S. pombegenome deletion project: An update

Abstract

IntroductionAlthough forward genetic screens have provided many important insights into various aspects of cell biology, they are limited by the large numbers of mutants that must be analyzed and inherent biases in mutagenesis techniques. Systematic reverse genetic screens using genome-wide gene deletion collections or RNAi libraries provide a powerful alternative to forward genetic screens (reviewed in refs. 1-5). Studies with the budding yeast Saccharomyces cerevisiae have shown the usefulness of such gene deletion collection for numerous studies including drug discovery (reviewed in refs. 6-9). The fission yeast S. pombe is an important model organism sharing many features with cells of higher eukaryotes. The availability of the S. pombe genome sequence 10 and PCR-based gene deletion technology 11 allowed researchers involved in the S. pombe genome deletion project (KRIBB-Bioneer-CRUK consortium) to generate a set of 4.836 heterozygous diploid deletion mutants covering 98.4% of the fission yeast open reading frames (Kim et al., in press). However, for some genes a deletion could either not be constructed or the analysis of mutant phenotypes gave ambiguous results. Here we use an efficient knock-out strategy to delete 29 of these genes. ResultsWe speculated that some of the genes which could not be deleted by the KRIBB-Bioneer-CRUK consortium may require longer regions of homology for efficient gene targeting 12,13 and may be the fission yeast Schizosaccharomyces pombe is a model organism used widely to study various aspects of eukaryotic biology. A collection of heterozygous diploid strains containing individual deletions in nearly all S. pombe genes has been created using a pCr based strategy. However, deletion of some genes has not been possible using this methodology.Here we use an efficient knockout strategy based on plasmids that contain large regions homologous to the target gene to delete an additional 29 genes. the collection of deletion mutants now covers 99% of the fission yeast open reading frames.

Splicing Functions and Global Dependency on Fission Yeast Slu7 Reveal Diversity in Spliceosome Assembly

“…Slu7 is essential in both budding and fission yeast (14,39; this study), while human cell lines knocked down for Slu7 are viable with likely physiological context-dependent splicing (20,51). In vitro splicing of model minitranscripts in budding yeast or human cell extracts showed the second step functions of Slu7, especially in the choice of a distal 3=ss (8,14,18,19).…”

Generation of a set of conditional analog-sensitive alleles of essential protein kinases in the fission yeastSchizosaccharomyces pombe

“…viability. 7,8 We created heterozygous diploid deletion strains for each of these 18 kinases and analyzed the haploid products after sporulation. We confirmed that 17 kinase genes are essential for viability.…”

PomBase: The Scientific Resource for Fission Yeast

“…This facilitates the comparison of results across different laboratories. Aside from the PomBase database, several organism specific resources are available to fission yeast researchers, including the genome-wide deletion mutant collection [3,4] and the Orfeome localization collection [5].…”