Expression in yeast of a cDNA copy of the K2 killer toxin gene.
A cDNA copy of the M2 dsRNA encoding the K2 killer toxin of Saccharomyces cerevisiae was expressed in yeast using the yeast ADH1 promoter. This construct produced K2-specific killing and immunity functions. Efficient K2-specific killing was dependent on the action of the KEX2 endopeptidase and the KEX1 carboxypeptidase, while K2-specific immunity was independent of these proteases. Comparison of the K2 toxin sequence with that of the K1 toxin sequence shows that although they share a common processing pathway and are both encoded by cytoplasmic dsRNAs of similar basic structure, the two toxins are very different at the primary sequence level. Site-specific mutagenesis of the cDNA gene establishes that one of the two potential KEX2 cleavage sites is critical for toxin action but not for immunity. Immunity was reduced by an insertion of two amino acids in the hydrophobic amino-terminal region which left toxin activity intact, indicating an independence of toxin action and immunity.
Dignard
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Genetic Engineering Section, Biotechnology Research Institute, Montreal, Quebec, Canada.
Whiteway
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Germain
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Tessier
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Thomas
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D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D004271:DNA, Fungal / Q000235:genetics* / Q000302:isolation & purification; D005656:Fungal Proteins / Q000235:genetics*; D005800:Genes, Fungal; D055646:Killer Factors, Yeast; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009183:Mycotoxins / Q000235:genetics* / Q000302:isolation & purification; D015345:Oligonucleotide Probes; D011487:Protein Conformation; D012330:RNA, Double-Stranded / Q000235:genetics; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins
D004271:DNA, Fungal; D005656:Fungal Proteins; D055646:Killer Factors, Yeast; D009183:Mycotoxins; D015345:Oligonucleotide Probes; D012330:RNA, Double-Stranded; D029701:Saccharomyces cerevisiae Proteins