Cloning and characterization of KNR4, a yeast gene involved in (1,3)-beta-glucan synthesis.
k9 killer toxin from Hansenula mrakii was used to select a number of resistant mutants from Saccharomyces cerevisiae. Preliminary biochemical and genetic studies showed that some of them acquired structural defects in the cell wall. One of these mutants, the knr4-1 mutant, displays a number of cell wall defects, including osmotic sensitivity; sensitivity to cercosporamide, a known antifungal agent; and resistance to Zymolyase, a (1,3)-beta-glucanase. We report here the isolation and analysis of the KNR4 gene. DNA sequence analysis revealed an uninterrupted open reading frame which contains five potential start codons. The longest coding template encodes a protein of 505 amino acids with a calculated molecular mass of 57,044 Da. A data base search revealed 100% identity with a nuclear protein, SMI1p. Disruption of the KNR4 locus does not result in cell death; however, it leads to reduced levels of both (1,3)-beta-glucan synthase activity and (1,3)-beta-glucan content in the cell wall. The gene was mapped to the right arm of chromosome VII.
Hong
Z
Z
Chemotherapy and Molecular Genetics, Schering-Plough Research Institute, Kenilworth, New Jersey 07033-0539.
Mann
P
P
Brown
N H
NH
Tran
L E
LE
Shaw
K J
KJ
Hare
R S
RS
DiDomenico
B
B
D000595:Amino Acid Sequence; D001483:Base Sequence; D001572:Benzofurans / Q000633:toxicity; D015153:Blotting, Western; D015825:Chromosomes, Fungal; D003001:Cloning, Molecular / Q000379:methods; D004271:DNA, Fungal / Q000302:isolation & purification / Q000378:metabolism; D004352:Drug Resistance, Microbial / Q000235:genetics*; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D005800:Genes, Fungal; D005936:Glucans / Q000096:biosynthesis*; D005964:Glucosyltransferases / Q000096:biosynthesis* / Q000378:metabolism; D006867:Hydrolases / Q000633:toxicity; D055646:Killer Factors, Yeast; D007700:Kinetics; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D010843:Pichia / Q000378:metabolism; D014176:Protein Biosynthesis; D011506:Proteins / Q000633:toxicity; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors; D047071:beta-Glucans
D001572:Benzofurans; D004271:DNA, Fungal; D005656:Fungal Proteins; D005936:Glucans; D055646:Killer Factors, Yeast; D008565:Membrane Proteins; D011506:Proteins; D011994:Recombinant Proteins; C085451:SMI1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D014157:Transcription Factors; D047071:beta-Glucans; C085452:cercosporamide; C037833:zymolyase; C033363:beta-1,3-glucan; D005964:Glucosyltransferases; C020570:1,3-beta-glucan synthase; D006867:Hydrolases