1887

Microbiology Society logo

Volume 147, Issue 5

Application of fibre-FISH (fluorescence hybridization) to filamentous fungi: visualization of the rRNA gene cluster of the ascomycete Free

Abstract

Fibre-FISH (fluorescence hybridization) has not been used in filamentous fungi before to the authors’ knowledge. In this study, this technique was applied to a filamentous ascomycete, , to visualize the organization of the rRNA gene clusters (rDNA). Using protoplasts embedded in agarose, DNA fibres were released from interphase nuclei and extended on a glass slide. Four kinds of probes (05–90 kb in size) that correspond to specific regions in the repeat unit of rDNA were hybridized singly or in combination to the DNA fibres, and the hybridization was detected with fluorescein- and/or rhodamine-conjugated antibodies after one round of signal amplification. The alternating arrangement of 18S and 28S rRNA genes as well as the tandem repetitive nature of the repeat units were clearly visualized by this single- or two-colour fibre-FISH. With a probe targeting the 58S or 18S rRNA gene, a region spanning over 800 kb could be visualized in a single fibre, allowing estimation of both the copy number of the repeat unit in rDNA and the stretching degree of the DNA fibre. It was shown that has more than 90 copies of the repeat unit in its rDNA and the stretching degree was similar to the value based on the Watson–Crick model. Visualization of individual genes on an extended DNA fibre was accomplished in filamentous fungi by this study.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): ascomycete , chromatin , FISH, fluorescence in situ hybridization , nucleolus , rDNA and ribosome
© Microbiology Society
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-147-5-1183
2001-05-01
2024-04-24
Loading full text...

Full text loading...

/deliver/fulltext/micro/147/5/1471183a.html?itemId=/content/journal/micro/10.1099/00221287-147-5-1183&mimeType=html&fmt=ahah

References

  1. Courseaux A., Grosgeorge J., Gaudray P. 14 other authors 1996; Definition of the minimal MEN1 candidate area based on a 5-Mb integrated map of proximal 11q13. Genomics 37:345–353
     [Google Scholar]
  2. Ersfeld K., Asbeck K., Gull K. 1998; Direct visualisation of individual gene organisation in Trypanosoma brucei by high-resolution in situ hybridisation. Chromosoma 107:237–240 [CrossRef]
     [Google Scholar]
  3. Fransz P. F., Alonso-Blanco C., Liharska T. B., Peeters A. J. M., Zabel P., de Jong J. H. 1996; High-resolution physical mapping in Arabidopsis thaliana and tomato by fluorescence in situ hybridization to extended DNA fibers. Plant J 9:421–430 [CrossRef]
     [Google Scholar]
  4. Garber R. C., Turgeon B. G., Selker E. U., Yoder O. C. 1988; Organization of ribosomal RNA genes in the fungus Cochliobolus heterostrophus . Curr Genet 14:573–582 [CrossRef]
     [Google Scholar]
  5. Heiskanen M., Karhu R., Hellsten E., Peltonen L., Kallioniemi O. P., Palotie A. 1994; High resolution mapping using fluorescence in situ hybridization to extended DNA fibers prepared from agarose-embedded cells. Biotechniques 17:928–934
     [Google Scholar]
  6. Heiskanen M., Hellsten E., Kallioniemi O. P., Alitalo K., Peltonen L., Palotie A, Mäkelä T. P. 1995; Visual mapping by fiber-FISH. Genomics 30:31–36 [CrossRef]
     [Google Scholar]
  7. Heiskanen M., Kallioniemi O., Palotie A. 1996; Fiber-FISH: experiences and a refined protocol. Genet Anal Biomol Eng 12:179–184 [CrossRef]
     [Google Scholar]
  8. Heng H. H. Q., Tsui L. C. 1998; High resolution free chromatin/DNA fiber fluorescent in situ hybridization. J Chromatogr A 806:219–229 [CrossRef]
     [Google Scholar]
  9. Heng H. H. Q., Squire J., Tsui L. C. 1992; High resolution mapping of mammalian genes by in situ hybridization to free chromatin. Proc Natl Acad Sci USA 89:9509–9513 [CrossRef]
     [Google Scholar]
  10. Jackson S. A., Wang M. L., Goodman H. M., Jiang J. 1998; Application of fiber-FISH in physical mapping of Arabidopsis thaliana . Genome 41:566–572 [CrossRef]
     [Google Scholar]
  11. de Jong J. H. Fransz P., Zabel P. 1999; High resolution FISH in plants – techniques and applications. Trends Plant Sci 4:258–263 [CrossRef]
     [Google Scholar]
  12. Leach J., Lang B. R., Yoder O. C. 1982; Methods for selection of mutants and in vitro culture of Cochliobolus heterostrophus . J Gen Microbiol 128:1719–1729
     [Google Scholar]
  13. Leppanen P., Isosomppi J., Schleutker J., Aulla P., Peltonen L. 1996; A physical map of the 6q14-q15 region harboring the locus for the lysosomal membrane sialic acid transport defect. Genomics 37:62–67 [CrossRef]
     [Google Scholar]
  14. Liu W. S., Soldatov N. M., Gustavsson I., Chowdhary B. P. 1998; Fiber-FISH analysis of the 3′-terminal region of the human L-type Ca2+ channel α1c subunit gene. Hereditas 129:169–175
     [Google Scholar]
  15. Maniatis T., Fritsch E. F., Sambrook J. 1982 Molecular Cloning: a Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  16. Parra I., Windle B. 1993; High resolution visual mapping of stretched DNA by fluorescent hybridization. Nat Genet 5:17–21 [CrossRef]
     [Google Scholar]
  17. Raap A. K., Florijn R. J., Blonden L. A. J., Wiegant J., Vaandrager J. W., Vrolijk H., Tanke H. J, den Dunnen J., van Ommen G. J. B. 1996; Fiber FISH as a DNA mapping tool. Methods 9:67–73 [CrossRef]
     [Google Scholar]
  18. Rosenberg C., Florijn R. J., Van De Rijke F. M., Blonden L. A. J., Raap A. K., Den Dunnen J, van Omen G. J. B. 1995; High resolution DNA fiber-FISH on yeast artificial chromosomes: direct visualization of DNA replication. Nat Genet 10:477–479 [CrossRef]
     [Google Scholar]
  19. Shiels C., Coutelle C., Huxley C. 1997; Analysis of ribosomal and alphoid repetitive DNA by fiber-FISH. Cytogenet Cell Genet 76:20–22 [CrossRef]
     [Google Scholar]
  20. Taga M., Murata M. 1994; Visualization of mitotic chromosomes in filamentous fungi by fluorescence staining and fluorescence in situ hybridization. Chromosoma 103:408–413 [CrossRef]
     [Google Scholar]
  21. Taga M., Murata M., VanEtten H. D. 1999; Visualization of a conditionally dispensable chromosome in the filamentous ascomycete Nectria haematococca by fluorescence in situ hybridization. Fungal Genet Biol 26:169–177 [CrossRef]
     [Google Scholar]
  22. Tsuge T., Kobayashi H., Nishimura S. 1989; Organization of ribosomal RNA genes in Alternaria alternata Japanese pear pathotype, a host-selective AK-toxin-producing fungus. Curr Genet 16:267–272 [CrossRef]
     [Google Scholar]
  23. White T. J., Bruns T., Lee S., Taylor J. 1990; Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: a Guide to Methods and Applications pp 225–234 Edited by Innis M. A., Gelfand D. H., Sninsky J. J., White T. J. San Diego, CA: Academic Press;
     [Google Scholar]
  24. Wiegant J., Kalle W., Mullenders L., Brookes S., Hoovers J. M. N., Dauwerse J. G., Raap A. K, van Omen G. J. B. 1992; High-resolution in situ hybridization using DNA halo preparations. Hum Mol Genet 1:587–591 [CrossRef]
     [Google Scholar]
  25. Zhong X. B., Fransz P. F., Ramanna M. S., Zabel P, van Eden J. W., van Kammen A., de Jong J. H. 1998; FISH studies reveal the molecular and chromosomal organization of individual telomere domains in tomato. Plant J 13:507–517 [CrossRef]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-147-5-1183
Loading
Application of fibre-FISH (fluorescence in situ hybridization) to filamentous fungi: visualization of the rRNA gene cluster of the ascomycete Cochliobolus heterostrophus
Microbiology 147, 1183 (2001); https://doi.org/10.1099/00221287-147-5-1183
/content/journal/micro/10.1099/00221287-147-5-1183
/content/journal/micro/10.1099/00221287-147-5-1183
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error