6533b7cefe1ef96bd125720a

RESEARCH PRODUCT

Cloning and expression of a cDNA copy of the viral K28 killer toxin gene in yeast

subject

description

The killer toxin K28, secreted by certain killer strains of the yeast Saccharomyces cerevisiae is genetically encoded by a 1.9 kb double-stranded RNA, M-dsRNA (M28), that is present within the cell as a cytoplasmically inherited virus-like particle (VLP). For stable maintenance and replication, M28-VLPs depend on a second dsRNA virus (LA), which has been shown to encode the major capsid protein (cap) and a capsid-polymerase fusion protein (cap-pol) that provides the toxin-coding M-satellites with their transcription and replicase functions. K28 toxin-coding M28-VLPs were isolated, purified and used in vitro for the synthesis of the single-stranded M28 transcript, which was shown to be of plus strand polarity and to bind to oligo(dT)-cellulose, indicating that M28(+)ssRNA contains an internal A-rich tract. Strand separation of the 1.9 kb M28-dsRNA and direct RNA sequencing of its 3' ends was performed in order to obtain specific DNA oligonucleotides that could be used as primers for cDNA synthesis. The nucleotide sequence of the toxin-coding M28-cDNA identified a single open reading frame (ORF) coding for a polypeptide of 345 amino acids, which contained two potential Kex2p/Kex1p processing sites and three potential sites for protein N-glycosylation. The toxin-coding cDNA was cloned and expressed in sensitive non-killer strains under the control of the yeast PGK promoter. Upon transformation, this construct conferred the complete K28 phenotype, demonstrating that both toxin and immunity determinants are contained within the cloned cDNA. In vitro translational analysis of the M28(+)ssRNA in vitro transcript identified the primary gene product of M28 as a K28 preprotoxin of 38 kDa (M-p38).