Replication of pRS64 Plasmids

We propose a possible model for the replication of pRS64, in which hairpins and terminal homologous regions have key roles. This model is modified from the model of vaccinia virus DNA replication (Baroudy et al. 1983). Replication of pRS64 DNAs might be initiated by site-specific nicking proximal to the loop structure and within the terminal homologous region. This results in the formation of a free 3'-hydroxyl end that can serve as a primer for DNA replication. If DNA replication proceeds from both ends simultaneously, mature molecules can be formed directly (Fig. 4, right). If DNA synthesis is initiated from a single end, replication will proceed around the opposite hairpin, leading to the formation of dimers (Fig. 4, left and center); one is a head-to-head dimer and the other is a tail-to-tail dimer (Miyashita et al. 1990). The finding of dimeric forms of pRS64-1 suggests that the replication of this plasmid is initiated from a single end.

The structures of the two dimer types shown in Fig. 4 are consistent with the model (Fig. 4, left and center); however, some molecules might replicate directly to monomers. In contrast, dimeric forms of pRS64-2 and -3 were not detected. Replication of these plasmid DNAs might be initiated from both ends. Another possibility is that these dimers are resolved into monomers too quickly to be detected (Miyashita et al. 1990). A terminal inverted repeat was found in vaccinia virus, and is thought to function as a recognition

Fig. 4 Possible models for replication of pRS64 DNA plasmids. Complementary sequences are indicated with capital and lowercase letters. The terminal hairpins are drawn as loops and designated ABC and FGH. The terminal homologous regions are designated DE and IJ

sequence for a single species of nicking enzyme. In pRS64, two different nicking enzymes might be involved in replication, although we cannot rule out the possibility of a short inverted repeat sufficient for recognition by a single species of nicking enzyme.

Replication of pFOXC2 and pFOXC3 Plasmids

A hypothetical model for the replication of pFOXC2 and pFOXC3 plasmids was constructed (Walther and Kennell 1999; Simpson et al. 2004; Galligan and Kennell 2007, this volume).

Transcription

Unique poly(A)-RNA, 0.5 kb in length and hybridizable with the pRS64 DNAs, was found in mycelial cells of the isolates RI-64 (containing all three plasmids), R101 (containing plasmid pRS64-1), and GM-11 (containing pRS64-2) (Chen et al. 1992). Comparison of the nucleotide sequence of the cDNA derived from poly(A)-RNA with those of pRS64-1, -2, and -3 showed 100, 73, and 84% homology, respectively. This suggests that the poly(A)-RNA hybridizing to pRS64 represents the transcription products of pRS64-1, -2, and -3, and that the genetic information in pRS64 DNAs is expressed (Hongo et al. 1994).

Unique poly(A)-RNA, 4.7 and 7.4 kb in length and hybridizing with the pRS224 DNA, are also found in the plasmid-containing R. solani H-16 isolate (Katsura et al. 2001). Transcript product-mapping allowed for the prediction of the locations of different expression signals. Of the two transcripts, one was approximately 2.7 kb longer than the 4.7-kb transcript. Transcripts longer than plasmids have also been detected from R. solani AG5 and AG6 (Kura-date et al. 1996). The striking finding in this study is that the 7.4-kb transcript is RNA containing the full-length transcript, which is generated from the left terminal region of the complementary strand via a full-length sense strand to the right terminal region of the complementary strand. In contrast, the 4.7-kb transcript is predicted to be transcribed from the center of the sense strand to the right terminal region of the complementary strand (Fig. 5).

887 aa

887 aa

Fig. 5 Mapping of pRS224-1 transcripts. The 7.4- and 4.7-kb RNAs are indicated with arrows. The black box indicates the ORF. Letters represent restriction sites
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