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. 1997 Jun 15;25(12):2409–2416. doi: 10.1093/nar/25.12.2409

Novel pattern of DNA methylation in Neurospora crassa transgenic for the foreign gene hph.

A C Codón 1, Y S Lee 1, V E Russo 1
PMCID: PMC146773  PMID: 9171093

Abstract

It has previously been reported that multiple copies of the hph gene integrated into the genome of Neurospora crassa are methylated at Hpa II sites (CCGG) during the vegetative life cycle of the fungus, while hph genes integrated as single copies are not methylated. Furthermore, methylation is correlated with silencing of the gene. We report here the methylation state of cytosine residues of the major part of the promoter region of the hph gene integrated into the genome of the multiple copy strain HTA5.7 during the vegetative stage of the life cycle. Cytosine methylation is sequence dependent, but the sequence specificity is complex and is different from the sequence specificity known for mammals and plants (CpG and CpNpG). The pattern of DNA methylation reported here is very different from that measured after meiosis in Neurospora or in Ascobulus . After the sexual cycle in those two fungi all the cytosines of multiple stretches of DNA are heavily methylated. This indicates that the still unknown methyltransferase in Neurospora has a different specificity in the sexual and the vegetative stages of the life cycle or that there are different methyltransferases. The pattern of methylation reported here is also different from the pattern of cytosine methylation of transgenes of Petunia , the only pattern published until now in plants that has DNA methylation at cytosines which are not in the canonical sequences CpG and CpNpG.

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Selected References

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  1. Bull J. H., Wootton J. C. Heavily methylated amplified DNA in transformants of Neurospora crassa. Nature. 1984 Aug 23;310(5979):701–704. doi: 10.1038/310701a0. [DOI] [PubMed] [Google Scholar]
  2. Clark S. J., Harrison J., Paul C. L., Frommer M. High sensitivity mapping of methylated cytosines. Nucleic Acids Res. 1994 Aug 11;22(15):2990–2997. doi: 10.1093/nar/22.15.2990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cogoni C., Irelan J. T., Schumacher M., Schmidhauser T. J., Selker E. U., Macino G. Transgene silencing of the al-1 gene in vegetative cells of Neurospora is mediated by a cytoplasmic effector and does not depend on DNA-DNA interactions or DNA methylation. EMBO J. 1996 Jun 17;15(12):3153–3163. [PMC free article] [PubMed] [Google Scholar]
  4. Feil R., Charlton J., Bird A. P., Walter J., Reik W. Methylation analysis on individual chromosomes: improved protocol for bisulphite genomic sequencing. Nucleic Acids Res. 1994 Feb 25;22(4):695–696. doi: 10.1093/nar/22.4.695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Frommer M., McDonald L. E., Millar D. S., Collis C. M., Watt F., Grigg G. W., Molloy P. L., Paul C. L. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1827–1831. doi: 10.1073/pnas.89.5.1827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Goyon C., Barry C., Grégoire A., Faugeron G., Rossignol J. L. Methylation of DNA repeats of decreasing sizes in Ascobolus immersus. Mol Cell Biol. 1996 Jun;16(6):3054–3065. doi: 10.1128/mcb.16.6.3054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Goyon C., Nogueira T. I., Faugeron G. Perpetuation of cytosine methylation in Ascobolus immersus implies a novel type of maintenance methylase. J Mol Biol. 1994 Jul 1;240(1):42–51. doi: 10.1006/jmbi.1994.1416. [DOI] [PubMed] [Google Scholar]
  8. Ingelbrecht I., Van Houdt H., Van Montagu M., Depicker A. Posttranscriptional silencing of reporter transgenes in tobacco correlates with DNA methylation. Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10502–10506. doi: 10.1073/pnas.91.22.10502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Martienssen R., Baron A. Coordinate suppression of mutations caused by Robertson's mutator transposons in maize. Genetics. 1994 Mar;136(3):1157–1170. doi: 10.1093/genetics/136.3.1157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. McClelland M., Nelson M., Raschke E. Effect of site-specific modification on restriction endonucleases and DNA modification methyltransferases. Nucleic Acids Res. 1994 Sep;22(17):3640–3659. doi: 10.1093/nar/22.17.3640. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Meyer P., Niedenhof I., ten Lohuis M. Evidence for cytosine methylation of non-symmetrical sequences in transgenic Petunia hybrida. EMBO J. 1994 May 1;13(9):2084–2088. doi: 10.1002/j.1460-2075.1994.tb06483.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Noyer-Weidner M., Trautner T. A. Methylation of DNA in prokaryotes. EXS. 1993;64:39–108. doi: 10.1007/978-3-0348-9118-9_4. [DOI] [PubMed] [Google Scholar]
  13. Olek A., Oswald J., Walter J. A modified and improved method for bisulphite based cytosine methylation analysis. Nucleic Acids Res. 1996 Dec 15;24(24):5064–5066. doi: 10.1093/nar/24.24.5064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Pandit N. N., Russo V. E. Reversible inactivation of a foreign gene, hph, during the asexual cycle in Neurospora crassa transformants. Mol Gen Genet. 1992 Sep;234(3):412–422. doi: 10.1007/BF00538700. [DOI] [PubMed] [Google Scholar]
  15. Raizis A. M., Schmitt F., Jost J. P. A bisulfite method of 5-methylcytosine mapping that minimizes template degradation. Anal Biochem. 1995 Mar 20;226(1):161–166. doi: 10.1006/abio.1995.1204. [DOI] [PubMed] [Google Scholar]
  16. Rein T., Natale D. A., Gärtner U., Niggemann M., DePamphilis M. L., Zorbas H. Absence of an unusual "densely methylated island" at the hamster dhfr ori-beta. J Biol Chem. 1997 Apr 11;272(15):10021–10029. doi: 10.1074/jbc.272.15.10021. [DOI] [PubMed] [Google Scholar]
  17. Rein T., Zorbas H., DePamphilis M. L. Active mammalian replication origins are associated with a high-density cluster of mCpG dinucleotides. Mol Cell Biol. 1997 Jan;17(1):416–426. doi: 10.1128/mcb.17.1.416. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Russell P. J., Wagner S., Rodland K. D., Feinbaum R. L., Russell J. P., Bret-Harte M. S., Free S. J., Metzenberg R. L. Organization of the ribosomal ribonucleic acid genes in various wild-type strains and wild-collected strains of Neurospora. Mol Gen Genet. 1984;196(2):275–282. doi: 10.1007/BF00328060. [DOI] [PubMed] [Google Scholar]
  19. Selker E. U., Fritz D. Y., Singer M. J. Dense nonsymmetrical DNA methylation resulting from repeat-induced point mutation in Neurospora. Science. 1993 Dec 10;262(5140):1724–1728. doi: 10.1126/science.8259516. [DOI] [PubMed] [Google Scholar]
  20. Tasheva E. S., Roufa D. J. A densely methylated DNA island is associated with a chromosomal replication origin in the human RPS14 locus. Somat Cell Mol Genet. 1995 Nov;21(6):369–383. doi: 10.1007/BF02310205. [DOI] [PubMed] [Google Scholar]
  21. Tasheva E. S., Roufa D. J. Densely methylated DNA islands in mammalian chromosomal replication origins. Mol Cell Biol. 1994 Sep;14(9):5636–5644. doi: 10.1128/mcb.14.9.5636. [DOI] [PMC free article] [PubMed] [Google Scholar]

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