Department of Biology, Faculty of Science, Okayama University, Tsushima-naka, Okayama 700-8530, Japan1
Author for correspondence: Masatoki Taga. Tel: +81 86 251 8501. Fax: +81 86 251 7876. e-mail: mtaga{at}cc.okayama-u.ac.jp
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ABSTRACT |
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Keywords: ascomycete, chromatin, nucleolus, rDNA, ribosome
Abbreviations: FISH, fluorescence in situ hybridization
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INTRODUCTION |
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In spite of the proven usefulness of fibre-FISH in higher eukaryotes, no report using this technique has been published so far for fungi. Considering the extreme smallness of their chromosomes and nuclei compared with those of higher organisms, it is not surprising that application of conventional FISH to the metaphase chromosomes or interphase nuclei has been very limited in this group of organisms (Taga & Murata, 1994 ; Taga et al., 1999
). Regarding fibre-FISH, however, there seems to be no innate hindrance to its use in fungi. Once DNA fibres are properly prepared, FISH techniques should be applicable to fungal DNA fibres without difficulty, as in other organisms.
In this study, we aimed to establish the fibre-FISH technique for filamentous fungi using an ascomycetous corn pathogen, Cochliobolus heterostrophus (Drechsler) Drechsler [anamorph, Bipolaris maydis (Nishikado and Miyake) Shoemaker]. This fungus serves as a model fungal pathogen in plant pathology with which extensive molecular genetic studies have been conducted. As the target DNA sequences, we chose the rRNA gene cluster, or rDNA, because relatively easy detection of this region, owing to its repetitive nature and occupation of a large part of a nucleolar chromosome (M. Taga, D. Tsuchiya & M. Murata, unpublished results), was thought to make it suitable for screening and establishing experimental conditions for fibre-FISH techniques in this fungus.
In this study, procedures for preparing DNA fibres for FISH were established in C. heterostrophus. With these fibres, the in situ organization of rDNA in terms of the arrangement of rRNA genes in the cluster was visually revealed by single- or two-colour fibre-FISH. Data for the number of repeat units in rDNA and the stretching degree of the DNA fibre were also obtained. To our knowledge, this is the first report of visualization of individual genes on an extended DNA fibre in fungi.
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METHODS |
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Preparation of DNA fibres.
Glass slides were coated with poly-L-lysine (Sigma) by soaking for 5 min and dried at room temperature overnight. A tiny protoplastagarose block (approx. 20 µl) was placed on a slide and mounted with 40 µl sterile water. The slide was placed on a heat block at 85 °C for 2030 s to melt the agarose. The liquified agarose drop was mechanically extended with a coverslip as described by Heiskanen et al. (1994) and air-dried. The slides were treated with 100 µg RNase A ml-1 in 2x SSC (1x SSC: 0·15 M NaCl, 0·015 M sodium citrate) for 45 min at 37 °C, dehydrated through an ethanol series (708099%), and air-dried.
DNA probes.
Four probes, each corresponding to a specific part of the repeat unit of rDNA, were used (Fig. 1). pABM2 and pABM4 are heterologous probes from the filamentous imperfect fungus Alternaria alternata, each of which contains XbaI fragments of the rDNA repeat unit of this fungus in Bluescribe M13 (Tsuge et al., 1989
). pLR59 contains a 9 kb PvuII fragment corresponding to the whole repeat unit of C. heterostrophus in pBR322 (Garber et al., 1988
). These three plasmid DNAs were isolated as described by Maniatis et al. (1982)
. ITS, which covers the whole region of the internal transcribed spacers and the 5·8S rRNA gene, was amplified by PCR with universal primers ITS1 and ITS4 (White et al., 1990
) using total genomic DNA of B30.A3.R.45 as template. The PCR reaction mixture contained approximately 100 ng template DNA, 200 µM each of dNTPs, 1·5 mM MgCl2, and 1·25 U Taq polymerase (Toyobo) in a 50 µl reaction volume. The thermal conditions were 10 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 30 s at 55 °C, and 1 min at 72 °C, then final extension for 10 min at 72 °C. After completion of the reaction, an aliquot of the mixture was directly used as a template for PCR labelling.
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In situ hybridization.
Hybridization mixture (50%, v/v, formamide; 10%, w/v, dextran sulfate; 100 ng sonicated salmon sperm DNA µl-1; and the proper amount of labelled probe DNA/2x SSC) was applied to the specimen, covered with a coverslip (18x32 mm), and sealed with rubber cement. The slide was heated for denaturation on a hot plate at 80 °C for 5 min, and incubated for hybridization at 37 °C for 1620 h. After hybridization, the coverslip was removed by floating it off in 2x SSC. Subsequently, the slide was washed once for 15 min in 50% formamide dissolved in 4x SSC at 37 °C, twice for 8 min in 2x SSC, once in 4x SSC for 5 min, and then blocked with 1% Block Ace (Dainippon Pharmaceutical)/4x SSC for 10 min at room temperature. Hybridization of biotin-labelled probes was detected with goat anti-biotin antibody (Vector Laboratories) followed by staining with fluorescein-conjugated rabbit anti-goat antibody (Boehringer Mannheim). Hybridization of digoxigenin-labelled probes was detected with mouse anti-digoxigenin (Boehringer Mannheim) followed by amplification with rhodamine-conjugated rabbit anti-mouse antibody (Chemicon International). All of the antibodies were diluted in 4x SSC containing 1% Block Ace. Incubations for detection and staining were 1 h each at 37 °C, followed by washing for 5 min in 4x SSC, for 5 min in 4x SSC containing 0·1% Triton X-100, and for 5 min in 4x SSC. Finally, the specimens were mounted with an antifading solution, Vectashield (Vector Laboratories).
Fluorescence microscopy.
Observations were made with an epifluorescence microscope (Olympus BHS-RFC) equipped with an IB excitation filter cube (Olympus BH2-DMIB) for fluorescein, and triple band pass filter (Chroma) for two-colour FISH. Photographs were taken on 800 ASA/ISO colour print film (Fujicolor Super HG800). The colour images on the negatives were digitized with a film scanner (Coolscan II; Nikon), and processed by personal computer software Photoshop version 5.0 (Adobe).
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RESULTS AND DISCUSSION |
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As to the interval length between signals, there was no significant difference between the two FISH experiments; i.e. the mean lengths in the FISH with pABM4 and that with ITS were 3·47±0·11 µm [observed number (n)=256] and 3·35±0·09 µm (n=221), respectively. Adopting 9·0 or 9·15 kb as the size of the repeat unit of this fungus (Garber et al., 1988 ), the stretching degree of DNA fibres was calculated to be 2·64 (=9·0/3·41) kb µm-1 (weighted mean length for both experiments) or 2·68 (=9·15/3·41) kb µm-1. These are in reasonable agreement with the extension of B-DNA of the WatsonCrick model (2·9 kb µm-1) as well as with the values obtained in other organisms, i.e. 2·77 kb µm-1 for mammalian rDNA (Shiels et al., 1997
) and 3·27 kb µm-1 for plant rDNA (Fransz et al., 1996
).
In addition, these experiments showed that preparation of DNA fibres exceeding 800 kb (9 kbx90 copies=810 kb) is possible in fungi. The upper limit of fibre length in fibre-FISH had been claimed to be approximately 500 kb in mammals (Heng & Tsui, 1998 ; Heiskanen et al., 1996
), whereas de Jong et al. (1999)
indicated that long fibres up to 1 Mb are obtainable in plants. Preparation of fibres 1 Mb long may be possible irrespective of the kind of organism.
Analysis of arrangement of 18S and 28S rRNA genes in rDNA
Two-colour FISH was carried out to analyse the arrangement of 18S and 28S rRNA genes in the cluster. The biotinylated insert that was excised from pABM2 and the digoxigenin-labelled pABM4 were simultaneously hybridized to the same specimen, and the hybridization was detected with fluorescein- and rhodamine-conjugated secondary antibodies. As shown in Fig. 2(c), alternating green and red signals corresponding to the sites of 28S and 18S rRNA genes were observed. Using a rather simpler detection system in which biotinylated and digoxigenin-labelled probes were detected with avidinFITC and rhodamine-conjugated anti-digoxigenin antibodies, respectively, signals were hardly visible. The result of this FISH presents proof for the head-to-tail tandem repetition of the units in rDNA. The space between neighbouring green or red signals was roughly constant, the mean of which was 3·89±0·22 µm (n=22). With this value, the stretching degree of the DNA fibre was calculated as above to be 2·31 or 2·35 kb µm-1.
Concluding remarks
In this study, the organization of rDNA in terms of the arrangement of rRNA genes was visualized for the first time in fungi. Except for 5S rDNA, as far as we know, there have been only two papers that have used fibre-FISH for visualizing the array of rDNA repeats. In a plant, Fransz et al. (1996) revealed the tandem repeated array of 18S and 28S rRNA genes in Arabidopsis thaliana by two-colour FISH. In a mammal, the repeated arrangement of 18S rRNA genes was shown by single-colour FISH with a probe containing a partial region of the gene (Shiels et al., 1997
). In contrast with the present study, counting of the copy number of repeat units was not performed in either paper. As to the FISH procedures used for the prepared DNA fibre, two rounds of signal amplification were done by Fransz et al. (1996)
, but a single round of amplification gave satisfactory results in the work of Shiels et al. (1997)
and our work.
This study was done using an ordinary epifluorescence microscope equipped with a camera for 35 mm film. As claimed for other organisms (Wiegant et al., 1992 ; Zhong et al., 1998
), instruments such as a cooled CCD camera are not essential for performing fibre-FISH in fungi as well. Therefore, studies similar to ours can be conducted in modestly equipped laboratories.
With the same procedures as described here, we have succeeded in visualizing 28S rRNA genes in another filamentous ascomycete, Nectria haematococca (anamorph, Fusarium solani), as well as detecting a single copy gene, DEC1, which encodes decarboxylase in C. heterostrophus (D. Tsuchiya & M. Taga, unpublished results). This indicates the versatility of our procedures in filamentous fungi. Thus the merits of fibre-FISH, such as higher mapping resolution and detection sensitivity, will be enjoyed in various aspects of genetic study for many fungi. The introduction of this method will especially accelerate high-resolution physical mapping of genomic clones and contribute to the elucidation of genome organization of fungi.
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ACKNOWLEDGEMENTS |
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Received 8 September 2000;
revised 18 December 2000;
accepted 15 January 2001.