1.8.7
THE COMPLETE NUCLEOTIDE SEQUENCE AND MAPPING OF TRANSCRIPTION OF THE LINEAR DNA PLASMID pRS224 FROM RHIZOCTONIA SOLANI

K KATSURA, M FUJI, K KURADATE, A SASAKI and T HASHIBA

Laboratory of Plant Pathology, Faculty of Agriculture, Tohoku University, Tsutsumidori-Amamiyamachi 1-1, Aoba-ku, Sendai 981, Japan

Background and objectives
Linear DNA plasmids from Rhizoctonia solani have been identified in isolates from nine anastomosis groups (AGs). Considerable sequence homology was observed among DNA plasmids obtained from isolates within the same AG [1]. The DNA plasmids pRS64 of R. solani AG4 have hairpin loops at both termini. We have already determined the complete nucleotide sequence of the pRS64 which contained both hairpin loop termini [2]. The plasmid pRS224s were in isolate H-16 belonging to AG2-2 of R. solani, and this plasmid size was about 4.8 kb. The complete nucleotide sequence of the DNA plasmid pRS224 was determined, and transcripts were analysed to discover more about their possible function.

Materials and methods
Fungal isolate: R. solani isolate H-16 belonging to AG2-2 was used in this study.
PCR amplification of the termini and nucleotide sequence: both termini were determined using the partly modified procedure of Katsura et al. [2].
(+/-)RNA probe: the (+)RNA probe was prepared from the sense chain of the cloned fragments by T7 RNA polymerase, and the (-)RNA probe was prepared from the complementary chain by the same method.

Results and conclusions
The complete nucleotide sequence of the linear DNA plasmids (pRS224) from the plant pathogenic fungus R. solani was determined. The pRS224 DNA consisted of 4986 nucleotides. A computer-based study of pRS224 DNA-folding at both termini predicted hairpin loop structures. The hairpin loops consisted of the left and right termini of 236 and 264 nucleotides, respectively, and shared no sequence homology. The hairpin loops form cruciform base-paired structures. Unique poly(A)- RNAs, 4.7 and 7.4 kb in length and hybridizable with the pRS224 DNAs, were found in mycelial cells of the isolate H-16. Transcript product mapping allowed prediction of the location of different expression signals. The primary transcripts of 7.4 and 4.7 kb are generated from the complementary chain (- strand) and sense chain (+ strand), respectively. It is not clear to which position primary transcripts are synthesized. One open reading frame (ORF) found in pRS224 is 888 amino acids long. The ORF has potential coding capacities of 102 kDa. The amino acid sequence showed no significant homology with known proteins. Further studies are necessary to determine the function of the products encoded by the linear DNA plasmid (pRS224) of R. solani.

References
1. Miyasaka A, Chen CL, Hashiba T, 1990. Journal of General Microbiology 136, 1791-1798.
2. Katsura K, Suzuki F, Miyashita S et al., 1997. Current Genetics 32, 431-435.