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      Histone variant H2A.Z promotes meiotic chromosome axis organization in Saccharomyces cerevisiae

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      , ,
      G3: Genes|Genomes|Genetics
      Oxford University Press
      meiosis, chromatin, histone H1, H2A.Z

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          Abstract

          Progression through meiosis is associated with significant reorganization of chromosome structure, regulated in part by changes in histones and chromatin. Prior studies observed defects in meiotic progression in yeast strains lacking the linker histone H1 or variant histone H2A.Z. To further define the contributions of these chromatin factors, we have conducted genetic and cytological analysis of cells undergoing meiosis in the absence of H1 and H2A.Z. We find that a spore viability defect observed in strains lacking H2A.Z can be partially suppressed if cells also lack histone H1, while the combined loss of both H1 and H2A.Z is associated with elevated gene conversion events. Cytological analysis of Red1 and Rec8 staining patterns indicates that a subset of cells lacking H2A.Z fail to assemble a proper chromosome axis, and the staining pattern of the synaptonemal complex protein Zip1 in htz1Δ/htz1Δ cells mimics that of cells deficient for Rec8-dependent meiotic cohesion. Our results suggest a role for H2A.Z in the establishment or maintenance of the meiotic chromosome axis, possibly by promoting the efficient chromosome cohesion.

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          Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae.

          Disruption-deletion cassettes are powerful tools used to study gene function in many organisms, including Saccharomyces cerevisiae. Perhaps the most widely useful of these are the heterologous dominant drug resistance cassettes, which use antibiotic resistance genes from bacteria and fungi as selectable markers. We have created three new dominant drug resistance cassettes by replacing the kanamycin resistance (kan(r)) open reading frame from the kanMX3 and kanMX4 disruption-deletion cassettes (Wach et al., 1994) with open reading frames conferring resistance to the antibiotics hygromycin B (hph), nourseothricin (nat) and bialaphos (pat). The new cassettes, pAG25 (natMX4), pAG29 (patMX4), pAG31 (patMX3), pAG32 (hphMX4), pAG34 (hphMX3) and pAG35 (natMX3), are cloned into pFA6, and so are in all other respects identical to pFA6-kanMX3 and pFA6-kanMX4. Most tools and techniques used with the kanMX plasmids can also be used with the hph, nat and patMX containing plasmids. These new heterologous dominant drug resistance cassettes have unique antibiotic resistance phenotypes and do not affect growth when inserted into the ho locus. These attributes make the cassettes ideally suited for creating S. cerevisiae strains with multiple mutations within a single strain. Copyright 1999 John Wiley & Sons, Ltd.
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            New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae.

            We have constructed and tested a dominant resistance module, for selection of S. cerevisiae transformants, which entirely consists of heterologous DNA. This kanMX module contains the known kanr open reading-frame of the E. coli transposon Tn903 fused to transcriptional and translational control sequences of the TEF gene of the filamentous fungus Ashbya gossypii. This hybrid module permits efficient selection of transformants resistant against geneticin (G418). We also constructed a lacZMT reporter module in which the open reading-frame of the E. coli lacZ gene (lacking the first 9 codons) is fused at its 3' end to the S. cerevisiae ADH1 terminator. KanMX and the lacZMT module, or both modules together, were cloned in the center of a new multiple cloning sequence comprising 18 unique restriction sites flanked by Not I sites. Using the double module for constructions of in-frame substitutions of genes, only one transformation experiment is necessary to test the activity of the promotor and to search for phenotypes due to inactivation of this gene. To allow for repeated use of the G418 selection some kanMX modules are flanked by 470 bp direct repeats, promoting in vivo excision with frequencies of 10(-3)-10(-4). The 1.4 kb kanMX module was also shown to be very useful for PCR based gene disruptions. In an experiment in which a gene disruption was done with DNA molecules carrying PCR-added terminal sequences of only 35 bases homology to each target site, all twelve tested geneticin-resistant colonies carried the correctly integrated kanMX module.
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              Epitope tagging of yeast genes using a PCR-based strategy: more tags and improved practical routines.

              Epitope tagging of proteins as a strategy for the analysis of function, interactions and the subcellular distribution of proteins has become widely used. In the yeast Saccharomyces cerevisiae, molecular biological techniques have been developed that use a simple PCR-based strategy to introduce epitope tags to chromosomal loci (Wach et al., 1994). To further employ the power of this strategy, a variety of novel tags was constructed. These tags were combined with different selectable marker genes, resulting in PCR amplificable modules. Only one set of primers is required for the amplification of any module. Furthermore, convenient laboratory techniques are described that facilitate the genetic manipulations of yeast strains, as well as the analysis of the epitope-tagged proteins. Copyright 1999 John Wiley & Sons, Ltd.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                G3 (Bethesda)
                Genetics
                g3journal
                G3: Genes|Genomes|Genetics
                Oxford University Press
                2160-1836
                August 2022
                24 May 2022
                24 May 2022
                : 12
                : 8
                : jkac128
                Affiliations
                Department of Molecular Biology and Biochemistry, Wesleyan University , Middletown, CT 06459, USA
                Department of Molecular Biology and Biochemistry, Wesleyan University , Middletown, CT 06459, USA
                Department of Molecular Biology and Biochemistry, Wesleyan University , Middletown, CT 06459, USA
                Author notes
                Corresponding author: Department of Molecular Biology and Biochemistry, 52 Lawn Avenue, Wesleyan University, Middletown CT 06459, USA. Email: sholmes@ 123456wesleyan.edu
                Author information
                https://orcid.org/0000-0001-6535-6663
                https://orcid.org/0000-0002-0919-7538
                https://orcid.org/0000-0002-0154-198X
                Article
                jkac128
                10.1093/g3journal/jkac128
                9339299
                35608312
                15e0a8fc-8a97-4b5a-b7c8-1820dcb1fe53
                © The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 24 August 2021
                : 07 May 2022
                : 11 June 2022
                Page count
                Pages: 11
                Funding
                Funded by: National Institutes of Health, DOI 10.13039/100000002;
                Award ID: 1R15GM102824-01
                Funded by: National Institutes of Health, DOI 10.13039/100000002;
                Award ID: GM116109
                Categories
                Investigation
                AcademicSubjects/SCI01180
                AcademicSubjects/SCI01140
                AcademicSubjects/SCI00010
                AcademicSubjects/SCI00960

                Genetics
                meiosis,chromatin,histone h1,h2a.z
                Genetics
                meiosis, chromatin, histone h1, h2a.z

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