Dataset Information

Identification of new players in cell division, DNA damage response, and morphogenesis through construction of Schizosaccharomyces pombe deletion strains.

ABSTRACT: Many fundamental biological processes are studied using the fission yeast, Schizosaccharomyces pombe. Here we report the construction of a set of 281 haploid gene deletion strains covering many previously uncharacterized genes. This collection of strains was tested for growth under a variety of different stress conditions. We identified new genes involved in DNA metabolism, completion of the cell cycle, and morphogenesis. This subset of nonessential gene deletions will add to the toolkits available for the study of biological processes in S. pombe.

SUBMITTER: Chen JS

PROVIDER: S-EPMC4349090 | biostudies-other | 2014 Dec

REPOSITORIES: biostudies-other

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Publications

Identification of new players in cell division, DNA damage response, and morphogenesis through construction of Schizosaccharomyces pombe deletion strains.

Chen Jun-Song JS Beckley Janel R JR McDonald Nathan A NA Ren Liping L Mangione MariaSanta M Jang Sylvia J SJ Elmore Zachary C ZC Rachfall Nicole N Feoktistova Anna A Jones Christine M CM Willet Alaina H AH Guillen Rodrigo R Bitton Danny A DA Bähler Jürg J Jensen Michael A MA Rhind Nick N Gould Kathleen L KL

G3 (Bethesda, Md.) 20141231 3

Many fundamental biological processes are studied using the fission yeast, Schizosaccharomyces pombe. Here we report the construction of a set of 281 haploid gene deletion strains covering many previously uncharacterized genes. This collection of strains was tested for growth under a variety of different stress conditions. We identified new genes involved in DNA metabolism, completion of the cell cycle, and morphogenesis. This subset of nonessential gene deletions will add to the toolkits availa ...[more]

PMID: 25552606

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Project description:BackgroundConstruction of plasmids is crucial in modern genetic manipulation. As of now, the common method for constructing plasmids is to digest specific DNA sequences with restriction enzymes and to ligate the resulting DNA fragments with DNA ligase. Another potent method to construct plasmids, known as gap-repair cloning (GRC), is commonly used in the budding yeast Saccharomyces cerevisiae. GRC makes use of the homologous recombination activity that occurs within the yeast cells. Due to its flexible design and efficiency, GRC has been frequently used for constructing plasmids with complex structures as well as genome-wide plasmid collections. Although there have been reports indicating GRC feasibility in the fission yeast Schizosaccharomyces pombe, this species is not commonly used for GRC as systematic studies of reporting GRC efficiency in S. pombe have not been performed till date.Methodology/principal findingsWe investigated GRC efficiency in S. pombe in this study. We first showed that GRC was feasible in S. pombe by constructing a plasmid that contained the LEU2 auxotrophic marker gene in vivo and showed sufficient efficiency with short homology sequences (>25 bp). No preference was shown for the sequence length from the cut site in the vector plasmid. We next showed that plasmids could be constructed in a proper way using 3 DNA fragments with 70% efficiency without any specific selections being made. The GRC efficiency with 3 DNA fragments was dramatically increased >95% in lig4Delta mutant cell, where non-homologous end joining is deficient. Following this approach, we successfully constructed plasmid vectors with leu1+, ade6+, his5+, and lys1+ markers with the low-copy stable plasmid pDblet as a backbone by applying GRC in S. pombe.Conclusions/significanceWe concluded that GRC was sufficiently feasible in S. pombe for genome-wide gene functional analysis as well as for regular plasmid construction. Plasmids with different markers constructed in this research are available from NBRP-yeast (http://yeast.lab.nig.ac.jp/).

Dataset Information

Identification of new players in cell division, DNA damage response, and morphogenesis through construction of Schizosaccharomyces pombe deletion strains.

Publications

Identification of new players in cell division, DNA damage response, and morphogenesis through construction of Schizosaccharomyces pombe deletion strains.

Similar Datasets

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