Covalently Closed Hairpin Ends Structure

Three linear DNA plasmids were isolated in RI-64 of AG4 of R. solani. These plasmids, designated pRS64-1, -2, and -3, had the same size (2.7 kb). Restriction mapping and Southern hybridization analysis of pRS64-1, -2, and -3 revealed the presence of homologous regions. The DNA plasmids were resistant to both 3'-exonuclease and 5'-exonuclease, even after treatment with proteinase K or alkali. The length of both terminal fragments generated by restriction endonuclease digestion was doubled under denaturation conditions, indicating that the linear DNA plasmids have hairpin loops at both termini (Miyashita et al. 1990; Hongo et al. 1994). A computer-based study of the pRS64-2 DNA folding at both termini predicted hairpin loop structures. The hairpin loops of the pRS64 DNA plasmid consist of the left- and right-hand terminal 113 and 105 nucleotides, and are therefore similar in length to the 104-nucleotide loop of vaccinia virus (Baroudy et al. 1982). Unlike the vaccinia virus DNA, however, they are not flip-flopped. The pRS64 plasmid hairpins form a cruciform base-paired structure (Katsura et al. 1997). They are not inverted and complementary (flip-flopped).

The linear DNA plasmid (pRS224-1) from isolate H-16 of AG2-2 of R. solani consists of 4986 nucleotides. A computer-based study of the folding of pRS224-1 at both termini predicted hairpin-loop structures. The hairpin

Fig. 2 Electron micrograph of DNA molecules from the isolate RI-64 of R. solani by the formamide and cytochrome c technique. The scale bar represents 1 kb
Fig. 3 Structure of plasmid DNA. The plasmid forms a single-stranded circle when completely denatured. Arrows show single-stranded circles that were produced. The scale bar indicates 1000 nucleotides

loops consisted of the left and right termini of 236 and 264 nucleotides, respectively, and share no sequence homology (Katsura et al. 2001).

Jabaji-Hare et al. (1994) also reported extrachromosomal DNA elements in field isolates of R. solani belonging to five AGs (1 to 5), and revealed a 2.4-kb plasmid (pRS104) in AG4 isolates and a 3.6-kb plasmid (pRS188) in AG5 isolates. Both plasmids were analyzed in detail and were determined to be linear with a knob structure at both termini, the same as the plasmids described by Miyashita et al. (1990).

Electron microscopy revealed that the pRS64 and pRS224 plasmid fractions purified from gels consisted of homogeneous populations of linear DNA (Fig. 2) (Miyasaka et al. 1990; Hashiba et al. 2001). The plasmid forms a single-stranded circle when completely denatured (Fig. 3). There were single-stranded DNA circles with a circumference twice the length of the duplex linear DNA (Katsura et al. 2001).

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