Skip to main content
PMC home page
Genetics logoLink to Genetics
. 1995 Dec;141(4):1299–1314. doi: 10.1093/genetics/141.4.1299

Isolation of the Ascobolus Immersus Spore Color Gene B2 and Study in Single Cells of Gene Silencing by Methylation Induced Premeiotically

V Colot 1, J L Rossignol 1
PMCID: PMC1206868  PMID: 8601475

Abstract

The ascomycete Ascobolus immersus has been extensively used as a model system for the genetic study of meiotic recombination. More recently, an epigenetic process, known as methylation induced premeiotically (MIP), that acts on duplicated sequences has been discovered in A. immersus and has raised a new interest in this fungus. To try and extend these studies, we have now cloned the A. immersus spore color gene b2, a well characterized recombination hot-spot. Isolation of the whole gene was verified by physical mapping of four large b2 alterations, followed by transformation and mutant rescue of a null b2 allele. Transformation was also used to duplicate b2 and subject it to MIP. As a result, we were able for the first time to observe gene silencing as early as just after meiosis and in single cells. Furthermore, we have found evidence for a modulating effect of MIP on b2 expression, depending on the region of the gene that is duplicated and hence subjected to MIP.

Full Text

The Full Text of this article is available as a PDF (9.8 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alani E., Reenan R. A., Kolodner R. D. Interaction between mismatch repair and genetic recombination in Saccharomyces cerevisiae. Genetics. 1994 May;137(1):19–39. doi: 10.1093/genetics/137.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barry C., Faugeron G., Rossignol J. L. Methylation induced premeiotically in Ascobolus: coextension with DNA repeat lengths and effect on transcript elongation. Proc Natl Acad Sci U S A. 1993 May 15;90(10):4557–4561. doi: 10.1073/pnas.90.10.4557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cao L., Alani E., Kleckner N. A pathway for generation and processing of double-strand breaks during meiotic recombination in S. cerevisiae. Cell. 1990 Jun 15;61(6):1089–1101. doi: 10.1016/0092-8674(90)90072-m. [DOI] [PubMed] [Google Scholar]
  4. Faugeron G., Goyon C., Grégoire A. Stable allele replacement and unstable non-homologous integration events during transformation of Ascobolus immersus. Gene. 1989 Mar 15;76(1):109–119. doi: 10.1016/0378-1119(89)90013-9. [DOI] [PubMed] [Google Scholar]
  5. Faugeron G., Rhounim L., Rossignol J. L. How does the cell count the number of ectopic copies of a gene in the premeiotic inactivation process acting in Ascobolus immersus? Genetics. 1990 Mar;124(3):585–591. doi: 10.1093/genetics/124.3.585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fincham J. R., Connerton I. F., Notarianni E., Harrington K. Premeiotic disruption of duplicated and triplicated copies of the Neurospora crassa am (glutamate dehydrogenase) gene. Curr Genet. 1989 May;15(5):327–334. doi: 10.1007/BF00419912. [DOI] [PubMed] [Google Scholar]
  7. Foss H. M., Selker E. U. Efficient DNA pairing in a Neurospora mutant defective in chromosome pairing. Mol Gen Genet. 1991 Dec;231(1):49–52. doi: 10.1007/BF00293820. [DOI] [PubMed] [Google Scholar]
  8. Goyon C., Faugeron G., Rossignol J. L. Molecular cloning and characterization of the met2 gene from Ascobolus immersus. Gene. 1988 Mar 31;63(2):297–308. doi: 10.1016/0378-1119(88)90533-1. [DOI] [PubMed] [Google Scholar]
  9. Goyon C., Faugeron G. Targeted transformation of Ascobolus immersus and de novo methylation of the resulting duplicated DNA sequences. Mol Cell Biol. 1989 Jul;9(7):2818–2827. doi: 10.1128/mcb.9.7.2818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Kohne D. E., Levison S. A., Byers M. J. Room temperature method for increasing the rate of DNA reassociation by many thousandfold: the phenol emulsion reassociation technique. Biochemistry. 1977 Nov 29;16(24):5329–5341. doi: 10.1021/bi00643a026. [DOI] [PubMed] [Google Scholar]
  12. Kunkel L. M., Monaco A. P., Middlesworth W., Ochs H. D., Latt S. A. Specific cloning of DNA fragments absent from the DNA of a male patient with an X chromosome deletion. Proc Natl Acad Sci U S A. 1985 Jul;82(14):4778–4782. doi: 10.1073/pnas.82.14.4778. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Laird P. W., Jaenisch R. DNA methylation and cancer. Hum Mol Genet. 1994;3(Spec No):1487–1495. doi: 10.1093/hmg/3.suppl_1.1487. [DOI] [PubMed] [Google Scholar]
  14. Leblon G., Rossignol J. L. Mechanism of gene conversion in Ascobolus immersus. 3. The interaction of heteroallelas in the conversion process. Mol Gen Genet. 1973 Apr 12;122(2):165–182. doi: 10.1007/BF00435189. [DOI] [PubMed] [Google Scholar]
  15. Li E., Bestor T. H., Jaenisch R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell. 1992 Jun 12;69(6):915–926. doi: 10.1016/0092-8674(92)90611-f. [DOI] [PubMed] [Google Scholar]
  16. Lisitsyn N., Lisitsyn N., Wigler M. Cloning the differences between two complex genomes. Science. 1993 Feb 12;259(5097):946–951. doi: 10.1126/science.8438152. [DOI] [PubMed] [Google Scholar]
  17. Martienssen R. A., Richards E. J. DNA methylation in eukaryotes. Curr Opin Genet Dev. 1995 Apr;5(2):234–242. doi: 10.1016/0959-437x(95)80014-x. [DOI] [PubMed] [Google Scholar]
  18. Moulton T., Crenshaw T., Hao Y., Moosikasuwan J., Lin N., Dembitzer F., Hensle T., Weiss L., McMorrow L., Loew T. Epigenetic lesions at the H19 locus in Wilms' tumour patients. Nat Genet. 1994 Jul;7(3):440–447. doi: 10.1038/ng0794-440. [DOI] [PubMed] [Google Scholar]
  19. Nag D. K., Petes T. D. Physical detection of heteroduplexes during meiotic recombination in the yeast Saccharomyces cerevisiae. Mol Cell Biol. 1993 Apr;13(4):2324–2331. doi: 10.1128/mcb.13.4.2324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Nag D. K., White M. A., Petes T. D. Palindromic sequences in heteroduplex DNA inhibit mismatch repair in yeast. Nature. 1989 Jul 27;340(6231):318–320. doi: 10.1038/340318a0. [DOI] [PubMed] [Google Scholar]
  21. Nicolas A., Petes T. D. Polarity of meiotic gene conversion in fungi: contrasting views. Experientia. 1994 Mar 15;50(3):242–252. doi: 10.1007/BF01924007. [DOI] [PubMed] [Google Scholar]
  22. Nicolas A., Treco D., Schultes N. P., Szostak J. W. An initiation site for meiotic gene conversion in the yeast Saccharomyces cerevisiae. Nature. 1989 Mar 2;338(6210):35–39. doi: 10.1038/338035a0. [DOI] [PubMed] [Google Scholar]
  23. RIZET G., ENGELMANN N., LEFORT C., LISSOUBA P., MOUSSEAU J. [On an Ascomycete of interest for the study of certain aspects of the problem of gene structure]. C R Hebd Seances Acad Sci. 1960 Mar 14;250:2050–2052. [PubMed] [Google Scholar]
  24. Rainier S., Feinberg A. P. Genomic imprinting, DNA methylation, and cancer. J Natl Cancer Inst. 1994 May 18;86(10):753–759. doi: 10.1093/jnci/86.10.753. [DOI] [PubMed] [Google Scholar]
  25. Rhounim L., Grégoire A., Salama S., Faugeron G. Clustering of multiple transgene integrations in highly-unstable Ascobolus immersus transformants. Curr Genet. 1994 Oct;26(4):344–351. doi: 10.1007/BF00310499. [DOI] [PubMed] [Google Scholar]
  26. Rhounim L., Rossignol J. L., Faugeron G. Epimutation of repeated genes in Ascobolus immersus. EMBO J. 1992 Dec;11(12):4451–4457. doi: 10.1002/j.1460-2075.1992.tb05546.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rossignol J. L., Faugeron G. Gene inactivation triggered by recognition between DNA repeats. Experientia. 1994 Mar 15;50(3):307–317. doi: 10.1007/BF01924014. [DOI] [PubMed] [Google Scholar]
  28. Rossignol J. L., Faugeron G. MIP: an epigenetic gene silencing process in Ascobolus immersus. Curr Top Microbiol Immunol. 1995;197:179–191. doi: 10.1007/978-3-642-79145-1_12. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Singer M. J., Selker E. U. Genetic and epigenetic inactivation of repetitive sequences in Neurospora crassa: RIP, DNA methylation, and quelling. Curr Top Microbiol Immunol. 1995;197:165–177. doi: 10.1007/978-3-642-79145-1_11. [DOI] [PubMed] [Google Scholar]
  31. Steenman M. J., Rainier S., Dobry C. J., Grundy P., Horon I. L., Feinberg A. P. Loss of imprinting of IGF2 is linked to reduced expression and abnormal methylation of H19 in Wilms' tumour. Nat Genet. 1994 Jul;7(3):433–439. doi: 10.1038/ng0794-433. [DOI] [PubMed] [Google Scholar]
  32. Straus D., Ausubel F. M. Genomic subtraction for cloning DNA corresponding to deletion mutations. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1889–1893. doi: 10.1073/pnas.87.5.1889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sun H., Treco D., Schultes N. P., Szostak J. W. Double-strand breaks at an initiation site for meiotic gene conversion. Nature. 1989 Mar 2;338(6210):87–90. doi: 10.1038/338087a0. [DOI] [PubMed] [Google Scholar]
  34. Weiner B. M., Kleckner N. Chromosome pairing via multiple interstitial interactions before and during meiosis in yeast. Cell. 1994 Jul 1;77(7):977–991. doi: 10.1016/0092-8674(94)90438-3. [DOI] [PubMed] [Google Scholar]
  35. Wieland I., Bolger G., Asouline G., Wigler M. A method for difference cloning: gene amplification following subtractive hybridization. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2720–2724. doi: 10.1073/pnas.87.7.2720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. de Massy B., Nicolas A. The control in cis of the position and the amount of the ARG4 meiotic double-strand break of Saccharomyces cerevisiae. EMBO J. 1993 Apr;12(4):1459–1466. doi: 10.1002/j.1460-2075.1993.tb05789.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES