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Saccharomyces cerevisiae has been widely used as a model organism in studies of replicative ageing and senescence. The relevance of these studies to ageing in other organisms has, however, been questioned, since this yeast divides by budding rather than fission, the more common pattern in higher organisms. Here we report that, contrary to popular belief, the fission yeast Schizosaccharomyces pombe also undergoes replicative senescence and in a manner superficially analogous to budding yeast. These experiments provide the first evidence of age asymmetry in cell fission and are consistent with the hypothesis of Jazwinski, that asymmetric division underlies culture immortality. Given their evolutionary divergence, comparison of the ageing determinants in fission and budding yeasts may help identify common mechanisms of the ageing process., (Copyright 1999 John Wiley & Sons, Ltd.)

Deletion via PCR-mediated gene replacement, together with basic functional and bioinformatic analyses, have been performed on six novel open reading-frames (ORFs) on the left arm of chromosome XII of Saccharomyces cerevisiae(YLL033w, YLL032c, YLL031c, YLL030c, YLL029w and YLL028w). ORF deletion was realized using either a short-flanking homology (SFH) or a long-flanking homology (LFH) replacement cassette in the diploid strain FY1679. Sporulation and tetrad analysis showed that YLL031c is the only essential gene of the six. Microscopic examination of the non-growing spores carrying a disrupted copy of the essential gene showed that most of them were blocked after one or two cell divisions with heterogeneous bud size. The standard EUROFAN growth tests failed to reveal any obvious phenotype resulting from the deletion of each the five non-essential ORFs. Bioinformatic analysis revealed that YLL029w is probably an aminopeptidase for mitochondrial or nuclear protein processing and YLL028w may be involved in drug resistance in S. cerevisiae. Replacement cassettes, comprising the promoter and terminator regions of each of the six ORFs, were cloned into pUG7 and demonstrated to efficiently mediate gene replacement in an alternative diploid strain, W303. All the cognate gene clones were constructed, using either PCR products amplified from genomic DNA, or gap-repair. All clones and strains generated have been deposited in the EUROFAN genetic stock centre (EUROSCARF, Frankfurt)., (Copyright 1999 John Wiley & Sons, Ltd.)

The green fluorescent protein (GFP) of Aequorea victoria is now an established marker for gene expression and subcellular localization in budding yeast. Relatively high expression (greater than 2500 copies per cell) of GFP is required for direct microscopic visualization. This report provides a method for studying the expression of less highly expressed genes by the analysis of crude cell extracts--a simple and cheap alternative to the fluorescent activated cell sorter (FACS). The utility of this marker is demonstrated in a study of the expression of the RAD54 gene. It is shown that the induction of the RAD54 promoter leads to the accumulation of Rad54p and of GFP and that the fluorescence induction is correctly regulated. This method should allow the screening of large numbers of novel gene disrupters for their effects on RAD54 expression and so identify trans-acting factors involved in the cellular response to DNA damage.