- Email: [email protected]
Location and direction of transcription of the 5S rRNA gene in Armillaria
Short Communications RAYNER, A. D. M. & BODDY, L. (1988). Fungal Decomposition of Wood. Chichester, New York, Brisbane, Toronto, Singapore: John Wiley & Sons. RAYNER, A. D. M., COATES, D., AINSWORTH, A. M., ADAMS, T. J. H., WILLIAMS, E. N. D. & TODD, N. K. (1984). The biological consequences of the individualistic mycelium. In The Ecology and Physiology of the Fungal Mycelium (ed. D. H . Jennings & A. D. M. Rayner), pp. 509-540. Cambridge, UK: Cambridge University Press.
266 SHARLAND, P. R., BURTON, J. L. & RAYNER, A. D. M. (1986). Mycelial dimorphism, interactions and pseudosclerotial plate formation in Hymenorhaeke corrugata. Transactions of the British Mycological Society 86, 158-163. WORRALL, J. J., CHET, I. & HUTTERMANN, A. (1986). Association of rhizomorph formation with laccase activity in Armillaria spp. Journal of General Microbiology 132, 2527-2533.
(Received for publication 11 April 1989)
Location and direction of transcription of the S S rRNA gene in Armillaria LUC C. DUCHESNE A N D JAMES B. A N D E R S O N Department of Botany, Erindale Campus, University of Toronto in Mississauga, Mississauga, Ontario, Canada L5L 1C6
Location and direction of transcription of the 5 s rRNA gene in Armillaria. Mycological Research 94 (2): 266269 (1990). For possible use as a phylogenetic character, the location and direction of transcription of the 5 s rRNA gene relative to the 18s RNA gene in Armilhria has been determined. Genomic DNAs of 33 isolates of Armillaria from 18 North American, European and Australasian species were digested with Bgl I1 and denatured with NaOH. Southern blots were probed with three end-labelled synthetic oligodeoxynucleotides complementary to the 5 s rRNA, the 18s rRNA or the coding strand of the 18s rRNA cistron respectively. For all 33 Armillaria isolates, the oligonucleotides complementary to the 5 s rRNA and the 18s rRNA both hybridized to the same DNA strand whereas the oligonucleotide complementary to the DNA strand coding for the 18s RNA gene hybridized to the other DNA strand. The direction of transcription of the 5 s rRNA cistron in Armillaria is, therefore, the same as that of the 18s rRNA cistron and is consistent across this genus for all the isolates investigated. Key words: Armillaria, Ribosomal RNA genes, 5 s rRNA, 18s rRNA. The location of the 5 s ribosomal R N A (rRNA) gene follows t w o different patterns in fungi. In some species, for example the filamentous ascomycetes Neurospora crassa Shear & Dodge (Selker et al., 1981) and Aspergillus nidulans (Eidam) Wint. (Lockington ef al., 1982), the 5S rRNA genes are found at several sites in the genome outside of the tandemly repeated segment carrying the 18S, 5-8s and 2 6 s rRNA genes. In other species such as Saccharomyces cerevisiae Meyen & Hans. (Bell ef al., 1977) and several species of basidiomycetes (Cassidy et al., 1984; Cassidy & Pukkila, 1987; Buckner, Novotny & Ullrich, 1988), the 5S rRNA gene is adjacent to the 18S, 5.8s and 2 6 s genes on each rDNA repeat unit. In these species the direction of transcription of the 5S gene may be the same, or may be opposite from the transcription of the large precursor carrying the 18S, 5.8s and 2 6 s rRNAs. For example in S. cerevisiae the 5 s and the 2 6 s precursors are transcribed in opposite directions, and are therefore coded o n different DNA strands (Bell ef al., 1977). In the basidiomycetes Schizophyllum commune Fr., Flarnmulina velutipes (Curt. : Fr.) Sing., Agaricus bisporus (Lange) Imbach, Coprinus cinereus (Schaeff. : Fr.) S. F. Gray, C. atmmenfarius (Bull.: Fr.) Fr. and C . micaceus (Bull. : Fr.) Fr. the 5 s rRNA genes are transcribed in the same direction as the rDNA repeats (Cassidy et al., 1984; Cassidy & Pukkila, 1987;
Buckner, Novotny & Ullrich, 1988). In C . comatw (Mull.: Fr.) S. F. Gray, however, the 5 s rRNA gene is oriented in the opposite direction from that of the large precursor transcript (Cassidy & Pukkila, 1987). These results suggest that the orientation of the 5 s rRNA cistron may b e of value for phylogenetic studies within genera. The genus Armillaria consists of several species, some of a group of which cause root-rot of trees (BPrubC & Dessureault, 1988; Anderson & Ullrich, 1979; Termorshuizen & Amolds, 1987; Kile & Watling, 1987). The r D N A repeat of Armillaria has been cloned and mapped with several restriction enzymes (Anderson, Bailey & Pukkila, 1989). Certain polymorphisms in rDNA were phylogenically informative. As in other basidiomycetes, the 5 s RNA gene of Armillaria occurs within the rDNA repeat but its exact location is unknown. The objective of this research was t o determine the location of the 5.5 rRNA gene within the rDNA repeat and to determine whether the orientation of the 5.5 rRNA genes is variable among different species of Armillaria.
Fungal cultures and DNA preparation. The origin, source, location of collection and host of most of the 33 Armillaria isolates used in this study have been described elsewhere (Anderson ef al., 1989). The 1 8 species investigated were
Short Communications represented as follows: North American biological species (NABS)I (isolates 27-1, 28-7, 113-4, 300-2), NABS I1 (isolates 35-5, 160-8), NABS 111 (Isolate 11-9), NABS V (isolates 486, 205-4), NABS VI (isolates 49-8, 97-I), NABS VII (isolates 90-4, 137-I), NABS IX (isolates 139-2, 207-4). NABS X (isolates 140-7, 206-1). A. cepisfipes Velen. (isolates 304-1,317I), A. borealis Marxm. & Korh. (isolates 330-1, 331-I), A. lutea Gillet (isolates 332-1, 333-I), A . mellea (Vahl.: Fr.) Kumm. (isolates 334-1, 335-1). A . osfoyae Romagn. (isolates 336-1, 337-I), A. tabascens (Scop.: Fr.) Sing. (isolates 416, 417), A. novae-zelandiae (Stev.) Herink (isolate 401), A . fumosa Kile & Watl. (isolate 403), A. hinnulea Kile & Watl. (isolate 404), A. limonea Stev. (isolates 406, 407). All isolates were haploid except for isolates 401, 403, 404, 406, 407, 416 and 417, which were diploid. Cultures were established in liquid Complete Yeast Medium (CYM) (Raper, Raper & Miller, 1972) for 4-6 wk in the dark at room temperature. The mycelium was washed with glass distilled water, frozen in liquid N,, lyophilized and stored at -20 OC. DNA extraction was carried out as in Murray & Thompson (1980) and ethidium bromide-CsCl density gradients as in Maniatis, Fritsch & Sambrook (1982). Aliquots of the DNA preparations were digested using the endonuclease Bgl I1 (Bio/Can, Mississauga, Canada) following the supplier's recommendations. The DNAs were denatured with f volume 0-5M-EDTA and & volume 10 N-NaOH (Cassidy & Pukkila, 1987) and subjected to electrophoresis in 0.75 % agarose gels (20 x 24 x 0.4 cm) in Tris-acetate buffer (Maniatis et al., 1982) at 1.5 V cm-' for 16 h. Capillary transfer of DNAs to nylon hybridization membranes was according to the supplier's direction (Genescreen Plus, Du Pont Canada, Inc.). Synthetic oligodeoxynu~leotideprobes. The membranes were probed with three 328/end-labelledsynthetic oligodeoxynucleotides designated d l , 0 - 2 and 0-3. End-labelling of the oligonucleotides was with T4 polynucleotide kinase (Bio/Can, Mississauga, Canada) and [y-32P]ATP (3000 Ci/mmol, Du 0-1 (5' Pont Canada Inc.). Oligonucleotide AGTCCTATGGCCGTGGAT 3') is complementary to the positions 1-18 of the 5 s rRNAs of a number of basidiom~cetes (Walker & Doolittle, 1982; Huysmans et al., 1983). Oligonucleotide 0 - 2 (5' GAATTACCGCGGCTGCTG 3') is complementary to the( 18s rRNA (Primer site 'A', Lane ef al., 1985) and appears to be highly conserved among eukaryotes (positions 791-808, Huysmans & De Wachter, 1986). Oligonucleotide 0 - 3 5' CAGCAGCCGCGGTAATTC 3') is complementary to th coding strand of the 18s rRNA and to oligonucleotide 0 - 2 . Oligonucleotides 0 - 1 and 0 - 3 were purchased from the Regional DNA synthesis laboratory, Calgary, Alberta, Canada. Oligonucleotide 0 - 2 was provided by Dr Masad Dahma, Department of Chemistry, University of Toronto. All three oligonucleotides were synthesized by the deoxynucleoside phosphoramidite method (Beaucage & Caruthers, 1981).Total RNA from Armillaria isolate 300-2 was probed with oligonucleotides 0 - 1 and 0 - 2 to ascertain their complementarity to Armillaria rRNA. Cellular RNA was extracted as described by Wu, Cassidy & Pukkila (1983) and subjected to electrophoresis in 0.7% agarose with formaldehyde (Maniatis ef a]., 1982).
1
267 For Southern hybridization of DNAs with oligonucleotide probes pre-hybridization was at 50° for 3 h in 6 x SSPE, 1% SDS and 10 x Denhart's and 166 pg sheared denatured Escherichia coli DNA (Wahl et al., 1981). Hybridization was overnight at 50° in 6 x SSPE, 1% SDS and end-labelled oligonucleotides. Blots were washed three times at room temperature in 6 x SSPE and 1 % SDS for 5-10 min with a final wash in 1 x SSPE and 1% SDS, dried in vacuo and baked for 15-20 min at 65O. Blots were probed in succession either with the end-labelled oligonucleotides 0 - 1 and 0 - 2 or with 0 - 1 and 0-3. Also, to further confirm the specificity of the probes, the blots were probed with a nick-translated plasmid (pUC9) ligated to the main rDNA repeat (designated pAM2) of Armillaria (Anderson ef al., 1989). Nick translation and probing with the pAM2 plasmid was as in Maniatis ef al. (1982). To determine the location of the 5s rRNA gene, plasmid pAM2 was digested with several enzymes, or combinations of enzymes and probed with end-labelled 0 - 1 in Southern hybridization. Autoradiography was with Kodak X-Omat AR Film with a Du Pont Cronex Lightning-Plus intensifying screen at - 70' for 1-2 d. Oligonucleotide 0 - 1 hybridized strongly with 5 s rRNA of Armillaria isolate 300-2 and also showed a weak reaction with a high molecular weight RNA molecule (Fig. 1). Oligonucleotide 0 - 2 hybridized with 18s rRNA from isolate 3002 (Fig. I). Restriction of genomic DNA of Armillaria with Bgl I1 yielded a single rDNA fragment of molecular weight ca 1013 kbp. This observation is consistent with a previous report (Anderson ef a]., 1989). Hybridization of the denatured DNAs with plasmid pAM2 showed the location of the two DNA strands (Fig. 2). Hybridization with the end-labelled oligonucleotides gave identical results for the 33 isolates investigated. Oligonucleotides 0 - 1 and 0 - 2 hybridized to the same DNA strand whereas oligonucleotide 0 - 3 hybridized with the opposite DNA strand (Fig. 2).
Fig. 1. Specificity of oligonucleotides 0 - 1 and 0 - 2 for rRNA of Armillaria isolate 300-2. Lane I, RNA preparation born isolate no. 301-2 after electrophoresis on agarose gel and staining with ethidium bromide. Lane 2, autoradiogram of northern blot probed with endlabelled oligonucleotide 0 - 1 . Lane 3, autoradiogram of northern blot probed with end-labelled oligonucleotide 0 - 2 .
Short Communications
268
Fig. 2. Autoradiograms of southern blot of 6 Amillaria isolates probed with (I) nick-translated plasmid pAM2 containing the main rDNA repeat of Armiliaria, (2) end-labelled oligonucleotide 0-1, and (3) end-labelled oligonucleotide 0-2. 1
3
2
Restriction mapping of plasmid pAM2 (Anderson et al., 1989) showed that the 5' end of the 5 s rRNA gene is o n a 1.5 kbp fragment approximately 0.8 kbp from the 3' end of the 2 6 s rRNA gene.
+
The hybridization of oligonucleotides 0 - 1 and 0 - 2 to denatured DNAs indicated that the 5 s rRNA gene and the 1 8 s rRNA gene are located on the same D N A strand of the rDNA repeat. Further confirmation was provided b y the observation that oligonucleotide 0 - 3 hybridized as expected t o the opposite D N A strand. These results show that the direction of transcription of the 5 s rRNA genes in Armillaria takes place in the same direction as does transcription of the 18S, 5.8s and 2 6 s rRNA genes. Phylogenetic relationships in Armillaria have been investigated b y comparing restriction maps of rDNA repeats (Anderson et al., 1989). Certain polymorphisms were phylogenically informative uniting sets of species while separating others. W e examined the 5S rRNA gene t o investigate possible differences in organization of rDNAs among Armillaria species, especially those showing insertions within the non-transcribed spacer. Unlike the genus Coprintls, however, the direction of transcription of the 5 s rRNA cistron is constant in all Armillaria examined and n o difference was detected. The nucleotide sequence of probe 0 - 1 , which is complementary to 5S rRNA, used for strand detection in this investigation is at least partially conserved in all the A r m i l h r i a species that were investigated. It remains to b e determined whether the exact sequence of all 18 nucleotides is conserved within the genus Armillaria. Moreover, the nucleotide sequence of other parts of the rRNA genes may provide valuable data for the phylogeny of Armillaria. It is possible that the presence of inversions in the 5 s RNA genes such as reported for the genus Coprintrs (Cassidy & Pukkila, 1987) is a phenomenon of rare occurrence. Altematively, such inversions may characterize large groups of basidiomycetes not yet investigated. This study demonstrates that the use of oligodeoxynucleotide probes may be advantageous t o investigate the direction of transcription of 5S ribosomal genes. The sequence for oligonucleotide 0 - 1 was the first 18 bases o n the 5' end of 5 s rRNA which are conserved in other basidiomycetes (Walker & Doolittle, 1982; Huysmans et al., 1983) and used in conjunction with two oligonucleotides conserved among the 1 8 s rRNA genes of eukaryotes. The use of such oligonucleotide probes allows
the rapid determination of rRNA gene transcription using genomic D N A and without the isolation of RNA molecules o r gene cloning. This technique can also be extended to other eukaryotes using 5S oligonucleotide probes that are complementary t o consensus sequences in their taxonomic groups. This research was supported b y a NSERC Operating grant to JBA and a NSERC Postdoctoral fellowship to LCD.
REFERENCES ANDERSON, J. B., BAILEY, S. S. & PUKKILA, P. J. (1989).Variation in ribosomal RNA among biological species of Armillaria, a genus of root-infecting fungi. Evolution (in the press). ANDERSON, J. B. & ULLRICH, R. C. (1979). Biological species of Armillaria mellea in North America. Mycologia 71, 402-414. BELL, G. I., DEGENNARD, L. J., GELFAND, D. H., BISHOP, R. J., VALENZUELA, P. & RUTTER, W. J. (1977). Ribosomal RNA genes of Saccharomyces cerevisiae. journal of Biological Chemistry 25, 8118-8125. BERUBE, J. A. & DESSUREAULT, M. (1988). Morphological characterization of Amillaria ostoyae and Amiliaria sinapina sp. nov. Canadian ]ournal of Botany 66, 2027-2034. BEAUCAGE, S. L. & CARUTHERS, M. H. (1981). De~x~nucleoside phosphoramidites - A new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Letters 22, 1856-1862. BUCKNER, B., NOVOTNY, C, P. & ULLRICH, R. C. (1988). Organization of the ribosomal RNA genes of Schizophyllum commune. Current Genetics 13, 417-424. CASSIDY, J. R., MOORE, D., LU, B. C. & PUKKILA, P. J. (1984). Unusual organization and lack of recombination in the ribosomal RNA genes of Coprinw cinereus. Current Genetics 8, 601-613. CASSIDY, J. R. & PUKKILA, P. J. (1987). Inversion of 5s ribosomal RNA genes within the genus Coprinw. Current Genetics 12,33-36. HUYSMANS, E. & DE WACHTER R. (1986). Compilation of small ribosomal subunit RNA sequences. Nucleic Acids Research 14, supplement r73-118. HUYSMANS, E., DAMS, E., VANDENBERGHE, A. & DE WACHTER, R. (1983). The nucleotide sequences of the 5 s rRNAs of four mushrooms and their use in studying the phylogenic position of basidiomycetes among the eukaryotes. Nucleic Acids Research 11, 2871-2880. KILE, G. A. & WATLING, R. (1987). Genetic analysis of the life cycle of Agaricw bisporus. Transactions of the British Mycological Society 91, 305-315. LOCKINGTON, R. A., TAYLOR, G. G., WINTHER, M., SCAZZOCCHIO, C. & DAVIES, W. (1982). A ~hysicalmap of the ribosomal RNA repeat unit of Aspergillw nidulans. Gene 20, 135-137.
Short Communications LANE, D. J., PACE, B., OLSEN, G. J., STAHL, D. A., SOGIN, M. L. & PACE, N. R. (1985). Rapid determination of 16s ribosomal RNA sequences for phylogenetic analyses. Proceedings of fhe National Academy of Sciences, U S A 82, 695545959. MANIATIS, T., FRITSCH, E. F. & SAMBROOK, J. (1982). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory: Cold Spring Harbor, New York. MURRAY, M. G. & THOMPSON, W. F. (1980). Rapid isolation of high molecular weight $ant DNA. Nucleic Acids Research 8, 4321-4325. RAPER, C. A., MPER, J. R. &MILLER, R. E. (1972).Genetic analysis of the life cycle of Agaricw bisponts. Mycologia 64, 1088-1117. SELKER, E. U., YANOFSKY, C., DRIFTMEIR, K., METZENBERG, R. L., ALZNER-DE WEED, B. & RAJ BHANDARY, U. L. (1981).
269 Dispersed 5 s RNA genes in Neurospora crassa : structure, expression and evolution. Cell 24, 819-828. TERMORSHUIZEN, A. & ARNOLDS, E. (1987). On the nomenclature of the European species of the Armillaria mellea group. Mycotaxon 30, 101-116. WALKER, W. F. & DOOLITTLE, W. F. (1982). Redividing the basidiomycetes on the basis of 5s rRNA sequences. Nature, London 299, 723-724. WAHL, G. M., ONG, E., MEINKOTIT, J., FRANCO, R. & BARANAGA, M. (1981). The Transfer of DNA, R N A and Proteins to Nifrocellulose and Diazotized Paper Solid Support. Schleicher & Schrell, Keene, NH. WU, M. M., CASSIDY, J. R. & PUKKILA, P. J. (1983).Polymorphisms in DNA of Coprinw cinerew. Current Genetics 7, 385-392.
(Received for publicafion 27 June 1989)
The genus Mundkurella us ti lag in ale^)^ K. V A N K Y Universitaf Tiibingen, Insfifut fiir Biologie I, Lehrsfuhl Spezielle Bofanik, Auf der Morgenstelle, 0-7400 Tiibingen, W. Germany
The genus Mundkttrella (Ustilaginales). Mycological Research 94 (2): 269-273 (1990). The smut genus Mundkurella is redescribed. In addition to the two known species ( M . heptapleuri, on Heptapleurum venulosum, from India, and M . mossii, on Aralia nudicaulis, from North America), a third species, M . kalopanacis, is described on Kalopanax pictus, originating from Korea. The three species parasitize hosts in the Araliaceae. Key words: Ustilaginales, Mundkurella, Taxonomy.
The genus Mundkurella was erected by Thirurnalachar (1944) for a peculiar fungus collected on Heptapleurum venulosum Seem., in India. Mundkurella (see also Vinky, 1987) produces sori in fruits, stems, petioles and leaves. Sori contain two kinds of spores: the first, I-celled, large, light-coloured, thin-walled, presumably sterile, and the second, 1- to 4-celled spores, darker-coloured,with rich cell contents. Following germination septate promycelia produce terminal and lateral sporidia. Thirumalachar (1944) considered that the 'heterosporous sori' and the presence of the 'constrictor cells' between the promycelial (metabasidial) cells are the most important characters of the genus Mundkurella. However, Thirumalachar's 'constrictor cells' (Thirumalachar 1944, Fig. 11)seem to be identical with the 'metabasidial fragmentation zones', which are common in the Ustilaginales. Fragmentation zones develop between primary and secondary septa of the metabasidium, or at the metabasidium-basidiospore isthmus between the sterigmal and hilar septa, studied among others in Usfilago maydis (DC.) Corda (O'Donnell & McLaughlin, 1984). The type species, Mundkurella heptapleuri Thirum., on Heptapleurum venulosum Seem. (Fig. 1a), was collected in India, Bangalore, Lal-Bagh, 15 Aug. 1942, M. J. Thirurnalachar Studies in Heter~basidiom~cetes, part 58.
(HCIO, BPI!). Its sori, formed in fruits, stems, petioles and leaves, contain a black, semi-agglutinated to powdery mass of spores. Spores are produced in large groups (Fig. 3), on the end of sporogenous filaments. Immature spores are all unicellular and light-coloured (Fig. 4). Mature spores (Fig. 5) are two kinds. Firstly, I-celled, large, 13-24 x 16-30 pm, subglobose or ovoid, usually collapsed, pale yellow or hyaline, thin-walled, verruculose and supposedly sterile. Secondly, 1- and 2-, rarely 3- or &celled spores, deep chocolate brown, with rich granular contents, subglobose to ovateoblong, 10-17 x 12-27 pm; wall thick, 1.5-2 pm excluding warts, composed of two layers : a dark brown, 0.5-1 pm thick inner layer, and a light yellowish-brown, 0.8-1.5 pm thick outer layer. Spore surface densely, rather coarsely verruculose, appearing serrate in profile. Warts 0.4-0.7 pm high x 0.30.8 pm diam, round or by confluences forming irregular groups or rows (Fig. 6). Intermediate spore forms may occur. After germination (Fig. Ib) septate promycelia with 'constrictor cells' produce terminal and lateral sporidia (Thirumalachar, 1944). The second species, Mundkurella mossii Savile (1975), was described on Aralia nudicaulis L., from Canada and U.S.A. (Type: Canada, Edmonton, 16 June 1931, E. H. Moss 2187; DAOM, BPI). It differs from M. heptapleuri in having slightly smaller, mostly 2- to $-celled spores rather than 1- to 2-celled
266 SHARLAND, P. R., BURTON, J. L. & RAYNER, A. D. M. (1986). Mycelial dimorphism, interactions and pseudosclerotial plate formation in Hymenorhaeke corrugata. Transactions of the British Mycological Society 86, 158-163. WORRALL, J. J., CHET, I. & HUTTERMANN, A. (1986). Association of rhizomorph formation with laccase activity in Armillaria spp. Journal of General Microbiology 132, 2527-2533.
(Received for publication 11 April 1989)
Location and direction of transcription of the S S rRNA gene in Armillaria LUC C. DUCHESNE A N D JAMES B. A N D E R S O N Department of Botany, Erindale Campus, University of Toronto in Mississauga, Mississauga, Ontario, Canada L5L 1C6
Location and direction of transcription of the 5 s rRNA gene in Armillaria. Mycological Research 94 (2): 266269 (1990). For possible use as a phylogenetic character, the location and direction of transcription of the 5 s rRNA gene relative to the 18s RNA gene in Armilhria has been determined. Genomic DNAs of 33 isolates of Armillaria from 18 North American, European and Australasian species were digested with Bgl I1 and denatured with NaOH. Southern blots were probed with three end-labelled synthetic oligodeoxynucleotides complementary to the 5 s rRNA, the 18s rRNA or the coding strand of the 18s rRNA cistron respectively. For all 33 Armillaria isolates, the oligonucleotides complementary to the 5 s rRNA and the 18s rRNA both hybridized to the same DNA strand whereas the oligonucleotide complementary to the DNA strand coding for the 18s RNA gene hybridized to the other DNA strand. The direction of transcription of the 5 s rRNA cistron in Armillaria is, therefore, the same as that of the 18s rRNA cistron and is consistent across this genus for all the isolates investigated. Key words: Armillaria, Ribosomal RNA genes, 5 s rRNA, 18s rRNA. The location of the 5 s ribosomal R N A (rRNA) gene follows t w o different patterns in fungi. In some species, for example the filamentous ascomycetes Neurospora crassa Shear & Dodge (Selker et al., 1981) and Aspergillus nidulans (Eidam) Wint. (Lockington ef al., 1982), the 5S rRNA genes are found at several sites in the genome outside of the tandemly repeated segment carrying the 18S, 5-8s and 2 6 s rRNA genes. In other species such as Saccharomyces cerevisiae Meyen & Hans. (Bell ef al., 1977) and several species of basidiomycetes (Cassidy et al., 1984; Cassidy & Pukkila, 1987; Buckner, Novotny & Ullrich, 1988), the 5S rRNA gene is adjacent to the 18S, 5.8s and 2 6 s genes on each rDNA repeat unit. In these species the direction of transcription of the 5S gene may be the same, or may be opposite from the transcription of the large precursor carrying the 18S, 5.8s and 2 6 s rRNAs. For example in S. cerevisiae the 5 s and the 2 6 s precursors are transcribed in opposite directions, and are therefore coded o n different DNA strands (Bell ef al., 1977). In the basidiomycetes Schizophyllum commune Fr., Flarnmulina velutipes (Curt. : Fr.) Sing., Agaricus bisporus (Lange) Imbach, Coprinus cinereus (Schaeff. : Fr.) S. F. Gray, C. atmmenfarius (Bull.: Fr.) Fr. and C . micaceus (Bull. : Fr.) Fr. the 5 s rRNA genes are transcribed in the same direction as the rDNA repeats (Cassidy et al., 1984; Cassidy & Pukkila, 1987;
Buckner, Novotny & Ullrich, 1988). In C . comatw (Mull.: Fr.) S. F. Gray, however, the 5 s rRNA gene is oriented in the opposite direction from that of the large precursor transcript (Cassidy & Pukkila, 1987). These results suggest that the orientation of the 5 s rRNA cistron may b e of value for phylogenetic studies within genera. The genus Armillaria consists of several species, some of a group of which cause root-rot of trees (BPrubC & Dessureault, 1988; Anderson & Ullrich, 1979; Termorshuizen & Amolds, 1987; Kile & Watling, 1987). The r D N A repeat of Armillaria has been cloned and mapped with several restriction enzymes (Anderson, Bailey & Pukkila, 1989). Certain polymorphisms in rDNA were phylogenically informative. As in other basidiomycetes, the 5 s RNA gene of Armillaria occurs within the rDNA repeat but its exact location is unknown. The objective of this research was t o determine the location of the 5.5 rRNA gene within the rDNA repeat and to determine whether the orientation of the 5.5 rRNA genes is variable among different species of Armillaria.
Fungal cultures and DNA preparation. The origin, source, location of collection and host of most of the 33 Armillaria isolates used in this study have been described elsewhere (Anderson ef al., 1989). The 1 8 species investigated were
Short Communications represented as follows: North American biological species (NABS)I (isolates 27-1, 28-7, 113-4, 300-2), NABS I1 (isolates 35-5, 160-8), NABS 111 (Isolate 11-9), NABS V (isolates 486, 205-4), NABS VI (isolates 49-8, 97-I), NABS VII (isolates 90-4, 137-I), NABS IX (isolates 139-2, 207-4). NABS X (isolates 140-7, 206-1). A. cepisfipes Velen. (isolates 304-1,317I), A. borealis Marxm. & Korh. (isolates 330-1, 331-I), A. lutea Gillet (isolates 332-1, 333-I), A . mellea (Vahl.: Fr.) Kumm. (isolates 334-1, 335-1). A . osfoyae Romagn. (isolates 336-1, 337-I), A. tabascens (Scop.: Fr.) Sing. (isolates 416, 417), A. novae-zelandiae (Stev.) Herink (isolate 401), A . fumosa Kile & Watl. (isolate 403), A. hinnulea Kile & Watl. (isolate 404), A. limonea Stev. (isolates 406, 407). All isolates were haploid except for isolates 401, 403, 404, 406, 407, 416 and 417, which were diploid. Cultures were established in liquid Complete Yeast Medium (CYM) (Raper, Raper & Miller, 1972) for 4-6 wk in the dark at room temperature. The mycelium was washed with glass distilled water, frozen in liquid N,, lyophilized and stored at -20 OC. DNA extraction was carried out as in Murray & Thompson (1980) and ethidium bromide-CsCl density gradients as in Maniatis, Fritsch & Sambrook (1982). Aliquots of the DNA preparations were digested using the endonuclease Bgl I1 (Bio/Can, Mississauga, Canada) following the supplier's recommendations. The DNAs were denatured with f volume 0-5M-EDTA and & volume 10 N-NaOH (Cassidy & Pukkila, 1987) and subjected to electrophoresis in 0.75 % agarose gels (20 x 24 x 0.4 cm) in Tris-acetate buffer (Maniatis et al., 1982) at 1.5 V cm-' for 16 h. Capillary transfer of DNAs to nylon hybridization membranes was according to the supplier's direction (Genescreen Plus, Du Pont Canada, Inc.). Synthetic oligodeoxynu~leotideprobes. The membranes were probed with three 328/end-labelledsynthetic oligodeoxynucleotides designated d l , 0 - 2 and 0-3. End-labelling of the oligonucleotides was with T4 polynucleotide kinase (Bio/Can, Mississauga, Canada) and [y-32P]ATP (3000 Ci/mmol, Du 0-1 (5' Pont Canada Inc.). Oligonucleotide AGTCCTATGGCCGTGGAT 3') is complementary to the positions 1-18 of the 5 s rRNAs of a number of basidiom~cetes (Walker & Doolittle, 1982; Huysmans et al., 1983). Oligonucleotide 0 - 2 (5' GAATTACCGCGGCTGCTG 3') is complementary to the( 18s rRNA (Primer site 'A', Lane ef al., 1985) and appears to be highly conserved among eukaryotes (positions 791-808, Huysmans & De Wachter, 1986). Oligonucleotide 0 - 3 5' CAGCAGCCGCGGTAATTC 3') is complementary to th coding strand of the 18s rRNA and to oligonucleotide 0 - 2 . Oligonucleotides 0 - 1 and 0 - 3 were purchased from the Regional DNA synthesis laboratory, Calgary, Alberta, Canada. Oligonucleotide 0 - 2 was provided by Dr Masad Dahma, Department of Chemistry, University of Toronto. All three oligonucleotides were synthesized by the deoxynucleoside phosphoramidite method (Beaucage & Caruthers, 1981).Total RNA from Armillaria isolate 300-2 was probed with oligonucleotides 0 - 1 and 0 - 2 to ascertain their complementarity to Armillaria rRNA. Cellular RNA was extracted as described by Wu, Cassidy & Pukkila (1983) and subjected to electrophoresis in 0.7% agarose with formaldehyde (Maniatis ef a]., 1982).
1
267 For Southern hybridization of DNAs with oligonucleotide probes pre-hybridization was at 50° for 3 h in 6 x SSPE, 1% SDS and 10 x Denhart's and 166 pg sheared denatured Escherichia coli DNA (Wahl et al., 1981). Hybridization was overnight at 50° in 6 x SSPE, 1% SDS and end-labelled oligonucleotides. Blots were washed three times at room temperature in 6 x SSPE and 1 % SDS for 5-10 min with a final wash in 1 x SSPE and 1% SDS, dried in vacuo and baked for 15-20 min at 65O. Blots were probed in succession either with the end-labelled oligonucleotides 0 - 1 and 0 - 2 or with 0 - 1 and 0-3. Also, to further confirm the specificity of the probes, the blots were probed with a nick-translated plasmid (pUC9) ligated to the main rDNA repeat (designated pAM2) of Armillaria (Anderson ef al., 1989). Nick translation and probing with the pAM2 plasmid was as in Maniatis ef al. (1982). To determine the location of the 5s rRNA gene, plasmid pAM2 was digested with several enzymes, or combinations of enzymes and probed with end-labelled 0 - 1 in Southern hybridization. Autoradiography was with Kodak X-Omat AR Film with a Du Pont Cronex Lightning-Plus intensifying screen at - 70' for 1-2 d. Oligonucleotide 0 - 1 hybridized strongly with 5 s rRNA of Armillaria isolate 300-2 and also showed a weak reaction with a high molecular weight RNA molecule (Fig. 1). Oligonucleotide 0 - 2 hybridized with 18s rRNA from isolate 3002 (Fig. I). Restriction of genomic DNA of Armillaria with Bgl I1 yielded a single rDNA fragment of molecular weight ca 1013 kbp. This observation is consistent with a previous report (Anderson ef a]., 1989). Hybridization of the denatured DNAs with plasmid pAM2 showed the location of the two DNA strands (Fig. 2). Hybridization with the end-labelled oligonucleotides gave identical results for the 33 isolates investigated. Oligonucleotides 0 - 1 and 0 - 2 hybridized to the same DNA strand whereas oligonucleotide 0 - 3 hybridized with the opposite DNA strand (Fig. 2).
Fig. 1. Specificity of oligonucleotides 0 - 1 and 0 - 2 for rRNA of Armillaria isolate 300-2. Lane I, RNA preparation born isolate no. 301-2 after electrophoresis on agarose gel and staining with ethidium bromide. Lane 2, autoradiogram of northern blot probed with endlabelled oligonucleotide 0 - 1 . Lane 3, autoradiogram of northern blot probed with end-labelled oligonucleotide 0 - 2 .
Short Communications
268
Fig. 2. Autoradiograms of southern blot of 6 Amillaria isolates probed with (I) nick-translated plasmid pAM2 containing the main rDNA repeat of Armiliaria, (2) end-labelled oligonucleotide 0-1, and (3) end-labelled oligonucleotide 0-2. 1
3
2
Restriction mapping of plasmid pAM2 (Anderson et al., 1989) showed that the 5' end of the 5 s rRNA gene is o n a 1.5 kbp fragment approximately 0.8 kbp from the 3' end of the 2 6 s rRNA gene.
+
The hybridization of oligonucleotides 0 - 1 and 0 - 2 to denatured DNAs indicated that the 5 s rRNA gene and the 1 8 s rRNA gene are located on the same D N A strand of the rDNA repeat. Further confirmation was provided b y the observation that oligonucleotide 0 - 3 hybridized as expected t o the opposite D N A strand. These results show that the direction of transcription of the 5 s rRNA genes in Armillaria takes place in the same direction as does transcription of the 18S, 5.8s and 2 6 s rRNA genes. Phylogenetic relationships in Armillaria have been investigated b y comparing restriction maps of rDNA repeats (Anderson et al., 1989). Certain polymorphisms were phylogenically informative uniting sets of species while separating others. W e examined the 5S rRNA gene t o investigate possible differences in organization of rDNAs among Armillaria species, especially those showing insertions within the non-transcribed spacer. Unlike the genus Coprintls, however, the direction of transcription of the 5 s rRNA cistron is constant in all Armillaria examined and n o difference was detected. The nucleotide sequence of probe 0 - 1 , which is complementary to 5S rRNA, used for strand detection in this investigation is at least partially conserved in all the A r m i l h r i a species that were investigated. It remains to b e determined whether the exact sequence of all 18 nucleotides is conserved within the genus Armillaria. Moreover, the nucleotide sequence of other parts of the rRNA genes may provide valuable data for the phylogeny of Armillaria. It is possible that the presence of inversions in the 5 s RNA genes such as reported for the genus Coprintrs (Cassidy & Pukkila, 1987) is a phenomenon of rare occurrence. Altematively, such inversions may characterize large groups of basidiomycetes not yet investigated. This study demonstrates that the use of oligodeoxynucleotide probes may be advantageous t o investigate the direction of transcription of 5S ribosomal genes. The sequence for oligonucleotide 0 - 1 was the first 18 bases o n the 5' end of 5 s rRNA which are conserved in other basidiomycetes (Walker & Doolittle, 1982; Huysmans et al., 1983) and used in conjunction with two oligonucleotides conserved among the 1 8 s rRNA genes of eukaryotes. The use of such oligonucleotide probes allows
the rapid determination of rRNA gene transcription using genomic D N A and without the isolation of RNA molecules o r gene cloning. This technique can also be extended to other eukaryotes using 5S oligonucleotide probes that are complementary t o consensus sequences in their taxonomic groups. This research was supported b y a NSERC Operating grant to JBA and a NSERC Postdoctoral fellowship to LCD.
REFERENCES ANDERSON, J. B., BAILEY, S. S. & PUKKILA, P. J. (1989).Variation in ribosomal RNA among biological species of Armillaria, a genus of root-infecting fungi. Evolution (in the press). ANDERSON, J. B. & ULLRICH, R. C. (1979). Biological species of Armillaria mellea in North America. Mycologia 71, 402-414. BELL, G. I., DEGENNARD, L. J., GELFAND, D. H., BISHOP, R. J., VALENZUELA, P. & RUTTER, W. J. (1977). Ribosomal RNA genes of Saccharomyces cerevisiae. journal of Biological Chemistry 25, 8118-8125. BERUBE, J. A. & DESSUREAULT, M. (1988). Morphological characterization of Amillaria ostoyae and Amiliaria sinapina sp. nov. Canadian ]ournal of Botany 66, 2027-2034. BEAUCAGE, S. L. & CARUTHERS, M. H. (1981). De~x~nucleoside phosphoramidites - A new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Letters 22, 1856-1862. BUCKNER, B., NOVOTNY, C, P. & ULLRICH, R. C. (1988). Organization of the ribosomal RNA genes of Schizophyllum commune. Current Genetics 13, 417-424. CASSIDY, J. R., MOORE, D., LU, B. C. & PUKKILA, P. J. (1984). Unusual organization and lack of recombination in the ribosomal RNA genes of Coprinw cinereus. Current Genetics 8, 601-613. CASSIDY, J. R. & PUKKILA, P. J. (1987). Inversion of 5s ribosomal RNA genes within the genus Coprinw. Current Genetics 12,33-36. HUYSMANS, E. & DE WACHTER R. (1986). Compilation of small ribosomal subunit RNA sequences. Nucleic Acids Research 14, supplement r73-118. HUYSMANS, E., DAMS, E., VANDENBERGHE, A. & DE WACHTER, R. (1983). The nucleotide sequences of the 5 s rRNAs of four mushrooms and their use in studying the phylogenic position of basidiomycetes among the eukaryotes. Nucleic Acids Research 11, 2871-2880. KILE, G. A. & WATLING, R. (1987). Genetic analysis of the life cycle of Agaricw bisporus. Transactions of the British Mycological Society 91, 305-315. LOCKINGTON, R. A., TAYLOR, G. G., WINTHER, M., SCAZZOCCHIO, C. & DAVIES, W. (1982). A ~hysicalmap of the ribosomal RNA repeat unit of Aspergillw nidulans. Gene 20, 135-137.
Short Communications LANE, D. J., PACE, B., OLSEN, G. J., STAHL, D. A., SOGIN, M. L. & PACE, N. R. (1985). Rapid determination of 16s ribosomal RNA sequences for phylogenetic analyses. Proceedings of fhe National Academy of Sciences, U S A 82, 695545959. MANIATIS, T., FRITSCH, E. F. & SAMBROOK, J. (1982). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory: Cold Spring Harbor, New York. MURRAY, M. G. & THOMPSON, W. F. (1980). Rapid isolation of high molecular weight $ant DNA. Nucleic Acids Research 8, 4321-4325. RAPER, C. A., MPER, J. R. &MILLER, R. E. (1972).Genetic analysis of the life cycle of Agaricw bisponts. Mycologia 64, 1088-1117. SELKER, E. U., YANOFSKY, C., DRIFTMEIR, K., METZENBERG, R. L., ALZNER-DE WEED, B. & RAJ BHANDARY, U. L. (1981).
269 Dispersed 5 s RNA genes in Neurospora crassa : structure, expression and evolution. Cell 24, 819-828. TERMORSHUIZEN, A. & ARNOLDS, E. (1987). On the nomenclature of the European species of the Armillaria mellea group. Mycotaxon 30, 101-116. WALKER, W. F. & DOOLITTLE, W. F. (1982). Redividing the basidiomycetes on the basis of 5s rRNA sequences. Nature, London 299, 723-724. WAHL, G. M., ONG, E., MEINKOTIT, J., FRANCO, R. & BARANAGA, M. (1981). The Transfer of DNA, R N A and Proteins to Nifrocellulose and Diazotized Paper Solid Support. Schleicher & Schrell, Keene, NH. WU, M. M., CASSIDY, J. R. & PUKKILA, P. J. (1983).Polymorphisms in DNA of Coprinw cinerew. Current Genetics 7, 385-392.
(Received for publicafion 27 June 1989)
The genus Mundkurella us ti lag in ale^)^ K. V A N K Y Universitaf Tiibingen, Insfifut fiir Biologie I, Lehrsfuhl Spezielle Bofanik, Auf der Morgenstelle, 0-7400 Tiibingen, W. Germany
The genus Mundkttrella (Ustilaginales). Mycological Research 94 (2): 269-273 (1990). The smut genus Mundkurella is redescribed. In addition to the two known species ( M . heptapleuri, on Heptapleurum venulosum, from India, and M . mossii, on Aralia nudicaulis, from North America), a third species, M . kalopanacis, is described on Kalopanax pictus, originating from Korea. The three species parasitize hosts in the Araliaceae. Key words: Ustilaginales, Mundkurella, Taxonomy.
The genus Mundkurella was erected by Thirurnalachar (1944) for a peculiar fungus collected on Heptapleurum venulosum Seem., in India. Mundkurella (see also Vinky, 1987) produces sori in fruits, stems, petioles and leaves. Sori contain two kinds of spores: the first, I-celled, large, light-coloured, thin-walled, presumably sterile, and the second, 1- to 4-celled spores, darker-coloured,with rich cell contents. Following germination septate promycelia produce terminal and lateral sporidia. Thirumalachar (1944) considered that the 'heterosporous sori' and the presence of the 'constrictor cells' between the promycelial (metabasidial) cells are the most important characters of the genus Mundkurella. However, Thirumalachar's 'constrictor cells' (Thirumalachar 1944, Fig. 11)seem to be identical with the 'metabasidial fragmentation zones', which are common in the Ustilaginales. Fragmentation zones develop between primary and secondary septa of the metabasidium, or at the metabasidium-basidiospore isthmus between the sterigmal and hilar septa, studied among others in Usfilago maydis (DC.) Corda (O'Donnell & McLaughlin, 1984). The type species, Mundkurella heptapleuri Thirum., on Heptapleurum venulosum Seem. (Fig. 1a), was collected in India, Bangalore, Lal-Bagh, 15 Aug. 1942, M. J. Thirurnalachar Studies in Heter~basidiom~cetes, part 58.
(HCIO, BPI!). Its sori, formed in fruits, stems, petioles and leaves, contain a black, semi-agglutinated to powdery mass of spores. Spores are produced in large groups (Fig. 3), on the end of sporogenous filaments. Immature spores are all unicellular and light-coloured (Fig. 4). Mature spores (Fig. 5) are two kinds. Firstly, I-celled, large, 13-24 x 16-30 pm, subglobose or ovoid, usually collapsed, pale yellow or hyaline, thin-walled, verruculose and supposedly sterile. Secondly, 1- and 2-, rarely 3- or &celled spores, deep chocolate brown, with rich granular contents, subglobose to ovateoblong, 10-17 x 12-27 pm; wall thick, 1.5-2 pm excluding warts, composed of two layers : a dark brown, 0.5-1 pm thick inner layer, and a light yellowish-brown, 0.8-1.5 pm thick outer layer. Spore surface densely, rather coarsely verruculose, appearing serrate in profile. Warts 0.4-0.7 pm high x 0.30.8 pm diam, round or by confluences forming irregular groups or rows (Fig. 6). Intermediate spore forms may occur. After germination (Fig. Ib) septate promycelia with 'constrictor cells' produce terminal and lateral sporidia (Thirumalachar, 1944). The second species, Mundkurella mossii Savile (1975), was described on Aralia nudicaulis L., from Canada and U.S.A. (Type: Canada, Edmonton, 16 June 1931, E. H. Moss 2187; DAOM, BPI). It differs from M. heptapleuri in having slightly smaller, mostly 2- to $-celled spores rather than 1- to 2-celled