Extending the Schizosaccharomyces pombe molecular genetic toolbox

D Fennessy, A Grallert, A Krapp, A Cokoja, A Bridge, Janni Petersen, A Patel, A Tallada, E Boke, B Hodgson, V Simanis, I Hagan

    Research output: Contribution to journalArticle

    29 Citations (Scopus)

    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.

    Original languageEnglish
    Article numbere97683
    Pages (from-to)e97683
    Number of pages16
    JournalPLoS One
    Volume9
    Issue number5
    DOIs
    Publication statusPublished - 2014

    Fingerprint Dive into the research topics of 'Extending the Schizosaccharomyces pombe molecular genetic toolbox'. Together they form a unique fingerprint.

  • Cite this

    Fennessy, D., Grallert, A., Krapp, A., Cokoja, A., Bridge, A., Petersen, J., Patel, A., Tallada, A., Boke, E., Hodgson, B., Simanis, V., & Hagan, I. (2014). Extending the Schizosaccharomyces pombe molecular genetic toolbox. PLoS One, 9(5), e97683. [e97683]. https://doi.org/10.1371/journal.pone.0097683