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      Two plasmid modules for introducing the auxin-inducible degron into the fission yeast Schizosaccharomyces pombe by PCR-based gene targeting

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          Abstract

          Targeted protein degradation is a powerful approach to study and inhibit protein function in vivo. Introduction of the auxin-inducible degron (AID) system to the fission yeast Schizosaccharomyces pombe was previously reported, but, to the best of our knowledge, no plasmid for constructing AID-tagged fission yeast strains has been described so far. Here, we describe two plasmids that facilitate the introduction of the mini auxin-inducible degron (mAID) tag with a FLAG epitope or GFP by the conventional PCR-based gene targeting method. Our experimental verification indicated that PCR-based mAID tagging is straightforward and that the auxin-degron system is useful for studying essential proteins in S. pombe.

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          Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe.

          We describe a straightforward PCR-based approach to the deletion, tagging, and overexpression of genes in their normal chromosomal locations in the fission yeast Schizosaccharomyces pombe. Using this approach and the S. pombe ura4+ gene as a marker, nine genes were deleted with efficiencies of homologous integration ranging from 6 to 63%. We also constructed a series of plasmids containing the kanMX6 module, which allows selection of G418-resistant cells and thus provides a new heterologous marker for use in S. pombe. The modular nature of these constructs allows a small number of PCR primers to be used for a wide variety of gene manipulations, including deletion, overexpression (using the regulatable nmt1 promoter), C- or N-terminal protein tagging (with HA, Myc, GST, or GFP), and partial C- or N-terminal deletions with or without tagging. Nine genes were manipulated using these kanMX6 constructs as templates for PCR. The PCR primers included 60 to 80 bp of flanking sequences homologous to target sequences in the genome. Transformants were screened for homologous integration by PCR. In most cases, the efficiency of homologous integration was > or = 50%, and the lowest efficiency encountered was 17%. The methodology and constructs described here should greatly facilitate analysis of gene function in S. pombe.
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            Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe.

            We report the construction and analysis of 4,836 heterozygous diploid deletion mutants covering 98.4% of the fission yeast genome providing a tool for studying eukaryotic biology. Comprehensive gene dispensability comparisons with budding yeast--the only other eukaryote for which a comprehensive knockout library exists--revealed that 83% of single-copy orthologs in the two yeasts had conserved dispensability. Gene dispensability differed for certain pathways between the two yeasts, including mitochondrial translation and cell cycle checkpoint control. We show that fission yeast has more essential genes than budding yeast and that essential genes are more likely than nonessential genes to be present in a single copy, to be broadly conserved and to contain introns. Growth fitness analyses determined sets of haploinsufficient and haploproficient genes for fission yeast, and comparisons with budding yeast identified specific ribosomal proteins and RNA polymerase subunits, which may act more generally to regulate eukaryotic cell growth.
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              A genome-wide resource of cell cycle and cell shape genes of fission yeast

              To identify near complete sets of genes required for the cell cycle and cell shape, we have visually screened a genome-wide gene deletion library of 4843 fission yeast deletion mutants (95.7% of total protein encoding genes) for their effects on these processes. A total of 513 genes have been identified as being required for cell cycle progression, 276 of which have not been previously described as cell cycle genes. Deletions of a further 333 genes lead to specific alterations in cell shape and another 524 genes result in generally misshapen cells. Here, we provide the first eukaryotic resource of gene deletions, which describes a near genome-wide set of genes required for the cell cycle and cell shape.
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                Author and article information

                Journal
                MicroPubl Biol
                MicroPubl Biol
                microPublication Biology
                Caltech Library
                2578-9430
                18 August 2021
                2021
                : 2021
                : 10.17912/micropub.biology.000442
                Affiliations
                [1 ] School of Life Science and Technology, ShanghaiTech University
                Author notes
                [§ ] Correspondence to: Tomoyasu Sugiyama ( tsugiyama@ 123456shanghaitech.edu.cn )

                XS: Investigation

                RX: Validation, Visualization, Writing - review and editing

                TS: Conceptualization, Funding acquisition, Methodology, Supervision, Writing - original draft, Writing - review and editing

                Article
                10.17912/micropub.biology.000442
                8374545
                34423281
                9aa06687-cf8b-4b33-90ce-5a0e98bc3c98
                Copyright: © 2021 by the authors

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 25 June 2021
                : 03 August 2021
                : 04 August 2021
                Categories
                New Finding
                Methods
                S. Pombe

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