Mutations sensitizing yeast cells to the start inhibitor nalidixic acid

Abstract

The regulatory step Start in the cell cycle of the budding yeast Saccharomyces cerevisiae is inhibited by nalidixic acid (Nal). To study this inhibition, mutations were identified that alter the sensitivity of yeast cells to Nal. Nal-sensitive mutations were sought because the inhibitory effects of Nal on wild-type cells are only transient, and wild-type cells naturally become refractory to Nal. Three complementation groups of Nal-sensitive mutations were found. Mutations in the first complementation group were shown to reside in the ARO7 gene, encoding chorismate mutase; tyrosine and phenylalanine synthesis was inhibited by Nal in these aro7 mutants, whereas wild-type chorismate mutase was unaffected, These aro7 alleles demonstrate 'recruitment', by mutation, of an innately indifferent protein to an inhibitor-sensitive form. The Nal-sensitive aro7 mutant cells were used to show that the resumption of Nal-inhibited nuclear activity and cell proliferation takes place while cytoplasmic Nal persists at concentrations inhibitory for the mutant chorismate mutase. Mutations in the second complementation group, nss2 (Nal-supersensitive), increased intracellular Nal concentrations, and may simply alter the permeability of cells to Nal. The third complementation group was found to be the ERG6 gene, previously suggested to encode the ergosterol biosynthetic enzyme sterol methyltransferase. Mutation or deletion of the ERG6 gene had little effect on the inhibition of Start by Nal, but prevented recovery from this inhibition. Mutation of ERG3, encoding another ergosterol biosynthetic enzyme, also caused Nal sensitivity, suggesting that plasma membrane sterol composition, and plasma membrane function, mediates recovery from Nal-mediated inhibition of Start.

Reduced Susceptibility to Polyenes Associated with a Missense Mutation in the ERG6 Gene in a Clinical Isolate of Candida glabrata with Pseudohyphal Growth

“…In addition, erg6 mutants grow as short chains of elongated cells and present a resistance to nystatin associated with a hypersensitivity to cycloheximide, anthracyclins, and brefeldin A (11,12,14). Likewise, inactivation of the ERG3 gene in S. cerevisiae results in changes in the susceptibility to ketoconazole (40), syringomycin (34), and nalidixic acid (27), and ERG3 null mutants are unable to grow on nonfermentable carbon sources and are cold sensitive (1,13,32). Since ergosterol is the polyene target, a qualitative and quantitative analysis of sterols was performed.…”

The Dna Replication Fork in Eukaryotic Cells

“…These proteins seem to compete with Ctf4p for binding to pol α. Biochemical and genetic interactions between pol α and these pol α-binding proteins [Ctf4p, Cdc68p (248)(249)(250) and Pob3p] have also been demonstrated (244). Furthermore, the cdc68-1 mutation was also synthetic lethal with the dna2-2 mutation (244).…”

“…It has not been determined whether initiation with the purified pol α/primase, RPA, and T antigen occurs at preferred sites for primer synthesis. A number of cellular proteins that associate or cooperate with pol α/primase, such as AAF (266,267), Ctf4 (Pob1) (245)(246)(247), Cdc68 (248)(249)(250), and Pob3 (244), might modulate the interaction of the primase with the DNA and affect primer-site selection. In addition, transcription factor activator domains can bind to RPA and stimulate replication in vitro (268,269), but the precise mechanism of this activation is unclear.…”

A C-methyltransferase involved in both ubiquinone and menaquinone biosynthesis: isolation and identification of the Escherichia coli ubiE gene

“…A search of the reported ORFs in this segment of the E. coli chromosome identified o251, which encoded the three methyltransferase motifs identified by Kagan and Clarke (17). Such motifs are present in a large family of methyltransferases that use AdoMet as the methyl donor and have also been identified in the eukaryotic Coq3 O-methyltransferase in Q biosynthesis (5,19), in the E. coli UbiG O-methyltransferase (42), and in the sequence encoded by ERG6, the presumed structural gene for AdoMet⌬ 24-sterol-C-methyltransferase in ergosterol synthesis (14,30). None of the other reported ORFs in this region were found to encode methyltransferase motifs.…”