The ORFs found in pRS64-1, -2, and -3 (ORF1-1, ORF2-1, and ORF3-1) encode predicted peptides that are 68 amino acids long. The deduced amino acid sequences had no significant homology with known proteins. Extracts from E. coli cells expressing ORF1-1 gave a specific 7-kDa protein, and anti-ORF1-1-encoded protein (RS64) antibodies raised against the synthetic fusion peptide cross-reacted with the specific 7-kDa protein from the mycelia

(Hongo et al. 1994). The other ORFs were not considered to be genes because the transcripts corresponding to these ORFs were not detected.

The complete nucleotide sequence of the hairpin-loop linear DNA plasmid (pRS224-1) from the plant pathogenic fungus R. solani AG2-2 isolate H-16 was determined, and its unique RNA transcripts were characterized. One ORF (RS224ORF) found in pRS224-1 encoded a peptide predicted to consist of 887 amino acids with an approximate molecular mass of 102 kDa (Katsura et al. 2001).

To determine whether the ORF of pRS224-1 is translated, we constructed pET11a-RS224orf carrying both the T7.Tag gene and the RS224ORF. Transformed E. coli cells were induced with IPTG, and the total extract was fractionated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fusion protein (T7.Tag-RS224orf, 92 kDa) was obtained in cells containing the pET11a-RS224orf. We detected a single protein with a molecular mass of approximately 92 kDa, a size slightly smaller than the 102 kDa calculated from the amino acid sequence coded by the ORF (Nagasaka et al. 2003).

Immunoblotting experiment results demonstrated that the antiserum raised against the RS224ORF recognized a specific polypeptide in the total protein extracts of mycelial cells. The putative polypeptide encoded by the ORF had no significant homology with polypeptides listed in various databases. The 92-kDa protein was associated with the mitochondrial fraction, as was the pRS224-1 DNA of R. solani which is also located in the mitochondria (Nagasaka et al. 2003). DNA plasmid pRS64 of R. solani AG4 is also associated with mitochondria (Chen et al. 1992), but the 7-kDa protein was detected in the cell wall fraction. A computer search using the DNA-SIS program revealed no significant homology between RS64 and RS224. In addition, RS64 and RS224 were detected in the cell wall and mitochon-drial fractions, respectively. Thus, RS224 are clearly different proteins with different functions.

The pRS224-1 ORF contains seven conserved domains characteristic of a reverse transcriptase (RT) (Fig. 6) (Katsura et al. 2001), including the highly-conserved YXDD (Try-xxx-asp-asp) sequence that is proposed to be part of the active site of RT (Varmus and Brown 1989; Xiong and Eickbush 1990). Pair-wise comparison of identical amino acids within the seven consensus sequences that were common to the RT sequence and included the highly conserved YXDD sequence had 57.8% identity to the Mauriceville retroplasmid of Neurospora crassa and 54.3% identity to the pFOXC2 of F. oxysporum (Fig. 6). We identified two regions between domains I and III, and IV and VII, of the amino acid domains with more than 25.6% amino acid sequence homology (Fig. 6a, boxes with a solid line) and an intervening region with less than 12.1% amino acid sequence homology. RT activity was assayed by the incorporation of [a-32P]dTTP into trichloroacetic acid-precipitable material (Farmerie et al. 1987); however, putative RT activity was not detected in mitochondrial lysates from R. solani isolate H-16.


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