Yeast autonomous linear plasmid pGKL2: ORF9 is an actively transcribed essential gene with multiple transcription start points

Abstract

A pair of linear plasmids, pGKL1 (8.9 kb) and pGKL2 (13.4 kb), resides in the cytoplasm of Kluyveromyces lactis killer strains. The smaller element, actually conferring the killer phenotype, strictly depends on the larger autonomous pGKL2. Here, we have examined the previously uncharacterized pGKL2 open reading frame (ORF)9 (1.34 kb). Northern analysis of a killer plasmid carrying Saccharomyces cerevisiae strain applying an ORF9-specific probe revealed a single transcript closely matching the size of the ORF9 coding region. Multiple transcriptional start points, determined by primer extension analysis, are located 16 nt downstream of a conserved sequence element regarded as the cytoplasmic promoter. In vivo disruption of pGKL2/ORF9 using the cytoplasmically expressible marker-gene LEU2* resulted in the establishment of a three-plasmid system composed of the native cytoplasmic elements pGKL1/2 and a hybrid of the latter, which only remained stable under selective conditions. The native pGKL2, however, did not segregate during prolonged subcultivations, proving an essential function of ORF9 for plasmid maintenance.

The primary target of the killer toxin from Pichia acaciae is tRNAGln

“…tRNA was isolated as described previously (Klassen et al ., 2008). Quantification and quality control of RNA was performed as described (Jeske et al ., 2006) by spectrophotometry using nano‐drop (NanoDrop Technologies, Wilmington, USA). tRNA samples were analysed on denaturing polyacrylamide gels (12%) containing 7.5 M urea and run in TBE (Sambrook and Russell, 2001).…”

Pichia acaciae Killer System: Genetic Analysis of Toxin Immunity

“…While target cell killing is the common purpose, the structures of toxins, their mechanisms of action, and the organizations of encoding genes are rather diverse (25,32). The Kluyveromyces lactis and Pichia acaciae toxin systems depend on double-stranded DNA elements (8,16,40). The two species each harbor a pair of extranuclear linear plasmids, i.e., pGKL1 (8.9 kb) and pGKL2 (13.5 kb) (K. lactis) and pPac1-1 (13.6 kb) and pPac1-2 (6.8 kb) (P. acaciae) (1,8,37).…”

Yeast Killer Toxins: Fundamentals and Applications

“…6.4 see below). Group I comprises the K. lactis and P. inositovora killer plasmids while group II harbours the P. acaciae and D. robertsiae killers (Schaffrath and Meinhardt 2004;Jeske et al 2006b; Klassen and Meinhardt 2007).…”