CLONING AND EXPRESSION OF A CDNA COPY OF THE VIRAL K-28 KILLER TOXIN GENE IN YEAST

The killer toxin K-28, secreted by certain killer strains of the yeast Saccharomyces cerevisiae is genetically encoded by a 1.9 kb double-st randed RNA, M-dsRNA (M(28)), that is present within the cell as a cyto plasmically inherited virus-like particle (VLP). For stable maintenanc e and replication, M(28)-VLPs depend on a second dsRNA virus (L(A)), w hich has been shown to encode the major capsid protein (cap) and a cap sid-polymerase fusion protein (cap-pol) that provides the toxin-coding M-satellites with their transcription and replicase functions. K-28 t oxin-coding M(28)-VLPs were isolated, purified and used in vitro for t he synthesis of the single-stranded M(28) transcript, which was shown to be of plus strand polarity and to bind to oligo(dT)-cellulose, indi cating that M(28)(+)ssRNA contains an internal A-rich tract. Strand se paration of the 1.9 kb M(28)-dsRNA and direct RNA sequencing of its 3' ends was performed in order to obtain specific DNA oligonucleotides t hat could be used as primers for CDNA synthesis. The nucleotide sequen ce of the toxin-coding M(28)-cDNA identified a single open reading fra me (ORF) coding for a polypeptide of 345 amino acids, which contained two potential Kex2p/Kex1p processing sites and three potential sites f or protein N-glycosylation. The toxin-coding cDNA was cloned and expre ssed in sensitive non-killer strains under the control of the yeast PG K promoter. Upon transformation, this construct conferred the complete K-28 phenotype, demonstrating that both toxin and immunity determinan ts are contained within the cloned cDNA. In vitro translational analys is of the M(28)(+)ssRNA in vitro transcript identified the primary gen e product of M(28) as a K-28 preprotoxin of 38 kDa (M-p38).