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Trasformation of Tetrahymena with hypermethylated ribosomal RNA genes
105
Gene, 74 (1988) 105-107 Elsevier GEN 02607
Transformation of ~e~a~~~~
with hy~rme~ylat~
ribosomal RNA genes *
(DNA replication; macronucleus; methyl adenine; microinjection, paromomycin; protozoa; restriction endonucleases)
Kathleen M. KarreP and Meng-Chao Yaob a~3io~o~, Brandeir Universe, Waltham, MA 02254 (U.S.A.) and b Divirion ofBasic Sciences, Fred~atchinsa~ Cancer Research Center, Seattle, WA 98104 (U.S.A.) Tel. (206)467-5005
n Depa~e~t
Received 24 May 1988 Accepted 12 June 1988 Received by publisher 28 June 1988
The ciliated protozoan, Te~~h~e~, has two nuclei: a diploid micronucleus and a polygenomic macronucleus. The macronucleus is the somatic nucleus in that it conducts the vast majority of the transcriptional activity in the cell. The micronucleus is the germ-line nucleus. During sexual reproduction the macronucleus degenerates and a new macronucleus develops from a mitotic product of the zygotic micronucleus (reviewed by Bruns, 1986). During molecular development there are a number of changes in the genome (reviewed by Karrer, 1986). Extensive DNA replication brings the DNA content of the macronucleus to 45 times that of the haploid genome. Micronuclear DNA is unmethylated (Pratt and Hattman, 198 1). During macronuclear development 0.8% of the adenine residues are de novo methylated to N6-methyladenine (mA) (Gorovsky et al., 1973). Methylation occurs in some subset of Correspondenceto: Dr. K.M. Karrer, Department of Biology, Brandeis University, 129 Bassine, Waltham, MA 02254 (U.S.A.) Tel. (617)736-3120. * Presented at the New England Biolabs Workshop on Biological DNA Modification, Gloucester, MA (U.S.A.) 20-23 May 1988. Abbreviations: bp, base pair(s); kb, 1000 bp; M 1, methyltransferase; N, any of the four deoxyribonucleotides; mA, N6* -methyladenine; rDNA, DNA coding for ribosomal RNA.
the sequence 5’-NAT-3’ (Bromberg et al., 1982) and is site-specific in that some sites are methylated in most or all of the 45 copies in the macronuclear genome (Harrison et al., 1986). A prevalent model for the maintenance of methylation patterns through cell division holds that fully methylated sites become hemimethylated as a result of DNA replication. A non~sc~a~g maintenance methyltransferase recognizes the hemimethylated site and methylates the daughter strand (Holliday and Pugh, 1975; Bird, 1978). According to the simplest form of the model, there is no significant enzymatic demethylation or de novo methylation and the strong preference of the maintenance metbyltransferase for a hemimethylated substrate propagates the established pattern. The model was tested by studying methylation of DNA at novel sites in vitro with bacterial methyltransferases. This hypermethylated DNA was microinjected into the macronuclei of host cells. After 20 to 25 cell fissions, DNA from clonal transformed cell lines was extracted and assayed by Southern blot hybridization for methylation at the novel site. The model predicts that hypermethylation will be maintained in the transformed cell lines. The molecule chosen for h~~e~ylation was the 21-kb extrachromosomal ribosomal RNA genes (rDNA) from TetTahymena. The rDNA was isolated
0378-l 119/88/$03.50 0 1988 Elsevier Science Publishers B.V. (Biomedical Division)
106
and methylated in vitro with the bacterial Eco Dam, M *EcoRI and M - ClaI methyltransferases. The rDNA was shown to be hypermethylated with an efficiency of greater than 99%, as judged by resistance to the appropriate bacterial restriction endonucleases. Hypermethylated rDNA was microinjected into the macronucleus of host B strain Tetrahymena (Tondravi and Yao, 1986). Control cells were injected with mock-methylated rDNA, which was incubated under the same conditions as the hypermethylated rDNA, but in the absence of methyltransferase. Approximately 100 clonal cell lines were established from cells microinjected with each DNA sample. Although only about 50 rDNA molecules were injected into each cell, the injected C3 strain rDNA is favored for DNA replication, and it rapidly replaces the 9000 endogenous rDNA molecules as the major constituent of the population. After six days of growth in axenic medium, the cells were replica plated into medium containing 100 pg paromomycin/ml. Transformed cell lines were identified on the basis of resistance to the drug, which is conferred by a mutation in the 17s rRNA gene of the injected rDNA from C3 strain cells (Larson et al., 1986). The average transformation efficiency from five experiments was 47%. Methylation of the injected rDNA had no effect on transformation efficiency relative to mock-methylated controls, demonstrating that the injected rDNA is both replicated and transcribed in the host cells. To ensure that transformation was not the result of a small percentage of molecules which might have escaped methylation, a second set of injections was done at a lower concentration of rDNA. In these experiments only about five molecules were injected into each cell. The transformation frequencies were slightly lower (with an average of 39%); and there was a greater increase in the proportion of clonal lines showing resistance to drug when challenged at six days vs. three days of clonal cell growth. These results were not unexpected. That is, since fewer molecules were injected into the cells, a longer period of time may be required for the injected molecules to become established as a fraction of the rDNA large enough to confer drug resistance. Again, there was no significant difference in transformation frequency between cells injected with hypermethylated rDNA vs. mock-methylated controls.
The proportion of injected C3 strain rDNA vs. host rDNA in the transformed cell lines was assessed by Southern-blot hybridization. After 20 to 25 cell fissions, DNA was isolated from clonal cell lines according to the method of Austerberry and Yao (1987). DNAs from four cell lines transformed with mock-methylated rDNA, and from eight cell lines transformed with DNA hypermethylated with each of the three enzymes, were digested with the restriction enzyme BarnHI. A restriction site polymorphism between the rDNA of the host strain and the injected C3 strain rDNA was exploited to distinguish the two. More than 95 y0 of the rDNA extracted from transformed cells was of the injected type. Methylation of the rDNA in transformants was assessed by Southern-blot hybridization. The complete sequence of the 21-kb rDNA molecule is known as the result of work in several laboratories, and it has been compiled by H. Nielsen and J. Engberg (personal communication). Computer analysis of the nucleotide sequence suggested that there are three ClaI sites and one EcoRI site on each half of the palindromic rDNA molecule. Southern blots of native rDNA and whole-cell DNA hybridized with a clone of the rDNA show all of the expected restriction fragments, suggesting that these sites were not methylated in vivo. The absence of detectable methylation in the rDNA from transformed cell lines suggests that the transformants were unable to maintain the methylation carried by the injected DNA. Relatively little is known regarding the sequence specificity of the Tetrahymena methyltransferase. Although one methylated EcoRI sequence (GAATTC) has been found in the Tetrahymena genome (Martindale et al., 1986) and one methylated C/a1 sequence (ATCGAT) has been identified in Oxytricha (Cartinhour and Herrick, 1984), it is possible that the Tetrahymena methyltransferase does not recognize the sites in rDNA. In this regard, Blackburn et al. (1983) found a low level of 5’-GATC-3’ methylation in Tetrahymena rDNA. Most molecules were not methylated at any of the 29 known GATC sites. About 10% of the molecules were methylated at a subset of the GATC sites, which results in a characteristic pattern of minor bands in DNA digested with DpnI. The same pattern of minor bands was observed in DNA from transformed cells digested with DpnI. Thus, the pat-
107
tern of methylation reverts to the in vivo pattern within 20 to 25 cell fissions because the methyltransferase was unable to maintain hypermethylation, even at a site which is methylated in vivo at a low level. These experiments suggest to us that the activity of a maintenance methyltransferase which depends primarily on a preference for a hemimethylated substrate for its specificity is insufXcient to account for maintenance of methylation patterns in Tefrahymena. Other factors such as chromatin structure must take precedence in maintaining the pattern.
ACKNOWLEDGEMENTS
This work was supported by Public Health Service grants GM 26210 and HD 00547 to M.-C. Y. and by GM 32989 and BRSG SO7 RR07044 to K.M.K. from the National Institutes of Health. The experiments are presented in detail in Karrer and Yao (1988).
REFERENCES Austerberry, C.F. and Yao, M.-C.: Nucleotide sequence structure and consistency of a developmentally regulated DNA deletion in Terrahymena thermophilu. Mol. Cell. Biol. 7 (1987) 435-443. Bird, A.P.: Use of restriction enzymes to study eukaryotic DNA methylation II. The symmetry of methylated sites supports semi-conservative copying ofthe methylation pattern. J. Mol. Biol. 118 (1978) 49-60.
Blackbum, E.H., Pan, W.-C. and Johnson, CC.: Methylation of ribosomal RNA genes in the macronucleus of Tetrahymeno thermophila. Nucleic Acids Res. 11 (1983) 5131-5145. Bromberg, S., Pratt, K. and Hattman, S.: Sequence specificity of the DNA-adenine methyltransferase in the protozoan, Tetrahymena rhermophila.J. Bacterial. 150 (1982) 993-996. Bruns, P.J.: Genetic organization of Tetruhymena. In Gall, J.G. (Ed.), The Molecular Biology of Ciliated Protozoa. Academic Press, Orlando, FL, 1986, pp. 27-44. Car&hour, S.W. and Herrick, G.A.: Three different macronucleus DNAs in Oxynicha fullux share a common sequence block. Mol. Cell Biol. 4 (1984) 931-938. Gorovsky, M.A., Hattman, S. and Pleger, G.L.: [N6] methyladenine in the nuclear DNA of a eucaryote, Tetruhymenu pyriformb. J. Cell Biol. 56 (1973) 697-701. Harrison, G.S., Findly, R.C. and Karrer, K.M.: Site specific methylation of adenine in the nuclear genome of a eucaryote, Tefruhymena thermophila. Mol. Cell Biol. 6 (1986) 2364-2370. Holliday, R. and Pugh, J.E.: DNA modification mechanisms and gene activity during development. Science 187 (1975) 226-232. Karrer, K.M.: The nuclear DNAs of holotrichous ciliates. In Gall, J.G. (Ed.), The Molecular Biology of Ciliated Protozoa. Academic Press, Orlando, FL, 1986, pp. 85-l 10. Karrer, K.M. and Yao, M.-C.: Transformation of Terrahymenu thermophila with hypermethylated rRNA genes. Mol. Cell. Biol. 8 (1988) 1664-1669. Larson, D.D., Blackbum, E.H., Yaeger, P.C. and Orias, E.: Control of DNA replication in Tetrahymena involves a cisacting upstream repeat of a promoter element. Cell 47 (1986) 229-240. Martindale, D.M., Martindale, H.M. and Bruns, P.J.: Terrahymenu conjugation-induced genes: structure and organization in macro- and micronuclei. Nucleic Acids Res. 14 (1986) 1341-1354. Pratt, K. and Hattman, S.: Deoxyribonucleic acid methylation and chromatin organization in Tetruhymena rhermophilu. Mol. Cell Biol. 1 (1981) 600-608. Tondravi, M.M. and Yao, M.-C.: Transformation of Tefruhymenu thermophila by microinjection of ribosomal RNA genes. Proc. Natl. Acad. Sci. USA 83 (1986) 4369-4373. Edited by S. Hattman.
Gene, 74 (1988) 105-107 Elsevier GEN 02607
Transformation of ~e~a~~~~
with hy~rme~ylat~
ribosomal RNA genes *
(DNA replication; macronucleus; methyl adenine; microinjection, paromomycin; protozoa; restriction endonucleases)
Kathleen M. KarreP and Meng-Chao Yaob a~3io~o~, Brandeir Universe, Waltham, MA 02254 (U.S.A.) and b Divirion ofBasic Sciences, Fred~atchinsa~ Cancer Research Center, Seattle, WA 98104 (U.S.A.) Tel. (206)467-5005
n Depa~e~t
Received 24 May 1988 Accepted 12 June 1988 Received by publisher 28 June 1988
The ciliated protozoan, Te~~h~e~, has two nuclei: a diploid micronucleus and a polygenomic macronucleus. The macronucleus is the somatic nucleus in that it conducts the vast majority of the transcriptional activity in the cell. The micronucleus is the germ-line nucleus. During sexual reproduction the macronucleus degenerates and a new macronucleus develops from a mitotic product of the zygotic micronucleus (reviewed by Bruns, 1986). During molecular development there are a number of changes in the genome (reviewed by Karrer, 1986). Extensive DNA replication brings the DNA content of the macronucleus to 45 times that of the haploid genome. Micronuclear DNA is unmethylated (Pratt and Hattman, 198 1). During macronuclear development 0.8% of the adenine residues are de novo methylated to N6-methyladenine (mA) (Gorovsky et al., 1973). Methylation occurs in some subset of Correspondenceto: Dr. K.M. Karrer, Department of Biology, Brandeis University, 129 Bassine, Waltham, MA 02254 (U.S.A.) Tel. (617)736-3120. * Presented at the New England Biolabs Workshop on Biological DNA Modification, Gloucester, MA (U.S.A.) 20-23 May 1988. Abbreviations: bp, base pair(s); kb, 1000 bp; M 1, methyltransferase; N, any of the four deoxyribonucleotides; mA, N6* -methyladenine; rDNA, DNA coding for ribosomal RNA.
the sequence 5’-NAT-3’ (Bromberg et al., 1982) and is site-specific in that some sites are methylated in most or all of the 45 copies in the macronuclear genome (Harrison et al., 1986). A prevalent model for the maintenance of methylation patterns through cell division holds that fully methylated sites become hemimethylated as a result of DNA replication. A non~sc~a~g maintenance methyltransferase recognizes the hemimethylated site and methylates the daughter strand (Holliday and Pugh, 1975; Bird, 1978). According to the simplest form of the model, there is no significant enzymatic demethylation or de novo methylation and the strong preference of the maintenance metbyltransferase for a hemimethylated substrate propagates the established pattern. The model was tested by studying methylation of DNA at novel sites in vitro with bacterial methyltransferases. This hypermethylated DNA was microinjected into the macronuclei of host cells. After 20 to 25 cell fissions, DNA from clonal transformed cell lines was extracted and assayed by Southern blot hybridization for methylation at the novel site. The model predicts that hypermethylation will be maintained in the transformed cell lines. The molecule chosen for h~~e~ylation was the 21-kb extrachromosomal ribosomal RNA genes (rDNA) from TetTahymena. The rDNA was isolated
0378-l 119/88/$03.50 0 1988 Elsevier Science Publishers B.V. (Biomedical Division)
106
and methylated in vitro with the bacterial Eco Dam, M *EcoRI and M - ClaI methyltransferases. The rDNA was shown to be hypermethylated with an efficiency of greater than 99%, as judged by resistance to the appropriate bacterial restriction endonucleases. Hypermethylated rDNA was microinjected into the macronucleus of host B strain Tetrahymena (Tondravi and Yao, 1986). Control cells were injected with mock-methylated rDNA, which was incubated under the same conditions as the hypermethylated rDNA, but in the absence of methyltransferase. Approximately 100 clonal cell lines were established from cells microinjected with each DNA sample. Although only about 50 rDNA molecules were injected into each cell, the injected C3 strain rDNA is favored for DNA replication, and it rapidly replaces the 9000 endogenous rDNA molecules as the major constituent of the population. After six days of growth in axenic medium, the cells were replica plated into medium containing 100 pg paromomycin/ml. Transformed cell lines were identified on the basis of resistance to the drug, which is conferred by a mutation in the 17s rRNA gene of the injected rDNA from C3 strain cells (Larson et al., 1986). The average transformation efficiency from five experiments was 47%. Methylation of the injected rDNA had no effect on transformation efficiency relative to mock-methylated controls, demonstrating that the injected rDNA is both replicated and transcribed in the host cells. To ensure that transformation was not the result of a small percentage of molecules which might have escaped methylation, a second set of injections was done at a lower concentration of rDNA. In these experiments only about five molecules were injected into each cell. The transformation frequencies were slightly lower (with an average of 39%); and there was a greater increase in the proportion of clonal lines showing resistance to drug when challenged at six days vs. three days of clonal cell growth. These results were not unexpected. That is, since fewer molecules were injected into the cells, a longer period of time may be required for the injected molecules to become established as a fraction of the rDNA large enough to confer drug resistance. Again, there was no significant difference in transformation frequency between cells injected with hypermethylated rDNA vs. mock-methylated controls.
The proportion of injected C3 strain rDNA vs. host rDNA in the transformed cell lines was assessed by Southern-blot hybridization. After 20 to 25 cell fissions, DNA was isolated from clonal cell lines according to the method of Austerberry and Yao (1987). DNAs from four cell lines transformed with mock-methylated rDNA, and from eight cell lines transformed with DNA hypermethylated with each of the three enzymes, were digested with the restriction enzyme BarnHI. A restriction site polymorphism between the rDNA of the host strain and the injected C3 strain rDNA was exploited to distinguish the two. More than 95 y0 of the rDNA extracted from transformed cells was of the injected type. Methylation of the rDNA in transformants was assessed by Southern-blot hybridization. The complete sequence of the 21-kb rDNA molecule is known as the result of work in several laboratories, and it has been compiled by H. Nielsen and J. Engberg (personal communication). Computer analysis of the nucleotide sequence suggested that there are three ClaI sites and one EcoRI site on each half of the palindromic rDNA molecule. Southern blots of native rDNA and whole-cell DNA hybridized with a clone of the rDNA show all of the expected restriction fragments, suggesting that these sites were not methylated in vivo. The absence of detectable methylation in the rDNA from transformed cell lines suggests that the transformants were unable to maintain the methylation carried by the injected DNA. Relatively little is known regarding the sequence specificity of the Tetrahymena methyltransferase. Although one methylated EcoRI sequence (GAATTC) has been found in the Tetrahymena genome (Martindale et al., 1986) and one methylated C/a1 sequence (ATCGAT) has been identified in Oxytricha (Cartinhour and Herrick, 1984), it is possible that the Tetrahymena methyltransferase does not recognize the sites in rDNA. In this regard, Blackburn et al. (1983) found a low level of 5’-GATC-3’ methylation in Tetrahymena rDNA. Most molecules were not methylated at any of the 29 known GATC sites. About 10% of the molecules were methylated at a subset of the GATC sites, which results in a characteristic pattern of minor bands in DNA digested with DpnI. The same pattern of minor bands was observed in DNA from transformed cells digested with DpnI. Thus, the pat-
107
tern of methylation reverts to the in vivo pattern within 20 to 25 cell fissions because the methyltransferase was unable to maintain hypermethylation, even at a site which is methylated in vivo at a low level. These experiments suggest to us that the activity of a maintenance methyltransferase which depends primarily on a preference for a hemimethylated substrate for its specificity is insufXcient to account for maintenance of methylation patterns in Tefrahymena. Other factors such as chromatin structure must take precedence in maintaining the pattern.
ACKNOWLEDGEMENTS
This work was supported by Public Health Service grants GM 26210 and HD 00547 to M.-C. Y. and by GM 32989 and BRSG SO7 RR07044 to K.M.K. from the National Institutes of Health. The experiments are presented in detail in Karrer and Yao (1988).
REFERENCES Austerberry, C.F. and Yao, M.-C.: Nucleotide sequence structure and consistency of a developmentally regulated DNA deletion in Terrahymena thermophilu. Mol. Cell. Biol. 7 (1987) 435-443. Bird, A.P.: Use of restriction enzymes to study eukaryotic DNA methylation II. The symmetry of methylated sites supports semi-conservative copying ofthe methylation pattern. J. Mol. Biol. 118 (1978) 49-60.
Blackbum, E.H., Pan, W.-C. and Johnson, CC.: Methylation of ribosomal RNA genes in the macronucleus of Tetrahymeno thermophila. Nucleic Acids Res. 11 (1983) 5131-5145. Bromberg, S., Pratt, K. and Hattman, S.: Sequence specificity of the DNA-adenine methyltransferase in the protozoan, Tetrahymena rhermophila.J. Bacterial. 150 (1982) 993-996. Bruns, P.J.: Genetic organization of Tetruhymena. In Gall, J.G. (Ed.), The Molecular Biology of Ciliated Protozoa. Academic Press, Orlando, FL, 1986, pp. 27-44. Car&hour, S.W. and Herrick, G.A.: Three different macronucleus DNAs in Oxynicha fullux share a common sequence block. Mol. Cell Biol. 4 (1984) 931-938. Gorovsky, M.A., Hattman, S. and Pleger, G.L.: [N6] methyladenine in the nuclear DNA of a eucaryote, Tetruhymenu pyriformb. J. Cell Biol. 56 (1973) 697-701. Harrison, G.S., Findly, R.C. and Karrer, K.M.: Site specific methylation of adenine in the nuclear genome of a eucaryote, Tefruhymena thermophila. Mol. Cell Biol. 6 (1986) 2364-2370. Holliday, R. and Pugh, J.E.: DNA modification mechanisms and gene activity during development. Science 187 (1975) 226-232. Karrer, K.M.: The nuclear DNAs of holotrichous ciliates. In Gall, J.G. (Ed.), The Molecular Biology of Ciliated Protozoa. Academic Press, Orlando, FL, 1986, pp. 85-l 10. Karrer, K.M. and Yao, M.-C.: Transformation of Terrahymenu thermophila with hypermethylated rRNA genes. Mol. Cell. Biol. 8 (1988) 1664-1669. Larson, D.D., Blackbum, E.H., Yaeger, P.C. and Orias, E.: Control of DNA replication in Tetrahymena involves a cisacting upstream repeat of a promoter element. Cell 47 (1986) 229-240. Martindale, D.M., Martindale, H.M. and Bruns, P.J.: Terrahymenu conjugation-induced genes: structure and organization in macro- and micronuclei. Nucleic Acids Res. 14 (1986) 1341-1354. Pratt, K. and Hattman, S.: Deoxyribonucleic acid methylation and chromatin organization in Tetruhymena rhermophilu. Mol. Cell Biol. 1 (1981) 600-608. Tondravi, M.M. and Yao, M.-C.: Transformation of Tefruhymenu thermophila by microinjection of ribosomal RNA genes. Proc. Natl. Acad. Sci. USA 83 (1986) 4369-4373. Edited by S. Hattman.