Extending the Schizosaccharomyces pombe Molecular Genetic Toolbox

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.

Traffic Through the Trans-Golgi Network and the Endosomal System Requires Collaboration Between Exomer and Clathrin Adaptors in Fission Yeast

“…These fragments and GFP were ligated into a KS+ vector bearing the ura4 + gene as a selectable marker; the plasmid was integrated into the gga22 + locus. The FYVE(EEA1) phosphatidylinositol-3-phosphate-binding probe was PCR-amplified from plasmid pRS424GFP-FYVE(EEA1) (#36096 Addgene; Burd and Emr 1998), ligated to the C-terminal end of mCherry, and cloned under the control of the nda2 + promoter and terminator into the pINTHA vector (Fennessy et al 2014). The construct was transformed into the hph.171K strain (Fennessy et al 2014;Yeast Genome Resource Center #FY23692).…”

A toolbox of Stable Integration Vectors (SIV) in the fission yeastSchizosaccharomyces pombe

“…Integrative plasmids that rely on two homology arms have previously been developed by the Sato and Hagan groups (Fennessy et al, 2014;Kakui et al, 2015). Whether these plasmids, which target different genomic loci than the ones used here, exhibit similar properties remains to be seen, as neither stability of integration nor number of integration events have been reported.…”

Architecture of the Mto1/2 microtubule nucleation complex

“…Fission yeast strains were constructed by targeted plasmid integration (Fennessy et al, 2014), PCR-based gene targeting (Bahler et al, 1998), and genetic crosses (Ekwall and Thon, 2017). Standard methods for were used culture (Petersen and Russell, 2016).…”

“…Mo2 NND and TCD domains were overexpressed (i.e. in separate strains) from transgenes integrated at the hph.171k locus on chromosome III by homologous recombination (Fennessy et al, 2014). NND and TCD sequences were amplified by PCR using plasmid pKS1258 as template and oligonucleotide primer pairs OKS3315/OKS3316 (for NND) and OKS3313/OKS3314 (for TCD).…”

“…PCR products were cloned into pJET1.2 (Thermo Fisher Scientific) by blunt-end ligation, to generate plasmids pKS1572 and pKS1573. NND and TCD inserts from these plasmids were then subcloned into fission yeast integration plasmid pINTH1 (Fennessy et al, 2014) (pKS1612 in our collection), using NdeI and BamHI sites, to generate plasmids pKS1574 and pKS1579.…”