Genome organization of the linear cytoplasmic element pPE1B from Pichia etchellsii

Abstract

The linear cytoplasmic element pPE1B from Pichia etchellsii CBS2011 (synonym Debaryomyces etchellsii) was totally sequenced. It consists of 12835 bp and has a remarkable high A+T content of 77.3%. The termini of pPE1B were found to consist of inversely orientated identical nucleotide repetitions 161 base pairs long, to which proteins are probably covalently linked at the 5k ends. Ten putative genes (open reading frames, ORFs) were identified, covering 96.5% of the total sequence. The predicted polypeptides correspond to proteins encoded by ORFs 2-11 of the linear plasmids pGKL2 of Kluyveromyces lactis and pSKL of Saccharomyces kluyveri. ORF1, existing on both latter elements, is lacking on pPE1B. An upstream conserved sequence motif (UCS) is located at the expected distance from the start codon of each of the 10 ORFs. As the arbitrarily chosen UCS6 was able to drive expression of a reporter gene in the heterologous pGKL-encoded killer system of K. lactis, extranuclear promoter function is probable. The almost congruent genome organization of pPE1B and other autonomous linear yeast plasmids sequenced so far, i.e. pGKL2 and pSKL, suggests a common, presumably viral, ancestor. The sequence of pPE1B has been submitted to the EMBL data library under Accession No. AJ278986.

Kluyveromyces lactis cytoplasmic plasmid pGKL2: heterologous expression of Orf3p and proof of guanylyltransferase and mRNA–triphosphatase activities

“…Thus, at least two enzymatic activities necessary for the capping of transcripts evidently reside in the ORF3 gene product. Predicted ORF3 gene products of all autonomous linear cytoplasmically localized yeast genetic elements that have been sequenced so far (Klassen, 2001) resemble capping enzymes known from cytoplasmic viruses (Poxviridae and Iridoviridae) in a way that putative guanylyltransferase motifs reside in the centre and triphosphatase motifs are located in the leading third of the polypeptide (Shuman, 1995;Larsen et al, 1998).…”

“…As disruption of ORF1 had no influence on replication and maintenance, it appeared to be dispensable (Schaffrath et al, 1992), a finding which was supported by elucidation of the structure of pPE1B from Pichia etchellsii, in which homologues of all pGKL2-based genes but ORF1 were found to exist (Klassen, 2001); ORF2 encodes the element specific DNA polymerase, including the terminal protein (Tommasino et al, 1988;Takeda et al, 1996); the predicted ORF3 polypeptide, which is under investigation here, was found to share similarities with capping enzymes (Larsen et al, 1998); ORF4 may encode a helicase (Tommasino et al, 1988;Stark et al, 1990); ORF5 presumably encodes a ss-DNA binding protein (Schaffrath, personal communication); the predicted ORF6 protein encodes the RNA-polymerase for transcribing pGKL-based genes (Wilson and Meacock, 1988); ORF7 may be a subunit of the latter ; although essential, a function is not known for ORFs 8 and 9 (unpublished data); ORF10 codes for a terminal recognition factor, probably involved in initiation of replication of both elements (McNeel and Tamanoi, 1991); ORF11, a long-unappreciated gene of unknown function, occupies a small gap between ORF3 and ORF4 (Larsen and Meinhardt, 2000). For detailed information on the killer system, we refer to a recent review (Meinhardt and Schaffrath, 2001).…”

“…In contrast to plants and filamentous fungi, almost all of the yeast linear dsDNA elements are cytoplasmically localized; they have been isolated from numerous genera, such as Botryoascus, Debaryomyces, Kluyveromyces, Pichia, Saccharomyces, Trichosporon and Wingea (Cong et al, 1994;Fukuhara, 1995). The genetic organization of yeast linear elements appeared to be quite uniform (Hishinuma and Hirai, 1991;Klassen, 2001) with the killer plasmid pair pGKL1 and pGKL2 of Kluyveromyces lactis (also referred to as k1 and k2) the most thoroughly characterized Gunge, 1995;Schaffrath et al, 1999;Meinhardt and Schaffrath, 2001). K. lactis cells containing pGKL1 and pGKL2 differ from plasmid-less cells by their ability to kill sensitive yeasts belonging either to the same or a different species .…”

Yeast Killer Toxins: Fundamentals and Applications

“…Replication of both autonomous and non-autonomous elements is initiated by protein priming, involving a viral B-type DNA polymerase fused to the terminal protein that remains covalently bound to the 5 0 ends of the plasmids (Hishinuma et al 1984;Stark et al 1984;Sor and Fukuhara 1985;Tommasino et al 1988;Hishinuma and Hirai 1991;Klassen et al 2001;Klassen and Meinhardt 2003;Jeske and Meinhardt 2006). Cytoplasmic transcription of linear plasmid-encoded genes involves a rather unique plasmid-encoded RNA polymerase and a mRNA-capping enzyme which resembles the capping enzyme of the cytoplasmic vaccinia virus (Wilson and Meacock 1988;Larsen et al 1998;Tiggemann et al 2001;.…”

Structural and functional analysis of the killer element pPin1-3 from Pichia inositovora

“…As killer activity is observable in a partially cured strain that lacks pPin1-2 (Hayman and Bolen 1991), and large linear plasmids in such systems routinely confer functions for maintenance of the elements, rather than toxin production (Schaffrath et al 1992;Klassen et al 2001), pPin1-3 was expected to encode the killer trait. In this study, we provide a characterization of the nonautonomous killer element pPin1-3 of P. inositovora and its encoded toxin.…”