New vectors for epitope tagging and gene disruption in Schizosaccharomyces pombe

Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA.
BioTechniques (Impact Factor: 2.95). 11/2013; 55(5):257-63. DOI: 10.2144/000114100
Source: PubMed


We describe a series of new vectors for PCR-based epitope tagging and gene disruption in the fission yeast Schizosaccharomyces pombe, an exceptional model organism for the study of cellular processes. The vectors are designed for amplification of gene-targeting DNA cassettes and integration into specific genetic loci, allowing expression of proteins fused to 12 tandem copies of the Pk (V5) epitope or 5 tandem copies of the FLAG epitope with a glycine linker. These vectors are available with various antibiotic or nutritional markers and are useful for protein studies using biochemical and cell biological methods. We also describe new vectors for fluorescent protein-tagging and gene disruption using ura4MX6, LEU2MX6, and his3MX6 selection markers, allowing researchers in the S. pombe community to disrupt genes and manipulate genomic loci using primer sets already available for the widely used pFA6a-MX6 system. Our new vectors may also be useful for gene manipulation in Saccharomyces cerevisiae.

Download full-text


Available from: Eishi Noguchi, May 19, 2014

  • No preview · Article · Jan 2015
  • [Show abstract] [Hide abstract]
    ABSTRACT: DNA replication is tightly coupled with DNA repair processes in order to preserve genomic integrity. During DNA replication, the replication fork encounters a variety of obstacles including DNA damage/adducts, secondary structures, and programmed fork-blocking sites, which are all difficult to replicate. The replication fork also collides with the transcription machinery, which shares the template DNA with the replisome complex. Under these conditions, replication forks stall, causing replication stress and/or fork collapse, ultimately leading to genomic instability. The mechanisms to overcome these replication problems remain elusive. Therefore, it is important to investigate how DNA repair and replication factors are recruited and coordinated at chromosomal regions that are difficult to replicate. In this chapter, we describe a chromatin immunoprecipitation method to locate proteins required for DNA repair during DNA replication in the fission yeast Schizosaccharomyces pombe. This method can also easily be adapted to study replisome components or chromatin-associated factors.
    No preview · Article · Apr 2015 · Methods in molecular biology (Clifton, N.J.)
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Histone H3 lysine 4 (K4) methylation is a dynamic modification. In budding yeast, H3K4 methylation is catalyzed by the Set1-COMPASS methyltransferase complex, and removed by Jhd2, a JMJC-domain family demethylase. The catalytic JmjC and JmjN domains of Jhd2 have the ability to remove all three degrees (mono-, di- and tri-) of H3K4 methylation. Jhd2 also contains a PHD finger required for its chromatin association and H3K4 demethylase functions. The Jhd2 PHD finger associates with chromatin independent of H3K4 methylation and the H3 N-terminal tail. Therefore, how Jhd2 associates with chromatin to perform H3K4 demethylation has remained unknown. We report a novel interaction between the Jhd2 PHD finger and histone H2A. Two residues in H2A (F26 and E57) serve as a binding site for Jhd2 in vitro, and mediate its chromatin association and H3K4 demethylase functions in vivo. Using RNA-seq, we have identified the functional target genes for Jhd2 and the H2A F26 and E57 residues. We demonstrate that H2A F26 and E57 residues control Jhd2's chromatin association and H3K4 demethylase functions during positive or negative regulation of transcription at target genes. Importantly, we show that H2BK123 ubiquitination blocks Jhd2 from accessing its binding site on chromatin, and thereby, we have uncovered a second mechanism by which H2B ubiquitination contributes to the trans-histone regulation of H3K4 methylation. Overall, our study provides novel insights into the chromatin binding dynamics and H3K4 demethylase functions of Jhd2.
    Full-text · Article · Oct 2015 · Journal of Biological Chemistry
Show more