The terminal protein of a linear mitochondrial plasmid is encoded in the N-terminus of the DNA polymerase gene in white-rot fungus Pleurotus ostreatus

Abstract

The gene structure and expression of the linear mitochondrial plasmids of the white-rot fungus Pleurotus ostreatus, pMLP1 and pMLP2, were analyzed. Cleavage by proteinase K and exonucleases indicated that the 5' ends of pMLP1 and pMLP2 DNAs were associated with terminal proteins. Nucleotide sequencing of the entire pMLP1 DNA revealed that it consists of 9,879 bp with terminal inverted repeat (TIR) sequences of 381 bp. The end sequence of TIR in pMLP1 is 3'-CCCCC-5', similar to those of Escherichia coli phage PRDI. The pMLP1 plasmid harbors two long open reading frames (ORFI and ORF2) and at least one minor ORF (mORF1). The deduced product of ORF1 is homologous to RNA polymerases of yeast mitochondria and several bacteriophages, whereas that of ORF2 is homologous to the protein-primed DNA polymerases of family B type. The mORF1 encodes a highly basic protein, most likely a TIR-binding protein, with no apparent sequence homology in the database. Expression of the predicted gene products from pMLP1 in mitochondria was demonstrated by Western blot analysis using antibodies against various expressed regions of pMLP1 ORFs. A plasmid-free strain, generated by curing with ethidium bromide, did not express any of these gene products. Terminal proteins of 70 kDa (TP1) and 73 kDa (TP2) were identified from pMLP1 and pMLP2, respectively. Western blot analysis indicated that TP1 was generated from the N-terminal half of the full-length product of ORF2 encoding a putative DNA polymerase.

Functional eukaryotic nuclear localization signals are widespread in terminal proteins of bacteriophages

“…The use of TP-mediated, protein-primed DNA replication has been reported or suggested in replicons from all domains of life, such as archaebacterial viruses, bacteriophages of Gram-positive and Gram-negative eubacterial hosts, Streptomyces chromosomes and plasmids, yeast plasmids, mitochondrial plasmids of plants and fungi, transposable elements, adenoviruses, and the recently described Mavirus virophage (6)(7)(8)(9)(10)(11). Besides priming DNA replication, TPs can perform other important roles in vivo.…”

Analysis of the structure and inheritance of a linear plasmid from the obligate biotrophic fungus Blumeria graminis f. sp. hordei

“…Kempken et al (1989) have provided evidence that a linear plasmid in Ascobolus immersus replicates by protein priming. The identification of a terminal protein with TIR binding capacity encoded by the N-terminal half of a putative DNA polymerase gene in a linear plasmid from Pleurotus ostreatus (Kim et al 2000) also supports a protein-primed mode of replication in this case. It has also been reported that ARS consensus sequences are present in two putative origins of replication in the yeast mitochondrial genome (Delouya and Nobrega 1991;Hyman and Garcia-Garcia 1993) and in some linear mitochondrial plasmids from basidiomycetes (Katayose et al 1990;Nakajima et al 1993).…”

“…If these represent transcription termination signals the predicted transcript sizes (4.2 and 5.6 kb) are in excellent agreement with the observed sizes. No significant homologies were found to terminal proteins identified in Escherichia coli phages or the N-terminal region of the DNA polymerase in the P. ostreatus plasmid (Kim et al 2000). Only mitochondrial codon usage resulted in long ORFs.…”

“…pC1K1 from C. purpurea (Gessner-Ulrich and Tudzynski 1992) and kalilo from N. crassa (Vickery and Griffiths 1993). Kim et al (2000) identified a sequence in the N-terminal region of an ORF encoding a DNA polymerase on plasmid pMLP1 of P. ostreatus that shows homology to the terminal replication proteins of bacteriophages. This substantiates the evidence provided by Kempken et al (1989) that linear plasmids replicate by protein priming at the 5¢ termini.…”

Putative DNA-dependent RNA polymerase in Mitochondrial Plasmid of Paramecium caudatum Stock GT704

“…Mitochondrial plasmid pMLPw from Pleurotus ostreatus has 5 0 -end TP. This plasmid has two ORFs, one of them encodes for protein product homolog to RNAP of yeast mitochondria and bacteriophages (Kim et al 2000). Mtplasmids found in fungi and plants have 5 0 -end TP and terminal inverted repeat, and encodes for RNAP (Meinhard et al 1990).…”