Abstract
5-Methyldeoxycytidine (5MC) was analyzed by high pressure liquid chromatography (HPLC) and by restriction enzyme digestion in rDNA isolated from Physarum polycephalum. rDNA from Physarum M3C strain microplasmodia has a significant 5MC content (about half that of the whole genomic DNA). This rDNA contains many C5MCGG sites because it is clearly digested further by Msp I than by Hpa II. However, most 5MC is in other sites. In particular, alternating CG sequences appear to be highly methylated. HPLC of deoxyribonucleosides shows tha most of the transcribed regions contain little or no 5MC. Restriction digestion indicates that there is little or no 5MC in any of the transcribed regions including the transcription origin and adjacent sequences. Over 90% of the total 5MC is in or near the central nontranscribed spacer and most methylated restriction sites are in inverted repeats of this spacer. rDNA is very heterogeneous with respect to 5MC. The 5MC pattern doesn't appear to change with inactivation of the rRNA genes during reversible differentiation from microplasmodia (growing) to microsclerotia (dormant), showing that inactivation is due to changes in other chromatin variables. The 5MC pattern is different between Physarum strains. The possible involvement of this 5MC in rDNA chromatin structure and in cruciform and Z-DNA formation is discussed.
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- Adams R. L., Burdon R. H. DNA methylation in eukaryotes. CRC Crit Rev Biochem. 1982;13(4):349–384. doi: 10.3109/10409238209108714. [DOI] [PubMed] [Google Scholar]
- Bird A. P., Taggart M. H., Gehring C. A. Methylated and unmethylated ribosomal RNA genes in the mouse. J Mol Biol. 1981 Oct 15;152(1):1–17. doi: 10.1016/0022-2836(81)90092-9. [DOI] [PubMed] [Google Scholar]
- Bird A. P., Taggart M. H. Variable patterns of total DNA and rDNA methylation in animals. Nucleic Acids Res. 1980 Apr 11;8(7):1485–1497. doi: 10.1093/nar/8.7.1485. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Boehm T. R., Drahovsky D. Enzymatic hypomethylation of inverted repeats in transcribed DNA regions of mouse P815 mastocytoma cells. Int J Biochem. 1981;13(2):153–158. doi: 10.1016/0020-711x(81)90150-6. [DOI] [PubMed] [Google Scholar]
- Busslinger M., deBoer E., Wright S., Grosveld F. G., Flavell R. A. The sequence GGCmCGG is resistant to MspI cleavage. Nucleic Acids Res. 1983 Jun 11;11(11):3559–3569. doi: 10.1093/nar/11.11.3559. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Campbell G. R., Littau V. C., Melera P. W., Allfrey V. G., Johnson E. M. Unique sequence arrangement of ribosomal genes in the palindromic rDNA molecule of Physarum polycephalum. Nucleic Acids Res. 1979 Apr;6(4):1433–1447. doi: 10.1093/nar/6.4.1433. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cooper D. N. Eukaryotic DNA methylation. Hum Genet. 1983;64(4):315–333. doi: 10.1007/BF00292363. [DOI] [PubMed] [Google Scholar]
- Courey A. J., Wang J. C. Cruciform formation in a negatively supercoiled DNA may be kinetically forbidden under physiological conditions. Cell. 1983 Jul;33(3):817–829. doi: 10.1016/0092-8674(83)90024-7. [DOI] [PubMed] [Google Scholar]
- Ehrlich M., Ehrlich K., Mayo J. A. Unusual properties of the DNA from Xanthomonas phage XP-12 in which 5-methylcytosine completely replaces cytosine. Biochim Biophys Acta. 1975 Jun 16;395(2):109–119. doi: 10.1016/0005-2787(75)90149-5. [DOI] [PubMed] [Google Scholar]
- Ehrlich M., Gama-Sosa M. A., Huang L. H., Midgett R. M., Kuo K. C., McCune R. A., Gehrke C. Amount and distribution of 5-methylcytosine in human DNA from different types of tissues of cells. Nucleic Acids Res. 1982 Apr 24;10(8):2709–2721. doi: 10.1093/nar/10.8.2709. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Evans H. H., Evans T. E. Methylation of the deoxyribonucleic acid of Physarum polycephalum at various periods during the mitotic cycle. J Biol Chem. 1970 Dec 10;245(23):6436–6441. [PubMed] [Google Scholar]
- Ferris P. J., Vogt V. M. Structure of the central spacer region of extrachromosomal ribosomal DNA in Physarum polycephalum. J Mol Biol. 1982 Aug 15;159(3):359–381. doi: 10.1016/0022-2836(82)90289-3. [DOI] [PubMed] [Google Scholar]
- Fradin A., Manley J. L., Prives C. L. Methylation of simian virus 40 Hpa II site affects late, but not early, viral gene expression. Proc Natl Acad Sci U S A. 1982 Sep;79(17):5142–5146. doi: 10.1073/pnas.79.17.5142. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Grainger R. M., Hazard-Leonards R. M., Samaha F., Hougan L. M., Lesk M. R., Thomsen G. H. Is hypomethylation linked to activation of delta-crystallin genes during lens development? Nature. 1983 Nov 3;306(5938):88–91. doi: 10.1038/306088a0. [DOI] [PubMed] [Google Scholar]
- Gross-Bellard M., Oudet P., Chambon P. Isolation of high-molecular-weight DNA from mammalian cells. Eur J Biochem. 1973 Jul 2;36(1):32–38. doi: 10.1111/j.1432-1033.1973.tb02881.x. [DOI] [PubMed] [Google Scholar]
- Gruenbaum Y., Cedar H., Razin A. Substrate and sequence specificity of a eukaryotic DNA methylase. Nature. 1982 Feb 18;295(5850):620–622. doi: 10.1038/295620a0. [DOI] [PubMed] [Google Scholar]
- Hall L., Braun R. The organisation of genes for transfer RNA and ribosomal RNA in amoebae and plasmodia of Physarum polycephalum. Eur J Biochem. 1977 Jun 1;76(1):165–174. doi: 10.1111/j.1432-1033.1977.tb11582.x. [DOI] [PubMed] [Google Scholar]
- Hori T. A. Induction of chromosome decondensation, sister-chromatid exchanges and endoreduplications by 5-azacytidine, an inhibitor of DNA methylation. Mutat Res. 1983 Jul;121(1):47–52. doi: 10.1016/0165-7992(83)90085-4. [DOI] [PubMed] [Google Scholar]
- Hsieh T. S., Wang J. C. Thermodynamic properties of superhelical DNAs. Biochemistry. 1975 Feb 11;14(3):527–535. doi: 10.1021/bi00674a011. [DOI] [PubMed] [Google Scholar]
- Johnson E. M., Allfrey V. G., Bradbury E. M., Matthews H. R. Altered nucleosome structure containing DNA sequences complementary to 19S and 26S ribosomal RNA in Physarum polycephalum. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1116–1120. doi: 10.1073/pnas.75.3.1116. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Johnson E. M., Campbell G. R., Allfrey V. G. Different nucleosome structures on transcribing and nontranscribing ribosomal gene sequences. Science. 1979 Dec 7;206(4423):1192–1194. doi: 10.1126/science.505006. [DOI] [PubMed] [Google Scholar]
- Johnson E. M., Matthews H. R., Littau V. C., Lothstein L., Bradbury E. M., Allfrey V. G. The structure of chromatin containing DNA complementary to 19 S and 26 S ribosomal RNA in active and inactive stages of Physarum polycephalum. Arch Biochem Biophys. 1978 Dec;191(2):537–560. doi: 10.1016/0003-9861(78)90392-2. [DOI] [PubMed] [Google Scholar]
- Judelson H. S., Vogt V. M. Accessibility of ribosomal genes to trimethyl psoralen in nuclei of Physarum polycephalum. Mol Cell Biol. 1982 Mar;2(3):211–220. doi: 10.1128/mcb.2.3.211. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Katz R. A., Erlanger B. F., Guntaka R. V. Evidence for extensive methylation of ribosomal RNA genes in a rat XC cell line. Biochim Biophys Acta. 1983 Apr 15;739(3):258–264. doi: 10.1016/0167-4781(83)90099-4. [DOI] [PubMed] [Google Scholar]
- Keshet E., Cedar H. Effect of CpG methylation on Msp I. Nucleic Acids Res. 1983 Jun 11;11(11):3571–3580. doi: 10.1093/nar/11.11.3571. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kovacic R. T., van Holde K. E. Sedimentation of homogeneous double-strand DNA molecules. Biochemistry. 1977 Apr 5;16(7):1490–1498. doi: 10.1021/bi00626a038. [DOI] [PubMed] [Google Scholar]
- Krumdieck C. L., Howard-Peebles P. N. On the nature of folic-acid-sensitive fragile sites in human chromosomes: an hypothesis. Am J Med Genet. 1983 Sep;16(1):23–28. doi: 10.1002/ajmg.1320160105. [DOI] [PubMed] [Google Scholar]
- Kunnath L., Locker J. Developmental changes in the methylation of the rat albumin and alpha-fetoprotein genes. EMBO J. 1983;2(3):317–324. doi: 10.1002/j.1460-2075.1983.tb01425.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kunnath L., Locker J. Variable methylation of the ribosomal RNA genes of the rat. Nucleic Acids Res. 1982 Jul 10;10(13):3877–3892. doi: 10.1093/nar/10.13.3877. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kuo K. C., McCune R. A., Gehrke C. W., Midgett R., Ehrlich M. Quantitative reversed-phase high performance liquid chromatographic determination of major and modified deoxyribonucleosides in DNA. Nucleic Acids Res. 1980 Oct 24;8(20):4763–4776. doi: 10.1093/nar/8.20.4763. [DOI] [PMC free article] [PubMed] [Google Scholar]
- La Volpe A., Taggart M., Macleod D., Bird A. Coupled demethylation of sites in a conserved sequence of Xenopus ribosomal DNA. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 2):585–592. doi: 10.1101/sqb.1983.047.01.069. [DOI] [PubMed] [Google Scholar]
- Mohberg J., Rusch H. P. Isolation and DNA content of nuclei of Physarum polycephalum. Exp Cell Res. 1971 Jun;66(2):305–316. doi: 10.1016/0014-4827(71)90682-3. [DOI] [PubMed] [Google Scholar]
- Molgaard H. V., Matthews H. R., Bradbury E. M. Organisation of genes for ribosomal RNA in Physarum polycephalum. Eur J Biochem. 1976 Sep 15;68(2):541–549. doi: 10.1111/j.1432-1033.1976.tb10842.x. [DOI] [PubMed] [Google Scholar]
- Pratt K., Hattman S. Deoxyribonucleic acid methylation and chromatin organization in Tetrahymena thermophila. Mol Cell Biol. 1981 Jul;1(7):600–608. doi: 10.1128/mcb.1.7.600. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Prior C. P., Cantor C. R., Johnson E. M., Littau V. C., Allfrey V. G. Reversible changes in nucleosome structure and histone H3 accessibility in transcriptionally active and inactive states of rDNA chromatin. Cell. 1983 Oct;34(3):1033–1042. doi: 10.1016/0092-8674(83)90561-5. [DOI] [PubMed] [Google Scholar]
- Reilly J. G., Braun R., Thomas C. A., Jr Methjylation in Physarum DNA. FEBS Lett. 1980 Jul 28;116(2):181–184. doi: 10.1016/0014-5793(80)80638-7. [DOI] [PubMed] [Google Scholar]
- SINSHEIMER R. L. The action of pancreatic deoxyribonuclease. II. Isomeric dinucleotides. J Biol Chem. 1955 Aug;215(2):579–583. [PubMed] [Google Scholar]
- Seebeck T., Stalder J., Braun R. Isolation of a minichromosome containing the ribosomal genes from Physarum polycephalum. Biochemistry. 1979 Feb 6;18(3):484–490. doi: 10.1021/bi00570a017. [DOI] [PubMed] [Google Scholar]
- Sinden R. R., Broyles S. S., Pettijohn D. E. Perfect palindromic lac operator DNA sequence exists as a stable cruciform structure in supercoiled DNA in vitro but not in vivo. Proc Natl Acad Sci U S A. 1983 Apr;80(7):1797–1801. doi: 10.1073/pnas.80.7.1797. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Singleton C. K., Klysik J., Stirdivant S. M., Wells R. D. Left-handed Z-DNA is induced by supercoiling in physiological ionic conditions. Nature. 1982 Sep 23;299(5881):312–316. doi: 10.1038/299312a0. [DOI] [PubMed] [Google Scholar]
- Stein R., Sciaky-Gallili N., Razin A., Cedar H. Pattern of methylation of two genes coding for housekeeping functions. Proc Natl Acad Sci U S A. 1983 May;80(9):2422–2426. doi: 10.1073/pnas.80.9.2422. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sturm K. S., Taylor J. H. Distribution of 5-methylcytosine in the DNA of somatic and germline cells from bovine tissues. Nucleic Acids Res. 1981 Sep 25;9(18):4537–4546. doi: 10.1093/nar/9.18.4537. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tantravahi U., Guntaka R. V., Erlanger B. F., Miller O. J. Amplified ribosomal RNA genes in a rat hepatoma cell line are enriched in 5-methylcytosine. Proc Natl Acad Sci U S A. 1981 Jan;78(1):489–493. doi: 10.1073/pnas.78.1.489. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Urieli-Shoval S., Gruenbaum Y., Sedat J., Razin A. The absence of detectable methylated bases in Drosophila melanogaster DNA. FEBS Lett. 1982 Sep 6;146(1):148–152. doi: 10.1016/0014-5793(82)80723-0. [DOI] [PubMed] [Google Scholar]
- Vardimon L., Kressmann A., Cedar H., Maechler M., Doerfler W. Expression of a cloned adenovirus gene is inhibited by in vitro methylation. Proc Natl Acad Sci U S A. 1982 Feb;79(4):1073–1077. doi: 10.1073/pnas.79.4.1073. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Viegas-Péquignot E., Dutrillaux B. Detection of G-C rich heterochromatin by 5-azacytidine in mammals. Hum Genet. 1981;57(2):134–137. doi: 10.1007/BF00282008. [DOI] [PubMed] [Google Scholar]
- Vogt V. M., Braun R. Structure of ribosomal DNA in Physarum polycephalum. J Mol Biol. 1976 Sep 25;106(3):567–587. doi: 10.1016/0022-2836(76)90252-7. [DOI] [PubMed] [Google Scholar]
- Waalwijk C., Flavell R. A. MspI, an isoschizomer of hpaII which cleaves both unmethylated and methylated hpaII sites. Nucleic Acids Res. 1978 Sep;5(9):3231–3236. doi: 10.1093/nar/5.9.3231. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yang R., Lis J., Wu R. Elution of DNA from agarose gels after electrophoresis. Methods Enzymol. 1979;68:176–182. doi: 10.1016/0076-6879(79)68012-6. [DOI] [PubMed] [Google Scholar]



