Abstract
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.
Highlights
The genetic malleability of the fission yeast S. pombe has helped it to maintain a prominent position alongside the more extensively exploited budding yeast Saccharomyces cerevisiae, as a powerful model system for the characterisation of the basic facets of eukaryotic cell and molecular biology
When used as a marker to select for site specific integration, multiple integration events can occur [10], suggesting either that the heterologous expression of the LEU2+ gene is barely sufficient for growth at low copy number or that the budding yeast enzyme is less attuned to fission yeast physiology than the native 3-isopropyl malate dehydrogenase enzyme, Leu1
We describe a number of the tools that we have developed to assist our efforts that exploit the molecular genetics of S. pombe to understand the signal transduction pathways that control cell division
Summary
The genetic malleability of the fission yeast S. pombe has helped it to maintain a prominent position alongside the more extensively exploited budding yeast Saccharomyces cerevisiae, as a powerful model system for the characterisation of the basic facets of eukaryotic cell and molecular biology This malleability is based upon an extensive repertoire of classical and molecular genetic techniques [1,2,3]. The ability to use this red pigmentation as a reporter for Ade function made this locus a major focus for studies of core genetic principles These studies led to the development of a number of useful genetic tools including ade.M210/ade.M216 hetero-allelic complementation for the selection and maintenance of diploid strains [5] and the use of the sup3.5 opal suppressor tRNAser mutation as a marker for selection in an ade6.704 mutant background [6,7,8]. The pJK210 uses a similar rescue of ura4.294 to target integration at the ura locus [25]
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