Abstract
The nmt1+ gene of the fission yeast Schizosaccharomyces pombe is subject to transcriptional repression mediated by thiamine. The promoter of nmt1+ has been used to construct a series of vectors that are now commonly used for the regulated expression of genes in S. pombe. In this report, we described ntf1+, a gene involved in regulating nmt1+ expression. The ntf1+ gene was cloned in a high copy suppressor screen that utilized a construct, nmt1:wee1+, in which expression of the mitotic inhibitor Wee1 tyrosine kinase was placed under the control of the nmt1 promoter. ntf1+ encodes a 706-amino acid protein that shares substantial homology with a family of Cys6 zinc finger-containing transcription factors typified by GAL4 from Saccharomyces cerevisiae. Increased gene dosage of ntf1+ greatly increases both the repressed and derepressed activity of the nmt1 promoter. Cells having a disrupted version of ntf1+ are viable thiamine prototrophs, but basal expression from the nmt1 promoter is greatly reduced. These data demonstrate that Ntf1 plays an important role in regulating nmt1 expression.
Highlights
In this reportw, e describentfl+,a gene involved in reguki-nase, which phosphorylates Cdc2 at Tyr-15 [10,11,12]
Weel tyrosine kinase was placed under the control of the nmtl promoter. ntfl' encodes a 706-amino acid protein that shares substantial homology with a family of Cys, zinc finger-containing transcription factors typified by GAL4 from Saccharomycee cereviuiae
In the presence of 1nm thiamine, which represses transcription from the nmtl promoter [16].A genetic screen was initiated to isolate multicopy suppressors of this mitotic catastrophe phenotype.Haploid cdc2-4wweel-50 nmt1:weel' cells were transformed by the lithium acetate method withan S.pombe genomiclibrary made in pDW232 [20]
Summary
E describentfl+,a gene involved in reguki-nase, which phosphorylates Cdc2 at Tyr-15 [10,11,12]. In the presence of 1nm thiamine, which represses transcription from the nmtl promoter [16].A genetic screen was initiated to isolate multicopy suppressors of this mitotic catastrophe phenotype.Haploid cdc2-4wweel-50 nmt1:weel' cells were transformed by the lithium acetate method withan S.pombe genomiclibrary made in pDW232 [20]. In a cdc2' strain containing an integrated copy of pMNSwee1, induction of the nmtl:weel+ construct by growth in medium lacking thiamine leads to cell cycle arrest inG, phase.
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