Extending the Schizosaccharomyces pombe molecular genetic toolbox.

Targeted alteration of the genome lies at the heart of the exploitation of S. pombe as a model system. The rate of analysis is often determined by the efficiency with which a target locus can be manipulated. For most loci this is not a problem, however for some loci, such as fin1+, rates of gene targeting below 5% can limit the scope and scale of manipulations that are feasible within a reasonable time frame. We now describe a simple modification of transformation procedure for directing integration of genomic sequences that leads to a 5-fold increase in the transformation efficiency when antibiotic based dominant selection markers are used. We also show that removal of the pku70+ and pku80+ genes, which encode DNA end binding proteins required for the non-homologous end joining DNA repair pathway, increases the efficiency of gene targeting at fin1+ to around 75-80% (a 16-fold increase). We describe how a natMX6/rpl42+ cassette can be used for positive and negative selection for integration at a targeted locus. To facilitate the evaluation of the impact of a series of mutations on the function of a gene of interest we have generated three vector series that rely upon different selectable markers to direct the expression of tagged/untagged molecules from distinct genomic integration sites. pINTL and pINTK vectors use ura4+ selection to direct disruptive integration of leu1+ and lys1+ respectively, while pINTH vectors exploit nourseothricin resistance to detect the targeted disruption of a hygromycin B resistance conferring hphMX6 cassette that has been integrated on chromosome III. Finally, we have generated a series of multi-copy expression vectors that use resistance to nourseothricin or kanamycin/G418 to select for propagation in prototrophic hosts. Collectively these protocol modifications and vectors extend the versatility of this key model system.

The Schizosaccharomyces pombe septation initiation network (SIN) is required for spore formation in meiosis.

When nutrients are abundant, S. pombe cells grow as rods, dividing by fission after formation of a medially placed cell wall or division septum. Septum formation is triggered by a group of proteins, called the septation initiation network or SIN, that trigger contraction of the acto-myosin contractile ring at the end of mitosis. Ectopic activation of the SIN can uncouple septum formation from other cell-cycle events, whereas loss of SIN signalling gives rise to multinucleated cells due to the failure of cytokinesis. When starved, S. pombe cells of opposite mating types fuse to form a diploid zygote that undergoes meiosis and produces four spores. No septa or contractile rings are formed during meiosis. In this study, we have investigated the role of the SIN in meiosis. Our data show that, whereas the meiotic divisions appear normal, SIN mutants cannot form spores. Forespore membrane formation is initiated, but the nuclei are not encapsulated properly. The SIN proteins localise to the spindle pole body in meiosis. The protein kinases Sid1p and Cdc7p do not associate with the spindle pole body until meiosis II, when forespore membrane deposition begins. These data indicate a role for the SIN in regulating spore formation during meiosis.