Double Hairpin Linear Retroplasmids

The pRS224-1 plasmid of Rhizoctonia solani isolate H-16 is a 5 kb linear double hairpin retroplasmid (Fig. 1; Katsura et al. 2001; see also Chap. 9, in this volume). Rhizoctonia solani is a soil-born basidiomycete that is capa ble of infecting a broad range of plants. The dual terminal hairpin structure of pRS224-1 was derived from sequence analysis, computer-assisted folding programs, and electron microscopy, which showed that the 5 kb linear plas-mid became a single-stranded circular molecule under denaturing conditions (Katsura et al. 2001). The plasmid is classified as a retroplasmid, based on the presence of a single large ORF that potentially encodes a polypeptide having the seven conserved amino acid motifs found among all RTs. Complete sequence analysis of pRS224-1 indicated that the ORF codes for an 887 amino acid polypeptide with a predicted mass of 102 kDa (Katsura et al. 2001; Na-gasaka et al. 2003). It shows 19% identity (30% similarity) with the pMaur-RT and 16% identity (27% similarity) with the pFOXC3-RT within the highly conserved domain region (Galligan and Kennell, unpublished observations). The coding sequence for this ORF was expressed in E. coli and a purified fusion protein was used to generate a polyclonal antibody. The antibody recognizes a ~ 90 kDa protein that is localized to the mitochondrion (Nagasaka et al. 2003).

The putative pRS224-1 RT is unusual in that it has a region of more than 200 amino acids between highly conserved domains III and IV that fail to match other RTs currently in the database (Katsura et al. 2001). Interestingly, in the cladogram shown in Fig. 2, pRS224 fails to group with either circular or linear retroplasmids, which suggests that it is highly divergent. Reverse transcriptase activity has not been demonstrated for the endogenous plasmid protein and little is known about the replication of pRS224-1. Two major plasmid transcripts have been identified, 4.7 kb and 7.4 kb in length. The smaller transcript begins near the center of the element and includes the RT-ORF and sequence of one of the terminal hairpins. The larger transcript encompasses the entire sense strand and both hairpins, and potentially serves as a template in plasmid replication (Katsura et al. 2001).

Several other linear retroplasmids have been reported based on partial sequence information (Mogen et al. 1991) or hybridization studies (Hirato et al. 1992), but the structures of the plasmid termini have not been determined. Conversely, another double hairpin linear DNA has also been reported in Rhi-zoctonia solani (pRS64-2; Katsura et al. 1997; see also Chap. 9, in this volume), yet it does not appear to encode a polymerase (the largest ORF is 163 amino acids), thus it is difficult to classify at this time.

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