Vol. 24, No. 2 Printed in U.S.A.
JOURNAL OF VIROLOGY, Nov. 1977, p. 695-700
Copyright i) 1977 American Society for Microbiology
Measurements of the Genome Sizes of Simian Virus 40 and Polyoma Virus LAUREN SOMPAYRAC AND KATHLEEN J. DANNA* Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309 Received for publication 21 June 1977
We have measured the genome sizes of simian virus 40 and polyoma virus and found them to be 5,010 ± 125 base pairs and 5,080 + 125 base pairs, respectively. respectively.
The genome sizes of two small DNA tumor viruses, simian virus 40 (SV40) and polyoma virus, have been estimated to be from 2.25 to 5.4 million daltons (1-4, 8, 10, 13, 14). This large range of values probably results from inaccuracies in some of the measuring techniques used, from strain differences that arise frequently during propagation of SV40 and polyoma (10, 11), and from the fact that size determinations for these DNA molecules have never been made relative to a standard of known molecular weight. Now, however, DNA of different virus strains can be characterized and compared by digestion with appropriate restriction enzymes and analysis of the products by gel electrophoresis. Also, Walter Fiers and his group have provided a molecular weight standard by determining the nucleotide sequence of the two contiguous HindII+HindIII fragments, H and I, of SV40 DNA, which together comprise the HindIII-D fragment (Fig. 1) (12; W. Fiers, personal communication). For these reasons, we decided to make a new determination of the genome sizes of well-characterized strains of SV40 and polyoma, using the HindIII-D fragment as a standard. Our stock of SV40 strain 776 was obtained from Harvey Ozer and is the same strain used in the sequence studies of Fiers et al. Largeplaque polyoma virus (Pasadena strain) was a kind gift from Tom Benjamin. Both viruses were extensively plaque purified, and stocks were made, using multiplicities of infection of less than 10' PFU per cell. DNA made from these stocks was characterized by restriction endonuclease digestion and gel electrophoresis (Fig. 2 and 3, Table 1). To obtain accurate length measurements, either polyoma or SV40 form I DNA was cleaved with the EcoRI enzyme (New England Biolabs) to yield full-length linear molecules. These were mixed with gel-purified HindIII-D fragments that had been circularized (see below) by treat-
ment with T4 DNA ligase (a kind gift of Marvin Caruthers) (7), spread for electron microscopy (Fig. 4), and measured with a Hewlett-Packard digitizer. Histograms of the results of these measurements are shown in Fig. 5. The HindIIID fragment is 10.5% as long as the SV40 genome and 10.35% as long as the polyoma genome. In preliminary experiments, we spread and measured mixtures of linear HindIII-D fragments and circularized HindIII-D fragments. Presumably because of difficulty in visualizing the ends of the linear molecules, they appeared about 2% shorter than the same molecules that had been circularized. To avoid these "end effects," we used the circularized HindIII-D fragments as a standard for all measurements presented here. We also checked that full-length EcoRI linear polyoma molecules could be cir-
695
"HIND II
D
FIG. 1. Physical map of the SV40genome showing cleavage sites for the EcoRI enzyme, HindIII enzyme (outer circle), and the combination ofHindII+HindIII enzymes (inner circle) (6, 15).
696
NOTES
J. VIROL.
cularized and that these circles were the same length as the linears. Since end effects are not significant for large molecules, these measurements indicate that the procedure of circularization itself does not change the apparent length of DNA molecules. Also, these experiments show that nuclease degradation did not occur during digestion with the EcoRI and HindIII enzymes, since such degradation would have prevented circularization under the conditions used for ligation. As a check on our electron microscopy results, two types of experiments were performed using gel electrophoresis. First, 32P-labeled SV40 DNA was digested with either the HindIII enzyme or HindII+HindIII enzymes and electrophoresed on a polyacrylamide slab gel. The counts per minute in the HindIII-D or HindII+HindIII-H plus I fragments were compared with the sum of the counts per minute in all the fragments. The percentage of counts per minute in a given fragment is taken to be equivalent to the fraction of the genome which that fragment represents. These results were consistent with the more accurate results obtained by electron microscopy (Table 2). As a second check, SV40 and polyoma form I DNAs were digested with the EcoRI FIG. 2. Major HindII+HindIII fragments of SV40 DNA and Hpa II fragments of polyoma DNA. BSC1 and NIH 3T3 cells were infected with SV40 and polyoma virus, respectively, at multiplicities of infection from 10 to 20 PFU/cell. Viral DNA isolated by the Hirt procedure (9) was phenol extracted, ethanol precipitated, and centrifuged to equilibrium in cesium chloride-ethidium bromide gradients. After thorough extraction with isopropanol to remove the ethidium bromide and dialysis to remove the cesium chloride, form I DNA was stored at -20'C. SV40 DNA I was digested with the combination of HindII+HindIII enzymes, and polyoma DNA I was digested with the Hpa II enzyme (New England Biolabs). The buffers used were 6.6 mM Tris (pH 7.5), 6.6 mM MgCl2, 50 mM NaCl, and 6.6 mM beta-mercaptoethanol for the Hind enzymes and 10 mM Tris (pH 7.5), 10 mM MgCl2, 6 mM KCl, 1 mM dithiothreitol, and 100 pg of gelatin per ml for the Hpa II enzyme. DNA was at a concentration of 2 to 20 pg/ml, and incubations were done for 6 to 12 h at 37°C in the presence of about 2 U of enzyme per mg of DNA. Digests were treated with RNase (heat treated, Worthington Biochemicals) at an enzyme concentration of 50 lpg/ml for 30 min at 37°C, extracted with phenol and chloroform, and run on a 40-cm, 4% polyacrylamide slab gel. These long gels were run typically for 36 h at 3 V/cm. The running buffer used in all gels was 0.04 M Tris (pH 7.7), 0.005 M sodium acetate, and 0.001 M EDTA. Gels were stained with ethidium bromide and photographed under UV light. Lane a shows the 11 major HindII+HindIII fragments of SV40 DNA, A through K. Lane b shows the 7 major Hpa II fragments ofpolyoma DNA, 1 through 7.
VOL. 24, 1977
NOTES
0 p No
*--
697
TABLE 1. Percentage of 32P counts per minute contained in HindII+HindIII fragments of SV40 DNA and in Hpa II fragments ofpolyoma DNA a SV40
HindII+
% cpm in
Polyoma HpaII
% cpm in
HindIII fragment
fragment"
fragment
fragment"
1 26.2 22.3 2 20.9 15.7 3 17.3 10.5 13.5 10.0 4 8.2 5 8.1 6 6.9 7.0 7 4.8 6.9 8 2.2 5.4 4.9 4.4 3.9 0.5 0.4 a Polyoma DNA and SV40 DNA were prepared as described in Fig. 2, except that 0.1 to 0.2 mCi of 32P was added to each 84-mm-diameter petri dish 24 h postinfection in low-phosphate medium. After electrophoresis of the digestion products on 4%/15% polyacrylamide step gels, the hands were excised, dissolved in 30% H202 at 80'C, and counted in Triton-toluene
A B C D E F G H I J K L M
counting solution. b Values given are averages of results obtained in several experiments.
enzyme and electrophoresed on a 0.7% agarose gel together with an Hha I digest of SV40 form I as a marker (Fig. 6). Hha I cleaves our strain of SV40 to yield two fragments, one 91% of the genome, and the other 9% (L. Sompayrac, unpublished data). Polyoma unit-length linear DNA migrated slightly slower than SV40 linear DNA (Fig. 6). Its position relative to the Hha I 91% marker indicated that the polyoma genome was approximately 2% larger than SV40 DNA. This again is consistent with our electron microscopy results. Our measurements indicate that the HindIIID fragment is 10.5% as large as the SV40 genome and 10.35% as large as the polyoma genome. Taken together with the results of Fiers et al. that there are 526 base pairs in the circularized HindIII-D fragment, we conclude that the genome of our strain of SV40 has 5,010 + 125 base pairs and that our strain of polyoma has a genome size of 5,080 ± 125 base pairs. Other ex-
8-
0
L FIG. 3. Minor HindII+HindIII fragments of SV40 DNA and Hpa H fragments of polyoma DNA. DNA , isQ, was prepared and digested as described in Fig. 2. Lane a is a 4% (upper portion)/12.5% polyacrylamide ^ N A step gel on which an Hpa II digest of polyoma DNA f I has been run to show minor fragment 8. Lane b is a ;%.; 4%/15% step gel overloaded with a HindII+HindIII brEmdigest of SV40 DNA in order to show the two minor U fragments, L and M.
698
J. VIROiL.
NOTES
5~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~4
4'.
10~
qr
1.-4
06.
It
9
s/.
* a 4a64 '*t**
r
~ ~ ~
4r
~
~
4
4a
~~
*~~~I ** ~~~~~~~~~~~~~~~~~~~~~~~ *5
a 94,t*
'5~~~~~~~~4
*
is~~~~~~~~~~~~~,C: t t ¼. ~ ~
C
* Ag
45
'S
4
*
a~~t',R ~~
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~4*
,
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
is I: c-*~~~~10
f~~~~~~~~~r~a.
nvj.o
~a~
4
of Hindill D fragments and EcoRI linears Circularized Hindill Dfragments~~~~~~~~~~~~~~~if 4 Electron micrograph FIG legthEcoI lnea 840DAmlceswrmidspado cabo-oatdgisi 0 andful a N (5)andphotgrahed itha Phlip EM0 lcrn irsoe I hspoedr formmid e fctcrm DA n 0v hs(H85 0 supede inaslto f5%fraie0 ae n abncae rd eedpe yohs fdstle mlTilitroa pedo te fim an drid afer a ethnol inse Grd wre hdwdwt ltnmplaimi throgh evaporator~~~~~~~~~~~~~~~I rotary
VOL. 24, 1977
NOTES
699
0)~~~~~~~~~~
.I 0
.55
.60
.65
inches
E z
6.0 inches
5.5
5.0
20
B) Polyomu Linears
~~~~~~~~~~~15 ~ ~ ~
~
~
~
~
~
~
~
~
~
pi 2SF Aft =t 55
60
.5
chess
E
6.0 inches FIG. 5. Histograms of polyoma and SV40 unitlength linear DNA measured with circularized HindIII-D fragments as a standard. Grids prepared as described in Fig. 4 with either SV40 (A) orpolyoma (B) unit-length linears were photographed, enlarged, and measured with a Hewlett-Packard digitizer and 9821 computer. For unit-length SV40 linears, the average values of these length measurements gave:
5.0
5.5
HindIII-D standard SV40 unit-length linear 0.591 inches (ca. 1.50 cm) 5.63 inches (ca. 14.30 cm)
=
10.5%
For polyoma: HindIII-D standard polyoma unit-length linear 0.590 inches (ca. 1.50 cm) 5.70 inches (ca. 14.48 cm)
10.35%
TABLE 2. Percentage of 32P counts per minute contained in HindIII-D and HindII+HindIII-H plus I fragments of SV40 DNA Enzyme used
Fragment studied
P cpm in fragment studied
cpm in frag32P total (all fragments) %mn tde 10.4 119,133
D HindIII 12,360 H plus I 10.3 HindII+HindIII 11,297 109,192 32P-labeled SV40 DNA was prepared, digested, electrophoresed, and counted as described in the legend of Fig. 2 and in Table 1. a
700
J. VIROL.
NOTES
100% Py 100% SV4O91 % SV40k~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
FIG. 6. Relative mobilities of SV40 DNA and polyoma DNA. Samples of polyoma and SV40 form I DNA (0.1 4) were digested with the EcoRI enzyme to yield unit-length linears. SV40 form I DNA (0.04 pg) was also digested with the Hha I enzyme to give a linear marker which is 91% of the SV40 genome. The buffer used for the EcoRI digestion was 0.1 M Tris (pH 7.5), 50 mM NaCl, and 5 mM MgCl2. For the Hha I digestions, a buffer of 6 mM Tris (pH 7.5), 50 mM NaCl, 6 mM MgCl2, 6 mM beta-mercaptoethanol, and 100 ,g of gelatin per ml was used. Digestions were done in a 50- d volume at an enzyme concentration of 50 U/ml for 2 h at 370C. Lane a: Polyoma unit-length linears alone. Lane b: Polyoma and SV40 unit-length linears and SV40 91% Hha I fragment. Lane c: SV40 unit-length linears alone. Top of gel is not shown. Actual migration distances were: polyoma unit-length linear DNA, 13.3 cm; SV40 unit-length linear DNA, 13.46 cm; SV40 Hha I 91% fragment, 14.10 cm. The 9% Hha I fragment of SV40 DNA ran off this gel.
perimenters should be able to adapt our size measurements to their variants of SV40 and polyoma either by direct comparison with our strains or by doing similar 32P labeling and enzyme digestions and comparing the fraction of counts in each of their fragments with our results in Table 1. We would like to thank John Heumann for introducing us to formamide spreading for electron microscopy and for writing the length measurement program for the Hewlett-Packard computer. We would also like to express appreciation to Joe Allen and Herbert Kroehl for allowing us to use the HewlettPackard digitizer and computer of the National Geophysical and Solar-Terrestrial Data Center, National Oceanic and At-
mospheric Administration. This research was supported by Public Health Service grant CA18212, awarded by the National Cancer Institute. L.S. is a postdoctoral fellow of the National Cancer Institute. LITERATURE CITED 1. Anderer, F. A., H. D. Schlumberger, M. A. Koch, H. Frank, and H. J. Eggers. 1967. Structure of simian virus 40. II. Symmetry and components of the virus particle. Virology 32:511-523. 2. Chen, M. C. Y., K. S. S. Chang, and N. P. Salzman. 1975. Studies of polyoma virus DNA: cleavage map of the polyoma virus genome. J. Virol. 15:191-198. 3. Crawford, L. V. 1964. The physical characteristics of polyoma virus. IV. The size of the DNA. Virology 22:149-152. 4. Crawford, L. V., and P. H. Black. 1964. The nucleic acid of simian virus 40. Virology 24:388-392.
5. Davis, R. W., M. Simon, and N. Davidson. 1971. Electron microscope heteroduplex methods for mapping regions of base sequence homology in nucleic acids.
Methods Enzymol. 21:413-428.
6. Danna, K. J., G. H. Sack, Jr., and D. Nathans. 1973. Studies of simian virus 40 DNA. VII. A cleavage map of the SV40 genome. J. Mol. Biol. 78:363-376. 7. Feunteun, J., L. Sompayrac, M. Fluck, and T. Benjamin. 1976. Localization of gene functions in polyoma virus DNA. Proc. Natl. Acad. Sci. U.S.A. 73:4169-4173. 8. Helping, R. B., H. M. Goodman, and H. W. Boyer. 1974. Analysis of endonuclease R EcoRI fragments of DNA from lambdoid bacteriophages and other viruses by agarose-gel electrophoresis. J. Virol. 14:1235-1244. 9. Shirt, B. 1967. Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26:365-369. 10. Tai, H. T., C. A. Smith, P. A. Sharp, and J. Vinograd. 1972. Sequence heterogeneity in closed simian virus 40 deoxyribonucleic acid. J. Virol. 9:317-325. 11. Thorne, H. V., J. Evans, and D. Warden. 1968. Detection of biologically defective molecules in component I of polyoma virus DNA. Nature (London) 219:728-730. 12. Volckaert, G., R. Contreras, E. Soeda, A. Van De Voorde, and W. Fiers. 1977. Nucleotide sequence of simian virus 40 hind H restriction fragment. J. Mol. Biol. 110:467-510. 13. Weil, R., and J. Vinograd. 1963. The cyclic helix and cyclic coil forms of polyoma viral DNA. Proc. Natl. Acad. Sci. U.S.A. 50:730-738. 14. Wetmur, J. G., and N. Davidson. 1968. Kinetics of renaturation of DNA. J. Mol. Biol. 31:349-370. 15. Yang, R., K. Danna, A. Van De Voorde, and W. Fiers. 1975. Location of the small restriction fragments, Hind-L, Hind-M, and Hpa-E, on the simian virus 40 genome. Virology 68:260-265.
JOURNAL OF VIROLOGY, Nov. 1977, p. 695-700
Copyright i) 1977 American Society for Microbiology
Measurements of the Genome Sizes of Simian Virus 40 and Polyoma Virus LAUREN SOMPAYRAC AND KATHLEEN J. DANNA* Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309 Received for publication 21 June 1977
We have measured the genome sizes of simian virus 40 and polyoma virus and found them to be 5,010 ± 125 base pairs and 5,080 + 125 base pairs, respectively. respectively.
The genome sizes of two small DNA tumor viruses, simian virus 40 (SV40) and polyoma virus, have been estimated to be from 2.25 to 5.4 million daltons (1-4, 8, 10, 13, 14). This large range of values probably results from inaccuracies in some of the measuring techniques used, from strain differences that arise frequently during propagation of SV40 and polyoma (10, 11), and from the fact that size determinations for these DNA molecules have never been made relative to a standard of known molecular weight. Now, however, DNA of different virus strains can be characterized and compared by digestion with appropriate restriction enzymes and analysis of the products by gel electrophoresis. Also, Walter Fiers and his group have provided a molecular weight standard by determining the nucleotide sequence of the two contiguous HindII+HindIII fragments, H and I, of SV40 DNA, which together comprise the HindIII-D fragment (Fig. 1) (12; W. Fiers, personal communication). For these reasons, we decided to make a new determination of the genome sizes of well-characterized strains of SV40 and polyoma, using the HindIII-D fragment as a standard. Our stock of SV40 strain 776 was obtained from Harvey Ozer and is the same strain used in the sequence studies of Fiers et al. Largeplaque polyoma virus (Pasadena strain) was a kind gift from Tom Benjamin. Both viruses were extensively plaque purified, and stocks were made, using multiplicities of infection of less than 10' PFU per cell. DNA made from these stocks was characterized by restriction endonuclease digestion and gel electrophoresis (Fig. 2 and 3, Table 1). To obtain accurate length measurements, either polyoma or SV40 form I DNA was cleaved with the EcoRI enzyme (New England Biolabs) to yield full-length linear molecules. These were mixed with gel-purified HindIII-D fragments that had been circularized (see below) by treat-
ment with T4 DNA ligase (a kind gift of Marvin Caruthers) (7), spread for electron microscopy (Fig. 4), and measured with a Hewlett-Packard digitizer. Histograms of the results of these measurements are shown in Fig. 5. The HindIIID fragment is 10.5% as long as the SV40 genome and 10.35% as long as the polyoma genome. In preliminary experiments, we spread and measured mixtures of linear HindIII-D fragments and circularized HindIII-D fragments. Presumably because of difficulty in visualizing the ends of the linear molecules, they appeared about 2% shorter than the same molecules that had been circularized. To avoid these "end effects," we used the circularized HindIII-D fragments as a standard for all measurements presented here. We also checked that full-length EcoRI linear polyoma molecules could be cir-
695
"HIND II
D
FIG. 1. Physical map of the SV40genome showing cleavage sites for the EcoRI enzyme, HindIII enzyme (outer circle), and the combination ofHindII+HindIII enzymes (inner circle) (6, 15).
696
NOTES
J. VIROL.
cularized and that these circles were the same length as the linears. Since end effects are not significant for large molecules, these measurements indicate that the procedure of circularization itself does not change the apparent length of DNA molecules. Also, these experiments show that nuclease degradation did not occur during digestion with the EcoRI and HindIII enzymes, since such degradation would have prevented circularization under the conditions used for ligation. As a check on our electron microscopy results, two types of experiments were performed using gel electrophoresis. First, 32P-labeled SV40 DNA was digested with either the HindIII enzyme or HindII+HindIII enzymes and electrophoresed on a polyacrylamide slab gel. The counts per minute in the HindIII-D or HindII+HindIII-H plus I fragments were compared with the sum of the counts per minute in all the fragments. The percentage of counts per minute in a given fragment is taken to be equivalent to the fraction of the genome which that fragment represents. These results were consistent with the more accurate results obtained by electron microscopy (Table 2). As a second check, SV40 and polyoma form I DNAs were digested with the EcoRI FIG. 2. Major HindII+HindIII fragments of SV40 DNA and Hpa II fragments of polyoma DNA. BSC1 and NIH 3T3 cells were infected with SV40 and polyoma virus, respectively, at multiplicities of infection from 10 to 20 PFU/cell. Viral DNA isolated by the Hirt procedure (9) was phenol extracted, ethanol precipitated, and centrifuged to equilibrium in cesium chloride-ethidium bromide gradients. After thorough extraction with isopropanol to remove the ethidium bromide and dialysis to remove the cesium chloride, form I DNA was stored at -20'C. SV40 DNA I was digested with the combination of HindII+HindIII enzymes, and polyoma DNA I was digested with the Hpa II enzyme (New England Biolabs). The buffers used were 6.6 mM Tris (pH 7.5), 6.6 mM MgCl2, 50 mM NaCl, and 6.6 mM beta-mercaptoethanol for the Hind enzymes and 10 mM Tris (pH 7.5), 10 mM MgCl2, 6 mM KCl, 1 mM dithiothreitol, and 100 pg of gelatin per ml for the Hpa II enzyme. DNA was at a concentration of 2 to 20 pg/ml, and incubations were done for 6 to 12 h at 37°C in the presence of about 2 U of enzyme per mg of DNA. Digests were treated with RNase (heat treated, Worthington Biochemicals) at an enzyme concentration of 50 lpg/ml for 30 min at 37°C, extracted with phenol and chloroform, and run on a 40-cm, 4% polyacrylamide slab gel. These long gels were run typically for 36 h at 3 V/cm. The running buffer used in all gels was 0.04 M Tris (pH 7.7), 0.005 M sodium acetate, and 0.001 M EDTA. Gels were stained with ethidium bromide and photographed under UV light. Lane a shows the 11 major HindII+HindIII fragments of SV40 DNA, A through K. Lane b shows the 7 major Hpa II fragments ofpolyoma DNA, 1 through 7.
VOL. 24, 1977
NOTES
0 p No
*--
697
TABLE 1. Percentage of 32P counts per minute contained in HindII+HindIII fragments of SV40 DNA and in Hpa II fragments ofpolyoma DNA a SV40
HindII+
% cpm in
Polyoma HpaII
% cpm in
HindIII fragment
fragment"
fragment
fragment"
1 26.2 22.3 2 20.9 15.7 3 17.3 10.5 13.5 10.0 4 8.2 5 8.1 6 6.9 7.0 7 4.8 6.9 8 2.2 5.4 4.9 4.4 3.9 0.5 0.4 a Polyoma DNA and SV40 DNA were prepared as described in Fig. 2, except that 0.1 to 0.2 mCi of 32P was added to each 84-mm-diameter petri dish 24 h postinfection in low-phosphate medium. After electrophoresis of the digestion products on 4%/15% polyacrylamide step gels, the hands were excised, dissolved in 30% H202 at 80'C, and counted in Triton-toluene
A B C D E F G H I J K L M
counting solution. b Values given are averages of results obtained in several experiments.
enzyme and electrophoresed on a 0.7% agarose gel together with an Hha I digest of SV40 form I as a marker (Fig. 6). Hha I cleaves our strain of SV40 to yield two fragments, one 91% of the genome, and the other 9% (L. Sompayrac, unpublished data). Polyoma unit-length linear DNA migrated slightly slower than SV40 linear DNA (Fig. 6). Its position relative to the Hha I 91% marker indicated that the polyoma genome was approximately 2% larger than SV40 DNA. This again is consistent with our electron microscopy results. Our measurements indicate that the HindIIID fragment is 10.5% as large as the SV40 genome and 10.35% as large as the polyoma genome. Taken together with the results of Fiers et al. that there are 526 base pairs in the circularized HindIII-D fragment, we conclude that the genome of our strain of SV40 has 5,010 + 125 base pairs and that our strain of polyoma has a genome size of 5,080 ± 125 base pairs. Other ex-
8-
0
L FIG. 3. Minor HindII+HindIII fragments of SV40 DNA and Hpa H fragments of polyoma DNA. DNA , isQ, was prepared and digested as described in Fig. 2. Lane a is a 4% (upper portion)/12.5% polyacrylamide ^ N A step gel on which an Hpa II digest of polyoma DNA f I has been run to show minor fragment 8. Lane b is a ;%.; 4%/15% step gel overloaded with a HindII+HindIII brEmdigest of SV40 DNA in order to show the two minor U fragments, L and M.
698
J. VIROiL.
NOTES
5~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~4
4'.
10~
qr
1.-4
06.
It
9
s/.
* a 4a64 '*t**
r
~ ~ ~
4r
~
~
4
4a
~~
*~~~I ** ~~~~~~~~~~~~~~~~~~~~~~~ *5
a 94,t*
'5~~~~~~~~4
*
is~~~~~~~~~~~~~,C: t t ¼. ~ ~
C
* Ag
45
'S
4
*
a~~t',R ~~
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~4*
,
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
is I: c-*~~~~10
f~~~~~~~~~r~a.
nvj.o
~a~
4
of Hindill D fragments and EcoRI linears Circularized Hindill Dfragments~~~~~~~~~~~~~~~if 4 Electron micrograph FIG legthEcoI lnea 840DAmlceswrmidspado cabo-oatdgisi 0 andful a N (5)andphotgrahed itha Phlip EM0 lcrn irsoe I hspoedr formmid e fctcrm DA n 0v hs(H85 0 supede inaslto f5%fraie0 ae n abncae rd eedpe yohs fdstle mlTilitroa pedo te fim an drid afer a ethnol inse Grd wre hdwdwt ltnmplaimi throgh evaporator~~~~~~~~~~~~~~~I rotary
VOL. 24, 1977
NOTES
699
0)~~~~~~~~~~
.I 0
.55
.60
.65
inches
E z
6.0 inches
5.5
5.0
20
B) Polyomu Linears
~~~~~~~~~~~15 ~ ~ ~
~
~
~
~
~
~
~
~
~
pi 2SF Aft =t 55
60
.5
chess
E
6.0 inches FIG. 5. Histograms of polyoma and SV40 unitlength linear DNA measured with circularized HindIII-D fragments as a standard. Grids prepared as described in Fig. 4 with either SV40 (A) orpolyoma (B) unit-length linears were photographed, enlarged, and measured with a Hewlett-Packard digitizer and 9821 computer. For unit-length SV40 linears, the average values of these length measurements gave:
5.0
5.5
HindIII-D standard SV40 unit-length linear 0.591 inches (ca. 1.50 cm) 5.63 inches (ca. 14.30 cm)
=
10.5%
For polyoma: HindIII-D standard polyoma unit-length linear 0.590 inches (ca. 1.50 cm) 5.70 inches (ca. 14.48 cm)
10.35%
TABLE 2. Percentage of 32P counts per minute contained in HindIII-D and HindII+HindIII-H plus I fragments of SV40 DNA Enzyme used
Fragment studied
P cpm in fragment studied
cpm in frag32P total (all fragments) %mn tde 10.4 119,133
D HindIII 12,360 H plus I 10.3 HindII+HindIII 11,297 109,192 32P-labeled SV40 DNA was prepared, digested, electrophoresed, and counted as described in the legend of Fig. 2 and in Table 1. a
700
J. VIROL.
NOTES
100% Py 100% SV4O91 % SV40k~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
FIG. 6. Relative mobilities of SV40 DNA and polyoma DNA. Samples of polyoma and SV40 form I DNA (0.1 4) were digested with the EcoRI enzyme to yield unit-length linears. SV40 form I DNA (0.04 pg) was also digested with the Hha I enzyme to give a linear marker which is 91% of the SV40 genome. The buffer used for the EcoRI digestion was 0.1 M Tris (pH 7.5), 50 mM NaCl, and 5 mM MgCl2. For the Hha I digestions, a buffer of 6 mM Tris (pH 7.5), 50 mM NaCl, 6 mM MgCl2, 6 mM beta-mercaptoethanol, and 100 ,g of gelatin per ml was used. Digestions were done in a 50- d volume at an enzyme concentration of 50 U/ml for 2 h at 370C. Lane a: Polyoma unit-length linears alone. Lane b: Polyoma and SV40 unit-length linears and SV40 91% Hha I fragment. Lane c: SV40 unit-length linears alone. Top of gel is not shown. Actual migration distances were: polyoma unit-length linear DNA, 13.3 cm; SV40 unit-length linear DNA, 13.46 cm; SV40 Hha I 91% fragment, 14.10 cm. The 9% Hha I fragment of SV40 DNA ran off this gel.
perimenters should be able to adapt our size measurements to their variants of SV40 and polyoma either by direct comparison with our strains or by doing similar 32P labeling and enzyme digestions and comparing the fraction of counts in each of their fragments with our results in Table 1. We would like to thank John Heumann for introducing us to formamide spreading for electron microscopy and for writing the length measurement program for the Hewlett-Packard computer. We would also like to express appreciation to Joe Allen and Herbert Kroehl for allowing us to use the HewlettPackard digitizer and computer of the National Geophysical and Solar-Terrestrial Data Center, National Oceanic and At-
mospheric Administration. This research was supported by Public Health Service grant CA18212, awarded by the National Cancer Institute. L.S. is a postdoctoral fellow of the National Cancer Institute. LITERATURE CITED 1. Anderer, F. A., H. D. Schlumberger, M. A. Koch, H. Frank, and H. J. Eggers. 1967. Structure of simian virus 40. II. Symmetry and components of the virus particle. Virology 32:511-523. 2. Chen, M. C. Y., K. S. S. Chang, and N. P. Salzman. 1975. Studies of polyoma virus DNA: cleavage map of the polyoma virus genome. J. Virol. 15:191-198. 3. Crawford, L. V. 1964. The physical characteristics of polyoma virus. IV. The size of the DNA. Virology 22:149-152. 4. Crawford, L. V., and P. H. Black. 1964. The nucleic acid of simian virus 40. Virology 24:388-392.
5. Davis, R. W., M. Simon, and N. Davidson. 1971. Electron microscope heteroduplex methods for mapping regions of base sequence homology in nucleic acids.
Methods Enzymol. 21:413-428.
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