Characterization of a Saccharomyces cerevisiae thermosensitive lytic mutant leads to the identification of a new allele of the NUD1 gene.
To improve our understanding of the factors involved in the osmotic stability of yeast cells, a search for novel conditional Saccharomyces cerevisiae cell lysis mutants was performed. Ten temperature-sensitive (ts) mutant strains of S. cerevisiae were isolated that lyse at the restrictive temperature on hypotonic, but not on osmotically supported medium. The ten mutants fell into four complementation groups: ts1 to ts4. To clone the wild-type gene corresponding to the ts4 mutation, a strategy aimed at complementing the thermosensitive phenotype-using low-copy and high-copy DNA libraries--was followed, but only two extragenic suppressors were identified. Another approach, in which classic genetic methods were combined with the use of yeast artificial chromosomes and traditional cloning procedures, allowed the identification of the NUD1 gene--which codes for a component of the spindle-pole body-as the wild-type gene corresponding to the ts4 mutation. Cloning and sequencing of the defective allele from the chromosome of the mutant cells resulted in the identification of a point mutation that produces a single amino acid change in the protein: a Gly-to-Glu change at position 585 (the nud1-G585E allele). Further analysis revealed that cells carrying this allele show a thermosensitive growth defect. At the restrictive temperature, the cells arrest with large buds, elongated spindles, and duplicated nuclei. In addition, with longer incubation times they are unable to maintain cellular integrity and lyse. Our results have allowed the identification of the first single amino acid mutation in NUD1, and suggest a link between cell cycle progression and cellular integrity.
Departamento de Microbiología y Genética, Instituto de Microbiología-Bioquímica, Universidad de Salamanca/CSIC, Campus Miguel de Unamuno, 37007 Salamanca, Spain.
Irina
I
Alexandar
Pedro
P
San Segundo
Pencho
P
Venkov
Francisco
F
del Rey
Carlos R
CR
Vázquez de Aldana
D000483:Alleles; D019943:Amino Acid Substitution / Q000235:genetics; D002453:Cell Cycle / Q000235:genetics* / Q000502:physiology; D003001:Cloning, Molecular; D003851:Deoxyribonucleases / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal / Q000235:genetics*; D015723:Gene Library; D009996:Osmotic Fragility / Q000235:genetics / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D012359:tRNA Methyltransferases
D029701:Saccharomyces cerevisiae Proteins; D012359:tRNA Methyltransferases; D003851:Deoxyribonucleases; C077501:TRM2 protein, S cerevisiae