S. pombe genome deletion project: an update

Abstract

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.

Figure 1

Maps of cloning vectors pCloneKan1 and pCloneBle1. Restriction sites used for cloning of the homology regions are indicated. Nucleotide sequence of pCloneKan1 (GQ354684) and pCloneBle1 (GQ354685) can be found in GenBank.

Figure 2

pCloneBle1 confers resistance to phleomycin and zeocin. To test the usefulness of the pCloneBle1, we used this plasmid to delete hhp1 gene. We first constructed hhp1 deletion plasmid (pCloneBle1-hhp1) according to protocol described in Gregan et al. After linearization of the pCloneBle1-hhp1 plasmid with the restriction enzyme XbaI, we transformed it into a wild-type S. pombe strain and selected for phleomycin-resistant colonies. Colony-pCr showed that we successfully deleted hhp1 in 7 out of 10 tested colonies. Transformants were resistant to both phleomycin and zeocin, which is a commercially produced antibiotic containing phleomycin. A 10-fold dilution series of wild-type (wt) (JG11318) and four transformants where hhp1 has been deleted with pCloneBle1-hhp1 construct (JG15352) were spotted onto YeS plates containing the indicated concentrations [mg/ml] of either phleomycin or zeocin. Plates were incubated at 32°C for 3 days.