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DNA methylation of viruses infecting a eukaryotic Chlorella -like green alga

DNA methylation of viruses infecting a eukaryotic Chlorella -like green alga Downloaded from https://academic.oup.com/nar/article/13/10/3471/2381446 by DeepDyve user on 20 August 2020 volume 13 Number 10 1985 Nucleic Acid s Research DNA methylation of viruses infecting a eukaryotk Chlorella-Uke green alga James L.Van Etten, Anne M.Schuster, Lois Girton, Dwight E.Burbank, David Swinton and Stanley Hattman1 Department of Plant Pathology, University of Nebraska, Lincoln, NE 68583-0722, and 'Department of Biology, University of Rochester, Rochester, NY 14627, USA Received 7 March 1985; Revised and Accepted 18 April 1985 ABSTRACT The genomic DNAB of the eukaryotic Chlorella-lilce green alga, strain NC64A, and eleven of its viruses all contain significant levels of 5-methyldeoxycytidine. In addition, the host DNA as well as six of the viral DNAs also contain N -methyldeoxyadenosine. At least some of the methylated bases in the host reside in different base sequences than the methylated bases in the viruses as shown by differential susceptibility to restriction endonuclease enzymes. This suggests that the viruses encode for DNA methyltransferases with sequence specificities different from that of the host enzyme. INTRODDCTIQN We have recently discovered a number of viruses in fresh water ponds and rivers in Illinois, North Carolina, and South Carolina which form plaques on lawns of a unicellular, eukaryotic, Chlorella-like green alga (1). All of these viruses are polyhedrons (160 to 190 nm in diameter) and contain large dsDNA genomes (ca. 300 kbp as estimated by summing restriction fragments). Although these viral DNAs exhibited extensive homology with the previously described PBCV-1 virus DNA (1, 2) , the viruses can be distinguished from one another and from PBCV-1 by at least one of the following characteristics: plaque size, DNA restriction patterns, or resistance to certain DNA restriction endonucleases. We have grouped the viruses into 5 classes based on the sensitivity or resistance of their DNAs to 15 restriction endonucleases (1). The resistance of the viral DNAs to certain restriction enzymes suggested that the viral DNAs might contain modified bases. The present report describes base analyses of eleven of these viral DNAs as well as the host Chlorella DNA and establishes the presence of 5-methyldeoxycytidine (m dC) in © IRL Press Limited, Oxford , England. 3471 Downloaded from https://academic.oup.com/nar/article/13/10/3471/2381446 by DeepDyve user on 20 August 2020 Nucleic Acids Research host nuclear DNA and all the viral DNAs. The host DNA, as well as six of the viral DNAs, also contain N -methyldeoxyadenoslne (m dA). Finally we show that at least some of the methylated bases in the host reside in different base sequences from those in the viruses. MATERIALS AND METHODS Preparation of viral and host DNAs. The production and purification of the viruses (PBCV-1, NC-1A, NC-1B, NC-1C, NC-1D, SC-1A, SC-1B, I1-2A, I1-2B, I1-3A, and I1-3D) and the growth of the host Chlorella NC64A on MBBM medium have been described (1, 2) . Viral DNAB were isolated on CsCl gradients from purified viruses as described (3). Host nuclear DNA was also isolated and separated from chloroplast DNA on CsCl gradients as described (4) except that the cells were disrupted in a mortar and pestle and the DNA extracts were not intentionally sheared. The purified DNAs were enzymatically digested (to avoid potential acid degradation of any unusual bases) and the resulting deoxynucleosides were analyzed by both ion-exclusion and reverse-phase high performance liquid chromatography as described (5). The DNAs were treated with restriction endonucleases according to manufacturers' recommendations and fragments were separated by electrophoresis on 1.2% agarose gels in 0.08 M Tris-phosphate and 0.008 M EDTA, pH 8.5. To determine if m dA was responsible for the resistance of PBCV-1 DNA to the restriction endonuclease Mbol. we used plasmid pLG164, which contains a 16 kbp BamHI fragment of PBCV-1 (designated B6) cloned into pBR322. This plasmid was purified from its original host (£. colj. strain LE392) and transformed into £. coll strain GM2163 using standard procedures (6). fi. coli GM2163, kindly provided by Dr. Martin Marinus, lacks both dam and den methylating activity (7). The plasmid was restricted with BamHI and the viral fragment B6 insert DNA was recovered from low melting agarose gels by repeated phenol and phenol:chloroform extractions. The purified fragment was nick-translated with d. - P dCTP (800 Ci/mmole) using a nick translation kit (Bethesda Research Labs) according to the manufacturer's recommendations. This labeled fragment was used to probe Southern blots (8) of PBCV-1 virus 3472 Downloaded from https://academic.oup.com/nar/article/13/10/3471/2381446 by DeepDyve user on 20 August 2020 Nucleic Acids Research DNA and pLG164/GH2163 DNA which had been sequentially digested with restriction endonucleases BamHI and either Dpnl. Mbol. or Sflu.3AI. RESDLTS Base compositional analysis. The percentages of modified bases found in eleven viral DNAs and the host nuclear DNA are summarized in Table 1. Although all the viral DNAa contained readily detectable levels of m dC, viruses NC-1A, NC-1B, I1-2A, I1-2B, I1-3A, and I1-3D contained high levels of this nucleoside (7.0 to 13.4%). With the exception of NC-ID (0.33% m dC), the remaining four viruses contained intermediate levels (1.7 to 2%) of m dC. The host Chlorella nuclear DNA contained 21.2% m dC. In addition to m dC, m dA was present in the host nuclear DNA (0.6%) and in six of the seven viruses from North and South Carolina (1.5 to 8.2%). The virus NC-ID and the four Illinois viruses lacked detectable m dA. The lower limit of detection in these experiments was about 0.02%. The overall G + C content of the host nuclear DNA was 67 mole percent and the viral DNAs ranged from 39 to 41 mole percent. There was no indication that the viruses contained modified bases other than m dC and m dA. Table 1. Concentration of methylated bases in the viral DNAs and their host, Chlorella NC64A. Percent Methylated Bases Virus m5dC a ,b PBCV-1 1.86 (6)c 1.45 (3) NC-1A 7.06 (5) 7.32 (2) NC-1B 13.36 (4) 8.19 (2) NC-1C 1.72 1.59 (4) (2) NC-ID 0.33 <0.08 (4) (2) SC-1A 1.94 7.34 (6) (3) SC-IB 2.04 7.45 (4) (2) I1-2A 9.38 <0.01 (4) (2) I1-2B 10.85 <0.08 (6) (4) 9.67 I1-3A (4) <0.03 (2) 12.62 I1-3D (5) <0.01 (3) Host nuclear 21.23 (6) 0.59 (4) + deoxyuridine. ^percen ^p f t ^ per dC + percent m dA per dA + nrdA + deoxyinosine. Number of determinations 3473 Downloaded from https://academic.oup.com/nar/article/13/10/3471/2381446 by DeepDyve user on 20 August 2020 Nucleic Acids Research 12345 6 Figure 1. Electrophoresis of genomic PBCV-1 DNA treated with BflmHI — 231 (lanes 1, 3, 5) or a 16 kbp PBCV-1 BaraHl fragment, B6, cloned in an — 9 4 E. coli dam~. dcm~ host (lanes 2, 4, 6). All of the DNAB were first — 6.7 treated with BamHi followed by digestion with either Hbol (lanes 1 . * and 2) , Dpnl (lanes 3 and 4) or Sflli3AI (lanes 5 and 6) . The bands were localized by probing with the 32P-labeled PBCV-1 B6 fragment after Southern blotting. Molecular weights in kbp are indicated on the 2 3 Bide o f th e figure. Note: the PBCV-1 2 O DNA fragment B6 is digested by QED.1 and resistant to Mboli after cloning fragment B6 in the dam-, dcm~ £. coli it is resistant to Dpnl and digested by Mbol. — 056 Sequence specificity of the methylation. We reported previously that the DNAs from PBCV-1, NC-1A, NC-1B, NC-1C, SC-1A, and SC-1B were resistant to cleavage by Mbol and sensitive to Dpnl (1) which suggests that m dA is present in the GATC sequence. To confirm that the resistance of PBCV-1 DNA to Hbfil and its sensitivity to Djjn.1 was due to GATC methylation, plasmid pLG164, containing a PBCV-1 BamHI fragment, was grown in an £. colj. dcm , dam strain. Following re-isolation, the PBCV-1 fragment was now sensitive to Hbfil but resistant to DJJJII (Fig. 1) . This result indicates that during propagation in the dam host, the cloned PBCV-1 segment lost the GATC methylation present in the genomic virion DNA. Host nuclear DNA shows a different pattern of 3474 Downloaded from https://academic.oup.com/nar/article/13/10/3471/2381446 by DeepDyve user on 20 August 2020 Nucleic Acids Research Figure 2. Electrophoresie of 1 2 3 4 5 6 Chlorella nuclear DNA after treat- ment with either Sau3AI (lane 2), Mbol (lane 3), Q£n.I (lane 4) , Hfifil (lane 6) or flEall (lane 7) . The DNA in lanes 1 and 5 was untreated. - 0 56 resistance/sensitivity to these restriction enzymes. The host DNA was cleaved by Mbol (and Sau3AI) but was partially resistant to Dpnl (Fig. 2, lanes 1 to 4). Likewise the viral DNAs which lack m dA exhibit the same sensitivity pattern as host DNA (1). These results clearly indicate that m dA is present in GATC sequences of PBCV-1, and probably in NC-1A, NC-1B, NC-1C, SC-1A and SC-1B viral DNAs but is absent in a major portion of the host nuclear GATC sequences. In a parallel experiment the susceptibility of the host DNA to the restriction endonucleases, flSEl and Hpall. was determined. These two enzymes recognize the sequence CCGG; however, MapI will not cleave this sequence if the external C is methylated, whereas Hpall will not cleave it if the internal C is methylated (9). All of the viral DNAs are cleaved by both 3475 Downloaded from https://academic.oup.com/nar/article/13/10/3471/2381446 by DeepDyve user on 20 August 2020 Nucleic Acids Research enzymes (1). In contrast, nuclear DNA was susceptible to restriction by Mspl but poorly restricted by Hpall (Fig. 2, lanes 5 to 7) . This suggests that at least some of the m dCs in the host DNA, but not in viral DNAs, are located in the internal C of the sequence CCGG. DISCDSSION These results establish that the genomic DNA of the eukaryotic green alga, Chlorella. strain NC64A, and eleven of its viruses contain significant levels of methylated bases. Chlorella nuclear DNA contains high levels of m dC (21.2%), as do certain higher plants (10, 11), but not other green algae (12, 13). This Chlorella strain also contains a low concentration (0.59%) of m dA. The nuclear DNA of several other eukaryotic protists also contains m dA. Examples include the green alga Chlamydomonas reinhardli (13), the protozoa, Tetrahvmena thermophila (previously named pyriformis) (14, 15) and Para.me.cium aurelia (16), and certain dinoflagellates (17). While some bacteriophages commonly contain small amounts of methylated bases, viruses that infect eukaryotic organisms typically lack methylated bases (18,19). One exception is frog virus 3 (FV 3) , an Iridovirus which contains approximately 20% m dC in its genome (20). Recent experiments suggest that FV 3 also encodes for a unique DNA methyltransferase (21). Another animal virus containing m dC is human papilloma virus (22, 23) ; in this instance, methylation occurs in specific CCGG and GCGC sequences. All the viruses described in this report contain m dC; the concentration of m dC varies from 0.33% to 13.4%. The viral DNAB are not methylated at most of the CCGG sequences, since they are equally susceptible to HEAII and HepI restriction endonucleases (1). This is in contrast to the host Chlorella DNA which is almost completely resistant to HQa.II, but sensitive to Mspl. indicating that it contains m dC in the sequence C CGG. Six of the Chlorella viruses also contain m dA. In contrast to the host Chlorella. the viral DNAs contain m dA in GATC sequences. These results suggest that the viruses may also encode one or more unique DNA methyltransferases. In addition, the viral DNA does not appear to be a substrate for the host DNA methyltransferase(s) in vivo, we have examined PBCV-1 infected 3476 Downloaded from https://academic.oup.com/nar/article/13/10/3471/2381446 by DeepDyve user on 20 August 2020 Nucleic Acids Research cells for DNA methytransferase activity and preliminary results indicate that infected cells contain much higher activity than uninfected cells. Characterization of this methyltransferase activity is in progress. ACKNOWLEDGMENTS We thank M. G. Narinus for kindly providing fi. coli strain GH2163. The helpful discussions with Myron Brakke, Les Lane, and Rues Heints are gratefully acknowledged. This investigation was supported, in part, by Public Health Service grants Gm-32441 (JVE) and GM-29227 (SH) from the National Institute of General Medical Sciences and grant DE-AC02- 82ER12086 (JVE) from the Department of Energy. Published with the approval of the Director as paper no. 7703, Journal Series, Nebraska Agricultural Research Division. REFERENCES 1. Van Etten, J.L., Burbank, D.E., Schuster, A.M. and Meints, R.H. (1985) Virology 140, 135-143. 2. Van Etten, J.L., Burbank, D.E., xia, Y. and Meints, R.H. (1983) Virology 126, 117-125. 3. Van Etten, J.L., Meints, R.H., Burbank, D.E., Kuczmarski, D., Cuppele, D.A. and Lane, L.C. (1981) Virology 113, 704-711. 4. Van Etten, J.L., Burbank, D.E., Joshi, J. and Meints, R.H. (1984) Virology 134, 443-449. 5. Proffitt, J.H., Davie, J.R., Swinton, D. and Hattman, S. (1984) Mol. Cell. Biol. 4, 985-987. 6. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory, New York. 7. Marinus, M.G. (1983) Mol. Gen. Genet. 192, 288-289. 8. Wahl, G.M., Stern, M. and Stark, G.R. (1979) Proc. Natl. Acad. Sci. USA 76, 3683-3687. 9. McClelland, M. (1983) Nucleic Acids Res. 11, rl69-rl73. 10. Wyatt, G.R. (1950) Nature 166, 237-238. 11. Vanyushin, B.F. and Belozerskii, A.N. (1959) Dokl. Akad. Nauk. SSSR 129, 944-946. 12. Pakhomova, M.W., Zaitseva, G.N. and Belozerskii, A.N. (1968) Dokl. Akad. Nauk. SSSR 182, 712-714. 13. Hattman, S., Kenny, C , Berger, L. and Pratt, K. (1978) J. Bacteriol. 135, 1156-1157. 14. Gorovsky, M.A., Hattman, S. and Pleger, G.L. (1973) J. Cell Biol. 56, 697-701. 15. Pratt, K. and Hattman, S. (1981) Mol. Cell. Biol. 1, 600-608. 16. Cunnnings, D.J., Tait, A. and Goddard, J. M. (1974) Biochim. Biophys. Acta 374, 1-11. 17. Rae, P.M.M. (1976) Science 194, 1062-1064. 18. Doerfler, W. (1981) J. Gen. Virol. 57, 1-20. 3477 Downloaded from https://academic.oup.com/nar/article/13/10/3471/2381446 by DeepDyve user on 20 August 2020 Nucleic Acids Research 19. Doerfler, W. (1983) Ann. Rev. Biochem. 52, 93-124. 20. Willis, D.B. and Granoff, A. (1980) Virology 107, 250-257. 21. Willie, D.B., Goorha, R. and Granoff, A. (1984) J. Virol. 49, 86-91. 22. Danoe, 0., Katinka, M. and Yaniv, M. (1980) Eur. J. Biochem. 109, 457-461. 23. Burnett, T.S. and Sleeman, J.P. (1984) Nucleic Acids Res. 12, 8847-8860. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nucleic Acids Research Oxford University Press

DNA methylation of viruses infecting a eukaryotic Chlorella -like green alga

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Downloaded from https://academic.oup.com/nar/article/13/10/3471/2381446 by DeepDyve user on 20 August 2020 volume 13 Number 10 1985 Nucleic Acid s Research DNA methylation of viruses infecting a eukaryotk Chlorella-Uke green alga James L.Van Etten, Anne M.Schuster, Lois Girton, Dwight E.Burbank, David Swinton and Stanley Hattman1 Department of Plant Pathology, University of Nebraska, Lincoln, NE 68583-0722, and 'Department of Biology, University of Rochester, Rochester, NY 14627, USA Received 7 March 1985; Revised and Accepted 18 April 1985 ABSTRACT The genomic DNAB of the eukaryotic Chlorella-lilce green alga, strain NC64A, and eleven of its viruses all contain significant levels of 5-methyldeoxycytidine. In addition, the host DNA as well as six of the viral DNAs also contain N -methyldeoxyadenosine. At least some of the methylated bases in the host reside in different base sequences than the methylated bases in the viruses as shown by differential susceptibility to restriction endonuclease enzymes. This suggests that the viruses encode for DNA methyltransferases with sequence specificities different from that of the host enzyme. INTRODDCTIQN We have recently discovered a number of viruses in fresh water ponds and rivers in Illinois, North Carolina, and South Carolina which form plaques on lawns of a unicellular, eukaryotic, Chlorella-like green alga (1). All of these viruses are polyhedrons (160 to 190 nm in diameter) and contain large dsDNA genomes (ca. 300 kbp as estimated by summing restriction fragments). Although these viral DNAs exhibited extensive homology with the previously described PBCV-1 virus DNA (1, 2) , the viruses can be distinguished from one another and from PBCV-1 by at least one of the following characteristics: plaque size, DNA restriction patterns, or resistance to certain DNA restriction endonucleases. We have grouped the viruses into 5 classes based on the sensitivity or resistance of their DNAs to 15 restriction endonucleases (1). The resistance of the viral DNAs to certain restriction enzymes suggested that the viral DNAs might contain modified bases. The present report describes base analyses of eleven of these viral DNAs as well as the host Chlorella DNA and establishes the presence of 5-methyldeoxycytidine (m dC) in © IRL Press Limited, Oxford , England. 3471 Downloaded from https://academic.oup.com/nar/article/13/10/3471/2381446 by DeepDyve user on 20 August 2020 Nucleic Acids Research host nuclear DNA and all the viral DNAs. The host DNA, as well as six of the viral DNAs, also contain N -methyldeoxyadenoslne (m dA). Finally we show that at least some of the methylated bases in the host reside in different base sequences from those in the viruses. MATERIALS AND METHODS Preparation of viral and host DNAs. The production and purification of the viruses (PBCV-1, NC-1A, NC-1B, NC-1C, NC-1D, SC-1A, SC-1B, I1-2A, I1-2B, I1-3A, and I1-3D) and the growth of the host Chlorella NC64A on MBBM medium have been described (1, 2) . Viral DNAB were isolated on CsCl gradients from purified viruses as described (3). Host nuclear DNA was also isolated and separated from chloroplast DNA on CsCl gradients as described (4) except that the cells were disrupted in a mortar and pestle and the DNA extracts were not intentionally sheared. The purified DNAs were enzymatically digested (to avoid potential acid degradation of any unusual bases) and the resulting deoxynucleosides were analyzed by both ion-exclusion and reverse-phase high performance liquid chromatography as described (5). The DNAs were treated with restriction endonucleases according to manufacturers' recommendations and fragments were separated by electrophoresis on 1.2% agarose gels in 0.08 M Tris-phosphate and 0.008 M EDTA, pH 8.5. To determine if m dA was responsible for the resistance of PBCV-1 DNA to the restriction endonuclease Mbol. we used plasmid pLG164, which contains a 16 kbp BamHI fragment of PBCV-1 (designated B6) cloned into pBR322. This plasmid was purified from its original host (£. colj. strain LE392) and transformed into £. coll strain GM2163 using standard procedures (6). fi. coli GM2163, kindly provided by Dr. Martin Marinus, lacks both dam and den methylating activity (7). The plasmid was restricted with BamHI and the viral fragment B6 insert DNA was recovered from low melting agarose gels by repeated phenol and phenol:chloroform extractions. The purified fragment was nick-translated with d. - P dCTP (800 Ci/mmole) using a nick translation kit (Bethesda Research Labs) according to the manufacturer's recommendations. This labeled fragment was used to probe Southern blots (8) of PBCV-1 virus 3472 Downloaded from https://academic.oup.com/nar/article/13/10/3471/2381446 by DeepDyve user on 20 August 2020 Nucleic Acids Research DNA and pLG164/GH2163 DNA which had been sequentially digested with restriction endonucleases BamHI and either Dpnl. Mbol. or Sflu.3AI. RESDLTS Base compositional analysis. The percentages of modified bases found in eleven viral DNAs and the host nuclear DNA are summarized in Table 1. Although all the viral DNAa contained readily detectable levels of m dC, viruses NC-1A, NC-1B, I1-2A, I1-2B, I1-3A, and I1-3D contained high levels of this nucleoside (7.0 to 13.4%). With the exception of NC-ID (0.33% m dC), the remaining four viruses contained intermediate levels (1.7 to 2%) of m dC. The host Chlorella nuclear DNA contained 21.2% m dC. In addition to m dC, m dA was present in the host nuclear DNA (0.6%) and in six of the seven viruses from North and South Carolina (1.5 to 8.2%). The virus NC-ID and the four Illinois viruses lacked detectable m dA. The lower limit of detection in these experiments was about 0.02%. The overall G + C content of the host nuclear DNA was 67 mole percent and the viral DNAs ranged from 39 to 41 mole percent. There was no indication that the viruses contained modified bases other than m dC and m dA. Table 1. Concentration of methylated bases in the viral DNAs and their host, Chlorella NC64A. Percent Methylated Bases Virus m5dC a ,b PBCV-1 1.86 (6)c 1.45 (3) NC-1A 7.06 (5) 7.32 (2) NC-1B 13.36 (4) 8.19 (2) NC-1C 1.72 1.59 (4) (2) NC-ID 0.33 <0.08 (4) (2) SC-1A 1.94 7.34 (6) (3) SC-IB 2.04 7.45 (4) (2) I1-2A 9.38 <0.01 (4) (2) I1-2B 10.85 <0.08 (6) (4) 9.67 I1-3A (4) <0.03 (2) 12.62 I1-3D (5) <0.01 (3) Host nuclear 21.23 (6) 0.59 (4) + deoxyuridine. ^percen ^p f t ^ per dC + percent m dA per dA + nrdA + deoxyinosine. Number of determinations 3473 Downloaded from https://academic.oup.com/nar/article/13/10/3471/2381446 by DeepDyve user on 20 August 2020 Nucleic Acids Research 12345 6 Figure 1. Electrophoresis of genomic PBCV-1 DNA treated with BflmHI — 231 (lanes 1, 3, 5) or a 16 kbp PBCV-1 BaraHl fragment, B6, cloned in an — 9 4 E. coli dam~. dcm~ host (lanes 2, 4, 6). All of the DNAB were first — 6.7 treated with BamHi followed by digestion with either Hbol (lanes 1 . * and 2) , Dpnl (lanes 3 and 4) or Sflli3AI (lanes 5 and 6) . The bands were localized by probing with the 32P-labeled PBCV-1 B6 fragment after Southern blotting. Molecular weights in kbp are indicated on the 2 3 Bide o f th e figure. Note: the PBCV-1 2 O DNA fragment B6 is digested by QED.1 and resistant to Mboli after cloning fragment B6 in the dam-, dcm~ £. coli it is resistant to Dpnl and digested by Mbol. — 056 Sequence specificity of the methylation. We reported previously that the DNAs from PBCV-1, NC-1A, NC-1B, NC-1C, SC-1A, and SC-1B were resistant to cleavage by Mbol and sensitive to Dpnl (1) which suggests that m dA is present in the GATC sequence. To confirm that the resistance of PBCV-1 DNA to Hbfil and its sensitivity to Djjn.1 was due to GATC methylation, plasmid pLG164, containing a PBCV-1 BamHI fragment, was grown in an £. colj. dcm , dam strain. Following re-isolation, the PBCV-1 fragment was now sensitive to Hbfil but resistant to DJJJII (Fig. 1) . This result indicates that during propagation in the dam host, the cloned PBCV-1 segment lost the GATC methylation present in the genomic virion DNA. Host nuclear DNA shows a different pattern of 3474 Downloaded from https://academic.oup.com/nar/article/13/10/3471/2381446 by DeepDyve user on 20 August 2020 Nucleic Acids Research Figure 2. Electrophoresie of 1 2 3 4 5 6 Chlorella nuclear DNA after treat- ment with either Sau3AI (lane 2), Mbol (lane 3), Q£n.I (lane 4) , Hfifil (lane 6) or flEall (lane 7) . The DNA in lanes 1 and 5 was untreated. - 0 56 resistance/sensitivity to these restriction enzymes. The host DNA was cleaved by Mbol (and Sau3AI) but was partially resistant to Dpnl (Fig. 2, lanes 1 to 4). Likewise the viral DNAs which lack m dA exhibit the same sensitivity pattern as host DNA (1). These results clearly indicate that m dA is present in GATC sequences of PBCV-1, and probably in NC-1A, NC-1B, NC-1C, SC-1A and SC-1B viral DNAs but is absent in a major portion of the host nuclear GATC sequences. In a parallel experiment the susceptibility of the host DNA to the restriction endonucleases, flSEl and Hpall. was determined. These two enzymes recognize the sequence CCGG; however, MapI will not cleave this sequence if the external C is methylated, whereas Hpall will not cleave it if the internal C is methylated (9). All of the viral DNAs are cleaved by both 3475 Downloaded from https://academic.oup.com/nar/article/13/10/3471/2381446 by DeepDyve user on 20 August 2020 Nucleic Acids Research enzymes (1). In contrast, nuclear DNA was susceptible to restriction by Mspl but poorly restricted by Hpall (Fig. 2, lanes 5 to 7) . This suggests that at least some of the m dCs in the host DNA, but not in viral DNAs, are located in the internal C of the sequence CCGG. DISCDSSION These results establish that the genomic DNA of the eukaryotic green alga, Chlorella. strain NC64A, and eleven of its viruses contain significant levels of methylated bases. Chlorella nuclear DNA contains high levels of m dC (21.2%), as do certain higher plants (10, 11), but not other green algae (12, 13). This Chlorella strain also contains a low concentration (0.59%) of m dA. The nuclear DNA of several other eukaryotic protists also contains m dA. Examples include the green alga Chlamydomonas reinhardli (13), the protozoa, Tetrahvmena thermophila (previously named pyriformis) (14, 15) and Para.me.cium aurelia (16), and certain dinoflagellates (17). While some bacteriophages commonly contain small amounts of methylated bases, viruses that infect eukaryotic organisms typically lack methylated bases (18,19). One exception is frog virus 3 (FV 3) , an Iridovirus which contains approximately 20% m dC in its genome (20). Recent experiments suggest that FV 3 also encodes for a unique DNA methyltransferase (21). Another animal virus containing m dC is human papilloma virus (22, 23) ; in this instance, methylation occurs in specific CCGG and GCGC sequences. All the viruses described in this report contain m dC; the concentration of m dC varies from 0.33% to 13.4%. The viral DNAB are not methylated at most of the CCGG sequences, since they are equally susceptible to HEAII and HepI restriction endonucleases (1). This is in contrast to the host Chlorella DNA which is almost completely resistant to HQa.II, but sensitive to Mspl. indicating that it contains m dC in the sequence C CGG. Six of the Chlorella viruses also contain m dA. In contrast to the host Chlorella. the viral DNAs contain m dA in GATC sequences. These results suggest that the viruses may also encode one or more unique DNA methyltransferases. In addition, the viral DNA does not appear to be a substrate for the host DNA methyltransferase(s) in vivo, we have examined PBCV-1 infected 3476 Downloaded from https://academic.oup.com/nar/article/13/10/3471/2381446 by DeepDyve user on 20 August 2020 Nucleic Acids Research cells for DNA methytransferase activity and preliminary results indicate that infected cells contain much higher activity than uninfected cells. Characterization of this methyltransferase activity is in progress. ACKNOWLEDGMENTS We thank M. G. Narinus for kindly providing fi. coli strain GH2163. The helpful discussions with Myron Brakke, Les Lane, and Rues Heints are gratefully acknowledged. This investigation was supported, in part, by Public Health Service grants Gm-32441 (JVE) and GM-29227 (SH) from the National Institute of General Medical Sciences and grant DE-AC02- 82ER12086 (JVE) from the Department of Energy. Published with the approval of the Director as paper no. 7703, Journal Series, Nebraska Agricultural Research Division. REFERENCES 1. Van Etten, J.L., Burbank, D.E., Schuster, A.M. and Meints, R.H. (1985) Virology 140, 135-143. 2. Van Etten, J.L., Burbank, D.E., xia, Y. and Meints, R.H. (1983) Virology 126, 117-125. 3. Van Etten, J.L., Meints, R.H., Burbank, D.E., Kuczmarski, D., Cuppele, D.A. and Lane, L.C. (1981) Virology 113, 704-711. 4. Van Etten, J.L., Burbank, D.E., Joshi, J. and Meints, R.H. (1984) Virology 134, 443-449. 5. Proffitt, J.H., Davie, J.R., Swinton, D. and Hattman, S. (1984) Mol. Cell. Biol. 4, 985-987. 6. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory, New York. 7. Marinus, M.G. (1983) Mol. Gen. Genet. 192, 288-289. 8. Wahl, G.M., Stern, M. and Stark, G.R. (1979) Proc. Natl. Acad. Sci. USA 76, 3683-3687. 9. McClelland, M. (1983) Nucleic Acids Res. 11, rl69-rl73. 10. Wyatt, G.R. (1950) Nature 166, 237-238. 11. Vanyushin, B.F. and Belozerskii, A.N. (1959) Dokl. Akad. Nauk. SSSR 129, 944-946. 12. Pakhomova, M.W., Zaitseva, G.N. and Belozerskii, A.N. (1968) Dokl. Akad. Nauk. SSSR 182, 712-714. 13. Hattman, S., Kenny, C , Berger, L. and Pratt, K. (1978) J. Bacteriol. 135, 1156-1157. 14. Gorovsky, M.A., Hattman, S. and Pleger, G.L. (1973) J. Cell Biol. 56, 697-701. 15. Pratt, K. and Hattman, S. (1981) Mol. Cell. Biol. 1, 600-608. 16. Cunnnings, D.J., Tait, A. and Goddard, J. M. (1974) Biochim. Biophys. Acta 374, 1-11. 17. Rae, P.M.M. (1976) Science 194, 1062-1064. 18. Doerfler, W. (1981) J. Gen. Virol. 57, 1-20. 3477 Downloaded from https://academic.oup.com/nar/article/13/10/3471/2381446 by DeepDyve user on 20 August 2020 Nucleic Acids Research 19. Doerfler, W. (1983) Ann. Rev. Biochem. 52, 93-124. 20. Willis, D.B. and Granoff, A. (1980) Virology 107, 250-257. 21. Willie, D.B., Goorha, R. and Granoff, A. (1984) J. Virol. 49, 86-91. 22. Danoe, 0., Katinka, M. and Yaniv, M. (1980) Eur. J. Biochem. 109, 457-461. 23. Burnett, T.S. and Sleeman, J.P. (1984) Nucleic Acids Res. 12, 8847-8860.

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Nucleic Acids Research – Oxford University Press

Published: May 24, 1985

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