Application of fibre-FISH (fluorescence in situ hybridization) to filamentous fungi: Visualization of the rRNA gene cluster of the ascomycete Cochliobolus heterostrophus

Abstract

Fibre-FISH (fluorescence in situ hybridization) has not been used in filamentous fungi before to the authors' knowledge. In this study, this technique was applied to a filamentous ascomycete, Cochliobolus heterostrophus, 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 (0·5-9·0 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 5·8S 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 C. heterostrophus 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.