*The Agricultural High-Tech Research Center,
Laboratory of Entomology,
Laboratory of Biological Chemistry, Meijo University, Tempaku-ku, Nagoya 468-8502, Japan
Group-I introns have been identified in the organellar and nuclear genomes of higher plants, fungi, amoebae, green algae, and archaea, and in the genomes of viruses and phages. This wide distribution and phylogenetically sporadic existence of group-I introns has led to the hypothesis of horizontal transfer. In the pyrenomycete fungi, Nikoh and Fukatsu (2001)
supposed that the common ancestor of Cordyceps prolifica and C. kanzashiana horizontally gained group-I introns of ribosomal DNA (rDNA) from distantly related fungi. An interkingdom transfer of group-I introns of rDNA between a plant, Youngia japonica, and a phytopathogenic fungus, Protomyces inouei, was also proposed by Nishida and Sugiyama (1995)
. Although the intron mobility of nuclear rDNA is widely accepted, few intron-encoded endonucleases are known. Because of the lack of the endonuclease gene, except for the cases of Nectria galligena (Johansen and Haugen 1999
) and the ericoid fungal isolates (Perotto et al. 2000
), most of the group-I introns in the fungal rDNA are principally thought to be immobile and vertically transmitted (Nikoh and Fukatsu 2001
). The present study deals with the group-I intron in the small subunit (SSU) rDNA of Beauveria bassiana IFO 31676, which has a putative endonuclease gene.
During the course of the phylogenetic analysis of the clavicipitalean fungi, we determined the nucleotide sequences of SSU rDNA of B. bassiana and related fungi. A polymorphism with a nucleotide length similar to that of SSU rDNA was observed in the B. bassiana species. This polymorphism was due to the inserted group-I introns. Group-I introns are abundant in the rDNAs of the clavicipitalean fungi, their specific insertion positions are characterized at SSU516, SSU943, SSU989, SSU1199, LSU1921, LSU2066, LSU2449, and LSU2563, relative to Escherichia coli (Nikoh and Fukatsu 2001
). Beauveria bassiana IFO 4848 (Nikoh and Fukatsu 2000
) and IFO 31953 have no group-I introns in their SSU rDNAs. Beauveria bassiana IFO 31676 has a large group-I intron (1,866 bases) at the position SSU943 (fig. 1
). Beauveria bassiana IFO 8554 and BCMU BB01 have two short group-I introns (about 380 bases) at positions SSU943 and SSU1199. All the SSU943 group-I introns have the conserved sequence elements P, Q, R, and S of the Cordyceps fungi (Cech 1988
; Nikoh and Fukatsu 2001
) (fig. 1
). The introns at the same inserted position are closely related (Nikoh and Fukatsu 2001
). The SSU943 introns of B. bassiana IFO 8554 and BCMU BB01 are identical. The nucleotide sequence (positions 1300 in fig. 1
) of the SSU943 intron of B. bassiana IFO 31676 has 92.0% identity with the sequences of the SSU943 introns of B. bassiana IFO 8554 and BCMU BB01. In fungi, except for N. galligena (Johansen and Haugen 1999
) and the ericoid fungi (Perotto et al. 2000
), the group-I introns reported so far were shorter by about 500 bases because of a lack of the endonuclease gene. The large SSU943 intron of B. bassiana IFO 31676 contains a putative homing endonuclease gene between the conserved elements R and S (fig. 1
). The predicted amino acid sequence of the B. bassiana endonuclease has a His-Cys box (Johansen et al. 1993
) conserved in the homing endonucleases (fig. 1
). Comparisons of the amino acid sequence of the B. bassiana endonuclease with databases were done using BLAST (Altschul et al. 1990
). High similarities were found at the His-Cys box, with the group-I intronencoded homing endonucleases in rDNAs of fungi N. galligena NI3 and NI109 (Johansen and Haugen 1999
), ericoid fungi isolate PSIV (Perotto et al. 2000
), amoebae Naegleria sp. NG874 (De Jonckheere and Brown 2001
), and Naegleria andersorni A2 (De Jonckheere 1994
), and slime mold Physarum polycephalum Carolina (Muscarella et al. 1990
). All homing endonucleases of the nuclear rDNA introns reported so far have the His-Cys box and belong to the ßß
-Me family (Gimble 2000
), although most of the homing endonucleases belong to the LAGLI-DADG family or to the GIY-YIG family (Gimble 2000
). His-Cys box endonucleases generally have two zinc-binding motifs (CysXnCysX4CysX4His and ArgX2CysX620CysXHisX3Cys) (Elde et al. 2000
). The homing endonuclease of B. bassiana IFO 31676 lacks the last Cys residue in the second zinc-binding motif (fig. 1
). The homing endonuclease of Didymium iridis Pan2 (Johansen and Vogt 1994
) also lacks the second zinc-binding motif and was proved to be functional (Johansen et al. 1997
). The three residues (Arg61, His98, and Asn119) of the homing endonuclease of P. polycephalum are important for DNA cleavage reaction (Mannino et al. 1999
). These three residues correspond to Arg192, His248, and Asn269 of the homing endonuclease of B. bassiana IFO 31676 (fig. 1
). But the possibility that the homing endonuclease of B. bassiana IFO 31676 is not functional cannot be excluded. The endonuclease genes of B. bassiana IFO 31676 and the ericoid isolate PSIV locate on the sense strand of their introns, whereas those of N. galligena NI3 and NI109 locate on the antisense strand. The direction of transcription of the endonuclease gene may not be important.
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Acknowledgements
This work was supported by the Agricultural High-Tech Research Center, Meijo University, under the "Environmental Control through the Function of Microorganisms" project.
Footnotes
Keywords: group-I intron
Beauveria bassiana
homing endonuclease
mobile element
small subunit ribosomal DNA
Address for correspondence and reprints: Akira Hara, Laboratory of Biological Chemistry, Meijo University, Tempaku-ku, Nagoya 468-8502, Japan. hara{at}ccmfs.meijo-u.ac.jp
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