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  • 1
    Electronic Resource
    Electronic Resource
    Expression of a dominant negative allele of cdc2 prevents activation of the endogenous p34cdc2 kinase (1991)
    Springer
    Molecular genetics and genomics 226 (1991), S. 432-440 
    Details
    ISSN: 1617-4623
    Keywords: Schizosaccharomyces pombe ; cdc2 protein kinase ; Dominant negative allele
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The cdc2 gene of the fission yeast Schizosaccharomyces pombe encodes a 34 kDa phosphoprotein with serine/threonine protein kinase activity that acts as the key component in regulation of the eukaryotic cell cycle. We used a repressible promoter fused to the cdc2 cDNA to isolate conditionally dominant negative mutants of cdc2. One of these mutants, DL5, is described in this paper. Overexpression of the mutant protein in a wild-type cdc2 background is lethal and confers cell cycle arrest with a typical cdc phenotype. Sequencing of the mutant cdc2 gene revealed a single amino acid substitution in a region highly conserved in cdc2-like proteins. The mutant protein exhibits no protein kinase activity, but is able to bind a component(s) required for an active protein kinase complex and thereby prevents binding of this component(s) to the co-existing wild-type cdc2 protein. We also demonstrate that S. pombe p34cdc2 contains no phosphoserine.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00260656
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  • 2
    Electronic Resource
    Electronic Resource
    A study of integrative transformation in Schizosaccharomyces pombe (1993)
    Springer
    Molecular genetics and genomics 238 (1993), S. 26-32 
    Details
    ISSN: 1617-4623
    Keywords: Integrative transformation ; Gene disruption ; Schizosaccharomyces pombe
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Using the one-step gene disruption technique, we studied the effect of various parameters on the disruption frequency (percentage of homologous integrants) and transformation efficiency (number of transformants per μg of input DNA) of integrative transformation in Schizosaccharomyces pombe. We used suc1 as the target gene for disruption and ura4 as the selectable marker. Our results are as follows. 1) Use of the strong adh1 promoter to drive the expression of ura4 did not affect the disruption frequency but modestly increased the transformation efficiency. 2) The transformation method had a profound effect, with the lithium acetate method yielding both a 10-fold higher disruption frequency compared to the protoplast method and a 5- to 10-fold higher transformation efficiency. 3) The presence of increasing amounts of non-homologous sequences at the ends of the transforming DNA decreased the disruption frequency by up to 5-fold but had no effect on the transformation efficiency. We also describe the use of the sup3–5 allele in an ade6-704 genetic background to discriminate between the products of homologous versus non-homologous integration, thereby promoting the identification of rare homologous integrants.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00279526
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    Paper (German National Licenses)
    Fulltext
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