JOURNAL OF VIROLOGY, Feb. 1977, p. 792-795 Copyright © 1977 American Society for Microbiology
Vol. 21, No. 2 Printed in U.S.A.
Electron Microscopic Studies of Circular DNA in Mouse Embryo Fibroblasts Infected by Rauscher Leukemia Virus TSUYOSKI KAKEFUDA,1 GERALD G. LOVINGER,2* RAYMOND V. GILDEN, AND MASAKAZU HATANAKA3 Frederick Cancer Research Center, Viral Oncology Program, Frederick, Maryland 21701
Received for publication 19 August 1976
Using electron microscopy, a closed circular form of DNA (4.3 ,um in contour length) was detected in the nucleus of mouse embryo fibroblasts 2.5 h after infection by Rauscher murine leukemia virus. These circles were distinguishable from mitochondrial DNA by various criteria, including size, absence of secondary features, and resistance to EcoRI endonuclease.
Newly synthesized, unintegrated viral DNA has been detected shortly after infection of permissive cells by type C virus using autoradiography (6, 12). More direct studies using nucleic acid hybridization have indicated that linear forms of viral DNA are synthesized early in the cytoplasm (7, 10, 14). At later times, supercoiled circular viral DNA has been detected both by cesium chloride-ethidium bromide centrifugation (4) and by electron microscopy (3). This circular form of DNA is presumed to be a late intermediate in the formation of integrated proviral DNA (5). Electron microscopic studies showing the presence of circular forms of DNA in the nucleus of mouse embryo fibroblasts 2.5 h after infection are described here. Mouse embryo fibroblast (MEF) cultures were initiated from 14- to 21-day-old BALB/c embryos. Primary cultures in 32-ounce (about 960-ml) trypsinized and and replated bottles were trypsinized 9b0-ml)bottles1xere oEalein bottles with growth medium consisting of Eagle minimal essential medium supplemented with 10% fetal bovine serum. After DEAE-dextran treatment, Rauscher murine leukemia virus (R-MuLV) was added in 20 ml of medium to subconfluent cell monolayers at a multiplicity of infection of 1 to 2 infectious units. At 60 min postinfection, the virus-containing medium was decanted ana and fresh growth medium was added. At 2.5 h postinfection, the cell monolayers were washed with cold Earle balanced salt solution containing 10-2 M sodium azide, and the cells were scraped from the glass with a
wadded.aectea5 rrestinfeciowtnte cellmolays
'Chemistry Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20014. 2 Send reprint requests to: Dr. G. G. Lovinger, Frederick Cancer Research Center, P.O. Box B, Frederick, MD
21701. 3 Laboratory of DNA Tumor Viruses, National Cancer Institute, National Institutes of Health, Bethesda, MD 20014.
T.
4 5x 10
Ad
' 300
u
l
200-
/
100
\ L
_
_ 5
_ 10
_
_ 15
FRACTION NUMBER FIG. 1. Neutral glycerol gradient sedimentation of DNA in BALBIc MEF 2.5 h after infection by RMuLV. The cells of three 32-ounce (about 960-ml) bottles of MEF harvested 2.5 h after R-MuLV infection were combined (-1.2 The x 107 cells) and packed by cells consisting were gently pipetcentrifugation. low-speed ted into 0.35 ml of lysing solution of 0.01 M Tris-hydrochloride, pH 7.4, 0.1 M NaCl, 1 mM EDTA, 2% sodium lauroyl sarcosinate (SLS), and 50 Mg ofproteinase K (EM Laboratories, Inc.) per ml. The lysing solution was layered on a linear 10 to 30% glycerol gradient in 0.01 M Tris-hydrochloride, pH 7.4, 0.1 M NaCl, 1 mM EDTA, and 0.05% SLS. Lysis was allowed to proceed 4 h at 20°C. Centrifugation was then performed for 10 h at 24,000 rpm in a SW41 rotor at 20°C. The following markers Spinco were also centrifuged: T4 bacteriophage DNA (61S); adenovirus 2 DNA (25S); and fragment 2 ofendonuclease EcoRI digestion of lambda DNA (4.5 x 106 daltons) (1). The DNA was fractionated and precipitated without DNA carrier in 2.5 volumes of ethanol. Solubilized DNA was allowed to hybridize with 800 to 1,200 cpm of single-stranded R-MuLV complementary [3H]DNA for 64 h in 2 x SSC (SSC = 0.15 M NaCI + 0.015 M sodium citrate) at 67°C in a talvolmeof10 sodiuPreparati o t Comple total volume of 100 Ml. Preparation of the complementary DNA and analysis for DNA-DNA hybrids
using single-strand-specific nuclease Si has been described (10). 792
VOL. 21, 1977 rubber policeman. Where indicated, the nuclear and cytoplasmic fractions were separated using 1% Nonidet P-40 (11). After lysis in hypotonic-detergent solution, the DNA was sedimented by rate zonal centrifugation at neutral pH. As reported previously (10), linear strands of viral DNA were found 60 to 90 min after infection by R-MuLV. At 2.5 h after infection, cellular DNA was analyzed by rate zonal centrifugation at neutral pH (Fig. 1). Nucleic acid hybridization using R-MuLV complementary DNA (cDNA) prepared from purified R-MuLV virions (10) showed three peaks of R-MuLV-specific se-
793 quences. Peak I, fractions 1 to 2, has been observed in uninfected BALB/c MEF (9) and is presumably integrated endogenous mouse viral sequences that cross-hybridize with R-MuLV. Peak II, fractions 11 to 13, contained a viral DNA species sedimenting faster than adenovirus DNA, with a sedimentation value of approximately 30 to 35S at neutral pH. Peak III, fractions 15 to 17, was presumably the unintegrated linear or open circular form of the provirus. Examination by electron microscopy of the three peaks containing R-MuLV sequences showed that peak II contained supercoiled cirNOTES
'V: N' ~ ~ ~
C
~
~
~
~
.~~~C
FIG. 2. BALE/c MEF cultures were harvested 2.5 h after R-MuLV infection. The cytoplasmic fraction was separated from the nuclear fraction by use of 1 % Nonidet P-40 (11). Each cellular fraction was lysed and centrifuged as described in Fig. 1. The peak II region of the gradient was pooled. To each pool sample (20 d) 50% (vol/vol) formamide, 0.1 M Tris-hydrochloride (pH 8.5), 10 mMEDTA, and 40 pg of cytochrome cper ml were added and spread over the surface of 10 mM Tris-hydrochloride (pH 8.5), 1 mM EDTA, and 1 7% (volI vol) formamide. The samples stained with uranyl acetate and platinum-palladium were observed under the electron microscope. Electron micrographs of circular DNA molecules isolated from nuclei of R-MuLVinfected cells (a) before and (b) after DNase I treatment (100 pg in 0.1 M Tris-hydrochloride, pH 8.0, 5 mM MgCl2) and (c) from infected cytoplasm. The arrow indicates a D-loop in one of the catenated mitochondrial DNA molecules. The bar represents 1.0 pum.
794
J. VIROL.
NOTES
cular DNA. In subsequent preparations of infected cells, nuclear and cytoplasmic fractions were separated before lysis and rate zonal centrifugation were performed. Electron micrographs of typical DNA molecules isolated from nuclei of virus-infected cells (Fig. 2a,b) and mitochondrial DNA from the cytoplasm of infected cells (Fig. 2c) are presented. Virtually no D-loop (8), replicative intermediate forms of semiconservative replication having forks (13) and no catenated dimer molecules were observed in the nuclear preparation of infected cells (Fig. 2a); however, they were frequently observed in mitochondrial DNA (Fig. 2c). The circular molecules isolated from nuclei 2.5 h after infection showed a mean contour length of 4.28 + 0.48 ,um (Fig. 3a). Treatment with the restriction enzyme EcoRI (Fig. 3b) did not change the distribution of contour lengths. Whereas mouse mitochondrial DNA is cleaved by this enzyme (2), closed circular viral DNA of Moloney virus is not (3). Figures 3c and 3d represent an infected cytoplasmic fraction and uninfected whole-cell preparations, respectively. The contour lengths of DNA molecules from these two preparations were 5.0 + 0.35 a value in agreement with that established for mouse mitochondrial DNA (2). The circular DNA molecules in both the cytoplasm of infected cells and uninfected whole-cell extracts were completely eliminated by treatment with the enzyme EcoRI. The following evidence suggests that the cir-
,tm,
cular DNA found in the nucleus of R-MuLV-
infected MEF is viral. First, the circles are found in gradient fractions of DNA that show . .....MuLV... Second, Secod the te hbridization to tocDN CDNA of Of R R-MuLV. hybridization small circles (4.3 ,um) are found in infected but not uninfected cells. Third, the 4.3-,um circles are distinguishable from mitochondrial DNA by smaller contour length, nuclear site, and insensitivity to EcoRI endonuclease. Gianni et al. (3) reported electron microscopic evidence of closed circular DNA in JLS-V9 cells 9 hhafe after infection by Moloney leukemia mia virus.
9enave ionfeionn we conlfrmea
tnat
lesu in result
vMrus R-MelulV-
infected cells and, in addition, have localized closed circular DNA to the nucleus; however, the size of the circles is apparently larger than that reported by Gianni et al. (3). The explanation for this difference is unclear. This work was performed at Flow Laboratories, Inc., Rockville, Md., and was supported by Public Health Service contract N01-CP-53530 with the National Cancer Institute. We thank T. M. Bak and H. P. Ling for technical assistance. We also thank Richard Roberts, Cold Spring Harbor Laboratory, for supplying the lambda bacteriophage DNA fragment and adenovirus DNA used as markers.
(a)
(b)
4
30
3 v
20
0
10
2
'Lu
o
(d)
_
V
g D Z
30 20
aa 10 2
3 4
5
6 2 3
4
5
6 7
LENGTH (y)
FIG. 3. Histogram showing distribution of the length of circular DNA molecules. Procedure for preparing, lysing, centrifuging, and isolating peak II region DNA is as described in Fig. 2. Cellular fractions from infected cell cultures and whole cells from uninfected cultures (approximately 2.4 x 107 cells)
were used for each preparation. The samples were prepared for electron microscopy also as described in Fig. 2. The magnification was calibrated by grating replica and Oxl74 DNA added to the sample as a known marker. The DNA preparations were incubated with DNase 1 to provide open circle molecules for length measurements. (a) Circular DNA isolated from nuclei of R-MuLV-infected cells. (b) Similar to (a) but the DNA was incubated with endonuclease EcoRI (New England Bio-Labs) in 100 mM Trishydrochloride (pH 7.5), 10 mM MgCl2, 50 mM NaCl at 370C for 30 min. The enzyme concentration was adjusted to be sufficient to introduce a single doublestranded break in more than 95% of the colicin El plasmid DNA (13) and simian virus 40 DNA. (c) Circular DNA from virus-infected cytoplasm. (d)
Sample from uninfected whole cells. LITERATURE
ITED CIE
1. Allet, B., P. G. N. Jeppesen, K. J. Katagiri, and H. Delius. 1973. Mapping the DNA fragments produced by cleavage of DNA with endonuclease RI. Nature (London) 241:120-122. 2. Brown, W. M., and J. Viongrad. 1974. Restriction endo-
VOL. 21, 1977
3.
4.
5.
6.
7.
8.
nuclease cleavage maps of animal mitochondrial DNAs. Proc. Natl. Acad. Sci. U.S.A. 71:4617-4621. Gianni, A. M., J. R. Hutton, D. Smotkin, and R. A. Weinberg. 1976. Proviral DNA of Moloney leukemia virus: purification and visualization. Science 191:569571. Gianni, A. M., D. Smotkin, and R. A. Weinberg. 1975. Murine leukemia virus: detection of unintegrated double-stranded DNA forms of provirus. Proc. Natl. Acad. Sci. U.S.A. 72:447-451. Guntaka, R. V., B. W. J. Mahy, J. M. Bishop, and H. Varmus. 1975. Ethidium bromide inhibits appearance of closed circular viral DNA and integration of virus-specific DNA in duck cells infected by avian sarcoma virus. Nature (London) 253:507-511. Hatanaka, M., T. Kakefuda, R. V. Gilden, E. A. 0. Callan. 1971. Cytoplasmic DNA synthesis induced by RNA tumor viruses. Proc. Natl. Acad. Sci. U.S.A. 68:1844-1847. Kakefuda, T., C. W. Dingman, T. M. Bak, M. Hatanaka, and Y. Kitano. 1974. Reverse transcription of the viral genome associated with the plasma membrane after infection with RNA tumor viruses. Cancer Res. 34:679-688. Kasamatsu, H., and J. Vinograd. 1973. Unidirectionality of replication in mouse mitochondrial DNA. Na-
NOTES
795
ture (London) New Biol. 241:103-105. 9. Lovinger, G. G., R. A. Klein, R. V. Gilden, and M. Hatanaka. 1974. Unintegrated murine leukemia viral DNA in newly infected cells. Virology 62:280283. 10. Lovinger, G. G., R. A. Klein, H. P. Ling, R. V. Gilden, and M. Hatanaka. 1975. Kinetics of murine type C viral specific DNA synthesis in newly infected cells. J. Virol. 16:824-831. 11. Penman, S. 1969. Preparation of purified nuclei and nucleoli from mammalian cells, p. 35-48. In K. Habel and N. P. Salzman (ed.), Fundamental techniques in virology. Academic Press Inc., New York. 12. Robin, M. S., S. Salzberg, and M. Green. 1974. Cytoplasmic synthesis of viral DNA early during infection and cell transformation by the murine sarcoma-leukemia virus. Intervirology 4:268-278. 13. Tomizawa, J., Y. Sakakibara, and T. Kakefuda. 1974. Replication of colicin El plasmid DNA in cell extracts. Origin and direction of replication. Proc. Natl. Acad. Sci. U.S.A. 71:2260-2264. 14. Varmus, H. E., R. V. Guntaka, W. J. W. Fan, S. Heasley, and M. J. Bishop. 1974. Synthesis of viral DNA in the cytoplasm ofduck embryo fibroblasts and in enucleated cells after infection by avian sarcoma virus. Proc. Natl. Acad. Sci. U.S.A. 71:3874-3878.
Vol. 21, No. 2 Printed in U.S.A.
Electron Microscopic Studies of Circular DNA in Mouse Embryo Fibroblasts Infected by Rauscher Leukemia Virus TSUYOSKI KAKEFUDA,1 GERALD G. LOVINGER,2* RAYMOND V. GILDEN, AND MASAKAZU HATANAKA3 Frederick Cancer Research Center, Viral Oncology Program, Frederick, Maryland 21701
Received for publication 19 August 1976
Using electron microscopy, a closed circular form of DNA (4.3 ,um in contour length) was detected in the nucleus of mouse embryo fibroblasts 2.5 h after infection by Rauscher murine leukemia virus. These circles were distinguishable from mitochondrial DNA by various criteria, including size, absence of secondary features, and resistance to EcoRI endonuclease.
Newly synthesized, unintegrated viral DNA has been detected shortly after infection of permissive cells by type C virus using autoradiography (6, 12). More direct studies using nucleic acid hybridization have indicated that linear forms of viral DNA are synthesized early in the cytoplasm (7, 10, 14). At later times, supercoiled circular viral DNA has been detected both by cesium chloride-ethidium bromide centrifugation (4) and by electron microscopy (3). This circular form of DNA is presumed to be a late intermediate in the formation of integrated proviral DNA (5). Electron microscopic studies showing the presence of circular forms of DNA in the nucleus of mouse embryo fibroblasts 2.5 h after infection are described here. Mouse embryo fibroblast (MEF) cultures were initiated from 14- to 21-day-old BALB/c embryos. Primary cultures in 32-ounce (about 960-ml) trypsinized and and replated bottles were trypsinized 9b0-ml)bottles1xere oEalein bottles with growth medium consisting of Eagle minimal essential medium supplemented with 10% fetal bovine serum. After DEAE-dextran treatment, Rauscher murine leukemia virus (R-MuLV) was added in 20 ml of medium to subconfluent cell monolayers at a multiplicity of infection of 1 to 2 infectious units. At 60 min postinfection, the virus-containing medium was decanted ana and fresh growth medium was added. At 2.5 h postinfection, the cell monolayers were washed with cold Earle balanced salt solution containing 10-2 M sodium azide, and the cells were scraped from the glass with a
wadded.aectea5 rrestinfeciowtnte cellmolays
'Chemistry Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20014. 2 Send reprint requests to: Dr. G. G. Lovinger, Frederick Cancer Research Center, P.O. Box B, Frederick, MD
21701. 3 Laboratory of DNA Tumor Viruses, National Cancer Institute, National Institutes of Health, Bethesda, MD 20014.
T.
4 5x 10
Ad
' 300
u
l
200-
/
100
\ L
_
_ 5
_ 10
_
_ 15
FRACTION NUMBER FIG. 1. Neutral glycerol gradient sedimentation of DNA in BALBIc MEF 2.5 h after infection by RMuLV. The cells of three 32-ounce (about 960-ml) bottles of MEF harvested 2.5 h after R-MuLV infection were combined (-1.2 The x 107 cells) and packed by cells consisting were gently pipetcentrifugation. low-speed ted into 0.35 ml of lysing solution of 0.01 M Tris-hydrochloride, pH 7.4, 0.1 M NaCl, 1 mM EDTA, 2% sodium lauroyl sarcosinate (SLS), and 50 Mg ofproteinase K (EM Laboratories, Inc.) per ml. The lysing solution was layered on a linear 10 to 30% glycerol gradient in 0.01 M Tris-hydrochloride, pH 7.4, 0.1 M NaCl, 1 mM EDTA, and 0.05% SLS. Lysis was allowed to proceed 4 h at 20°C. Centrifugation was then performed for 10 h at 24,000 rpm in a SW41 rotor at 20°C. The following markers Spinco were also centrifuged: T4 bacteriophage DNA (61S); adenovirus 2 DNA (25S); and fragment 2 ofendonuclease EcoRI digestion of lambda DNA (4.5 x 106 daltons) (1). The DNA was fractionated and precipitated without DNA carrier in 2.5 volumes of ethanol. Solubilized DNA was allowed to hybridize with 800 to 1,200 cpm of single-stranded R-MuLV complementary [3H]DNA for 64 h in 2 x SSC (SSC = 0.15 M NaCI + 0.015 M sodium citrate) at 67°C in a talvolmeof10 sodiuPreparati o t Comple total volume of 100 Ml. Preparation of the complementary DNA and analysis for DNA-DNA hybrids
using single-strand-specific nuclease Si has been described (10). 792
VOL. 21, 1977 rubber policeman. Where indicated, the nuclear and cytoplasmic fractions were separated using 1% Nonidet P-40 (11). After lysis in hypotonic-detergent solution, the DNA was sedimented by rate zonal centrifugation at neutral pH. As reported previously (10), linear strands of viral DNA were found 60 to 90 min after infection by R-MuLV. At 2.5 h after infection, cellular DNA was analyzed by rate zonal centrifugation at neutral pH (Fig. 1). Nucleic acid hybridization using R-MuLV complementary DNA (cDNA) prepared from purified R-MuLV virions (10) showed three peaks of R-MuLV-specific se-
793 quences. Peak I, fractions 1 to 2, has been observed in uninfected BALB/c MEF (9) and is presumably integrated endogenous mouse viral sequences that cross-hybridize with R-MuLV. Peak II, fractions 11 to 13, contained a viral DNA species sedimenting faster than adenovirus DNA, with a sedimentation value of approximately 30 to 35S at neutral pH. Peak III, fractions 15 to 17, was presumably the unintegrated linear or open circular form of the provirus. Examination by electron microscopy of the three peaks containing R-MuLV sequences showed that peak II contained supercoiled cirNOTES
'V: N' ~ ~ ~
C
~
~
~
~
.~~~C
FIG. 2. BALE/c MEF cultures were harvested 2.5 h after R-MuLV infection. The cytoplasmic fraction was separated from the nuclear fraction by use of 1 % Nonidet P-40 (11). Each cellular fraction was lysed and centrifuged as described in Fig. 1. The peak II region of the gradient was pooled. To each pool sample (20 d) 50% (vol/vol) formamide, 0.1 M Tris-hydrochloride (pH 8.5), 10 mMEDTA, and 40 pg of cytochrome cper ml were added and spread over the surface of 10 mM Tris-hydrochloride (pH 8.5), 1 mM EDTA, and 1 7% (volI vol) formamide. The samples stained with uranyl acetate and platinum-palladium were observed under the electron microscope. Electron micrographs of circular DNA molecules isolated from nuclei of R-MuLVinfected cells (a) before and (b) after DNase I treatment (100 pg in 0.1 M Tris-hydrochloride, pH 8.0, 5 mM MgCl2) and (c) from infected cytoplasm. The arrow indicates a D-loop in one of the catenated mitochondrial DNA molecules. The bar represents 1.0 pum.
794
J. VIROL.
NOTES
cular DNA. In subsequent preparations of infected cells, nuclear and cytoplasmic fractions were separated before lysis and rate zonal centrifugation were performed. Electron micrographs of typical DNA molecules isolated from nuclei of virus-infected cells (Fig. 2a,b) and mitochondrial DNA from the cytoplasm of infected cells (Fig. 2c) are presented. Virtually no D-loop (8), replicative intermediate forms of semiconservative replication having forks (13) and no catenated dimer molecules were observed in the nuclear preparation of infected cells (Fig. 2a); however, they were frequently observed in mitochondrial DNA (Fig. 2c). The circular molecules isolated from nuclei 2.5 h after infection showed a mean contour length of 4.28 + 0.48 ,um (Fig. 3a). Treatment with the restriction enzyme EcoRI (Fig. 3b) did not change the distribution of contour lengths. Whereas mouse mitochondrial DNA is cleaved by this enzyme (2), closed circular viral DNA of Moloney virus is not (3). Figures 3c and 3d represent an infected cytoplasmic fraction and uninfected whole-cell preparations, respectively. The contour lengths of DNA molecules from these two preparations were 5.0 + 0.35 a value in agreement with that established for mouse mitochondrial DNA (2). The circular DNA molecules in both the cytoplasm of infected cells and uninfected whole-cell extracts were completely eliminated by treatment with the enzyme EcoRI. The following evidence suggests that the cir-
,tm,
cular DNA found in the nucleus of R-MuLV-
infected MEF is viral. First, the circles are found in gradient fractions of DNA that show . .....MuLV... Second, Secod the te hbridization to tocDN CDNA of Of R R-MuLV. hybridization small circles (4.3 ,um) are found in infected but not uninfected cells. Third, the 4.3-,um circles are distinguishable from mitochondrial DNA by smaller contour length, nuclear site, and insensitivity to EcoRI endonuclease. Gianni et al. (3) reported electron microscopic evidence of closed circular DNA in JLS-V9 cells 9 hhafe after infection by Moloney leukemia mia virus.
9enave ionfeionn we conlfrmea
tnat
lesu in result
vMrus R-MelulV-
infected cells and, in addition, have localized closed circular DNA to the nucleus; however, the size of the circles is apparently larger than that reported by Gianni et al. (3). The explanation for this difference is unclear. This work was performed at Flow Laboratories, Inc., Rockville, Md., and was supported by Public Health Service contract N01-CP-53530 with the National Cancer Institute. We thank T. M. Bak and H. P. Ling for technical assistance. We also thank Richard Roberts, Cold Spring Harbor Laboratory, for supplying the lambda bacteriophage DNA fragment and adenovirus DNA used as markers.
(a)
(b)
4
30
3 v
20
0
10
2
'Lu
o
(d)
_
V
g D Z
30 20
aa 10 2
3 4
5
6 2 3
4
5
6 7
LENGTH (y)
FIG. 3. Histogram showing distribution of the length of circular DNA molecules. Procedure for preparing, lysing, centrifuging, and isolating peak II region DNA is as described in Fig. 2. Cellular fractions from infected cell cultures and whole cells from uninfected cultures (approximately 2.4 x 107 cells)
were used for each preparation. The samples were prepared for electron microscopy also as described in Fig. 2. The magnification was calibrated by grating replica and Oxl74 DNA added to the sample as a known marker. The DNA preparations were incubated with DNase 1 to provide open circle molecules for length measurements. (a) Circular DNA isolated from nuclei of R-MuLV-infected cells. (b) Similar to (a) but the DNA was incubated with endonuclease EcoRI (New England Bio-Labs) in 100 mM Trishydrochloride (pH 7.5), 10 mM MgCl2, 50 mM NaCl at 370C for 30 min. The enzyme concentration was adjusted to be sufficient to introduce a single doublestranded break in more than 95% of the colicin El plasmid DNA (13) and simian virus 40 DNA. (c) Circular DNA from virus-infected cytoplasm. (d)
Sample from uninfected whole cells. LITERATURE
ITED CIE
1. Allet, B., P. G. N. Jeppesen, K. J. Katagiri, and H. Delius. 1973. Mapping the DNA fragments produced by cleavage of DNA with endonuclease RI. Nature (London) 241:120-122. 2. Brown, W. M., and J. Viongrad. 1974. Restriction endo-
VOL. 21, 1977
3.
4.
5.
6.
7.
8.
nuclease cleavage maps of animal mitochondrial DNAs. Proc. Natl. Acad. Sci. U.S.A. 71:4617-4621. Gianni, A. M., J. R. Hutton, D. Smotkin, and R. A. Weinberg. 1976. Proviral DNA of Moloney leukemia virus: purification and visualization. Science 191:569571. Gianni, A. M., D. Smotkin, and R. A. Weinberg. 1975. Murine leukemia virus: detection of unintegrated double-stranded DNA forms of provirus. Proc. Natl. Acad. Sci. U.S.A. 72:447-451. Guntaka, R. V., B. W. J. Mahy, J. M. Bishop, and H. Varmus. 1975. Ethidium bromide inhibits appearance of closed circular viral DNA and integration of virus-specific DNA in duck cells infected by avian sarcoma virus. Nature (London) 253:507-511. Hatanaka, M., T. Kakefuda, R. V. Gilden, E. A. 0. Callan. 1971. Cytoplasmic DNA synthesis induced by RNA tumor viruses. Proc. Natl. Acad. Sci. U.S.A. 68:1844-1847. Kakefuda, T., C. W. Dingman, T. M. Bak, M. Hatanaka, and Y. Kitano. 1974. Reverse transcription of the viral genome associated with the plasma membrane after infection with RNA tumor viruses. Cancer Res. 34:679-688. Kasamatsu, H., and J. Vinograd. 1973. Unidirectionality of replication in mouse mitochondrial DNA. Na-
NOTES
795
ture (London) New Biol. 241:103-105. 9. Lovinger, G. G., R. A. Klein, R. V. Gilden, and M. Hatanaka. 1974. Unintegrated murine leukemia viral DNA in newly infected cells. Virology 62:280283. 10. Lovinger, G. G., R. A. Klein, H. P. Ling, R. V. Gilden, and M. Hatanaka. 1975. Kinetics of murine type C viral specific DNA synthesis in newly infected cells. J. Virol. 16:824-831. 11. Penman, S. 1969. Preparation of purified nuclei and nucleoli from mammalian cells, p. 35-48. In K. Habel and N. P. Salzman (ed.), Fundamental techniques in virology. Academic Press Inc., New York. 12. Robin, M. S., S. Salzberg, and M. Green. 1974. Cytoplasmic synthesis of viral DNA early during infection and cell transformation by the murine sarcoma-leukemia virus. Intervirology 4:268-278. 13. Tomizawa, J., Y. Sakakibara, and T. Kakefuda. 1974. Replication of colicin El plasmid DNA in cell extracts. Origin and direction of replication. Proc. Natl. Acad. Sci. U.S.A. 71:2260-2264. 14. Varmus, H. E., R. V. Guntaka, W. J. W. Fan, S. Heasley, and M. J. Bishop. 1974. Synthesis of viral DNA in the cytoplasm ofduck embryo fibroblasts and in enucleated cells after infection by avian sarcoma virus. Proc. Natl. Acad. Sci. U.S.A. 71:3874-3878.
