedimentation coefficient as t’he double helical structure collapses. This rise is followed by a sharp fall as the two strands separate. Similar experiments under identical conditions with the 21 S component of polyoma virus DNA gave different results. The sedimentation coefficient increased after exposure to elevated temperatures, but this rise was not followed by a fall. Even when heated in the presence of 12% HCHO to 70” for 10 minutes, more than 15” above the temperature at which strand separation would be expected to occur, the sedimentation coefficient of the main boundary remained high, 48 S. Apparently the double helical structure of the DNA collapsed, but the strands remained intertwined with each other owing to the circularity of the structure. Equilibrium Density Gradient Centrifugation. When the 21 S component of polyoma virus DNA, isolated by sucrose density gradient centrifugation, was centrifuged in CsCl, the band obtained was symmetrical and approximately Gaussian. The band profile and a graph of the square of band width against DNA concentration are shown in
PHYSICAL
CHARACTERISTICS
OF
POLYOMA
VIRUS.
151
IV
lecular weight by Burgi and Hershey (1963) has removed one of the main difficulties in the interpretation of sedimentation studies. Previously the values obtained from the equations of Doty et al. (1958) and Rubenstein et al. (1961) were widely different in the region considered here. The molecular weight now obtained for the 15.5 S component is 2.9 million (Doty et al., 1958) or 3.4 million (Burgi and Hershey, 1963). The molecular weight of the 21 S component is probably the same since the more compact shape of this component, shown by Dulbecco and Vogt (1963) to be circular, would result in more rapid sedimentation. The relation between sedimentation coefficient and molecular weight for circular molecules of this type may be given by SZOW= 0.105 MO.35, a
I
0 T
.a FIG. 1. Equilibrium density gradient of 21 S component. Approximately 2 rg of the 21 S component of polyoma virus DNA extracted with phenol was spun at 31,410 rpm as described under Materials and Methods. The microdensitometer trace is shown inset on the graph of the square of band width against DNA concentration derived from it.
Fig. 1. The method of presentation is taken from Thomas and Berns (1961). The molecular weight calculated for the 21 S component by this method is close to 2.5 X lo6 both for phenol-extracted and detergentextracted DNA. A similar experiment with the 15.5 S component produced the profile shown in Fig. 2. The profile is symmetrical but not Gaussian. This could be due to the presence of two types of molecule of similar shape but different density, forming overlapping bands. It is not possible to estimate molecular weight from band width in this case. The difference in density between the two types of slow component was estimated to be 0.01 g/ml. DISCUSSION
Recently a reassessment of the relation between sedimentation coefficient and mo-
L
.6 c /cMAX .4
.o
.2
‘c \
FIG. 2. Equilibrium density gradient of 15.5 S component. Approximately 3 rg of the 15.5 S component of polyoma virus DNA extracted with phenol was spun at 35,666 rpm as described under Materials and Methods. The microdensitometer trace is shown inset on the graph of the square of band width against DNA concentration derived from it.
152
CRAWFORD
modification of the equation of Burgi and Hershey (1963). The equation is based on studies of polyoma DNA and papilloma DNA (Crawford, in preparation) and does not appear to apply to molecules larger than papilloma DNA. The molecular weight of the 21 S cornponent of polyoma virus DNA, estimated from band-width measurements in equilibrium density gradients, was 2.5 million, as compared to the figure of 3.4 million above. The reason for this difference is not known. Other DNA’s have been found to give bandwidth molecular weights lower than those derived from other methods (Thomas and Berns, 1961). The band profile of the 15.5 S component was such as to suggest that two types of slow component exist, similar in size but differing in density. This density difference could result from the presence of material other than DNA in one type of slow component. If this were so it may be calculated that a protein with a molecular weight of about lo5 attached to the lighter type would be sufficient to account for the difference in density between the two types. This speculation may be of interest in connection with the nature of the linkage which holds the two ends of the linear molecule in the form of a ring. It is conceivable that some protein concerned with the replication of the DNA might remain attached to the molecule, linking the two ends to form a ring. The value proposed for the molecular weight of polyoma DTU’A, 3.4 million, is lower than the value reported for the DNA content of the virus particle, 6 to 8.5 million (Crawford et al., 1962). This would seem to suggest that the virus particle contains two molecules of DNA. A much more likely explanation would be that the estimate of the DNA content of the particle is too high. The value reported depended on particle counting and systematic errors, including the loss of particles onto glassware during dilution, are difficult to detect or avoid in this method. In view of this uncertainty it seemsmore reasonable to suggest that polyoma virus contains a single DNA molecule, with a molecular weight of approximately 3.4 million.
REFERENCES BRITTEN,
R. J.,
and
ROBERTS,
resolutiondensity gradient
R. B. (1960). sedimentation
High anal-
ysis. BURGI,
Science 131, 32-33. E., and HERSHEY, A. D. (1963). Sedimentation rate as a measure of molecular weight of DNA. Biophys. J. 3,309-321. CRdWFORD, L. V. (1962). The adsorption of polyoma virus. virology 18, 177-181. CRAWFORD, L. V. (1963). The physical characteristics of polyoma virus. II. The nucleic acid. Virology 19, 279-282. CRAWFORD, L. V., CRAWFORD, E. M., and W.~TSON, D. H. (1962). The physical characteristics of polyoma virus. I. Two types of particle. ViTiroZogy 18, 170-176. DIMAYORCA, G. A., EDDY, B. E., STEWART, S. E., HUNTER, W. S., FRIEND, C., and BENDICH, A. (1959). Isolation of infectious deoxyribonucleic acid from SE polyoma-infected tissue cultures. Proc. Natl. Acad. Sci. U.S. 45, 1805-1808. DOTY, P., MCGILL? B. B., and RICE, S. (1958).
The propertiesof sonicfragmentsof deoxyribonucleic acid. Proc. Natl. Acad. Sci. U.S. 44, 432-438. DULBECCO, R., and VOGT, M. (1963). Evidence for a ring structure of polyoma virus Dh’A. Proc. Natl. Acad. Sci. U.S. 50, 236-243. FREIFELDER, D., and DAVISON, P. F. (1963). Physicochemical studies on the reaction between formaldehyde and DNA. Biophys. J. 3, 49-63. MCCULLOCH, E. A., HOWATSON, A. F., SIMINOVITCH, L., AXELRAD, A. A., and HIM, A. W. (1959). A cytopathogenic agent from mammary tumour in a C3H mouse that produces tumours in Swiss mice and hamsters. Nature 183, 15351536. MESELSON, M., STAHL, F., and VINOGRAD, J. (1957). Equilibrium sedimentation of macromolecules in density gradients. Proc. Natl. Acad. Sci. U.S. 43, 581-588. RUBENSTEIN, I., THOMAS, C. A., JR., and HERSHEY, A. D. (1961). The molecular weights of T2 bacteriophage DNA and its first and second breakage products. Proc. Natl. Acad. Sci. lid’. 47, 1113-1122. THOMAS, C. A., JR., and BERNS, K. I. (1961). The physical characterization of DNA molecules released from T2 and T4 bacteriophage. J. Mol. Biol. 3, 277-288. WATSON, J. D., and LITTLEFIELD, J. W. (1960). Some properties of DNA from Shope papilloma virus. J. Mol. Biol. 2, 161-165. WEIL, R. (1961). A quantitative assay for a subviral infective agent related to polyoma virus. Virology 14, 46-53.