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      CTD kinase I is required for the integrity of the rDNA tandem array

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      Nucleic Acids Research

      Oxford University Press

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          Abstract

          The genomic stability of the rDNA tandem array is tightly controlled to allow sequence homogenization and to prevent deleterious rearrangements. In this report, we show that the absence of the yeast CTD kinase I (CTDK-I) complex in null mutant strains leads to a decrease in the number of tandem rDNA repeats. Reintroduction of the missing gene induces an increase of rDNA repeats to reach a copy number similar to that of the original strain. Interestingly, while expansion is dependent on Fob1, a protein required for replication fork blocking activity in rDNA, contraction occurs in the absence of Fob1. Furthermore, silencing of class II genes at the rDNA, a process connected to rDNA stability, is not affected. Ctk1, the kinase subunit of the CTDK-I complex is involved in various steps of mRNA synthesis. In addition, we have recently shown that Ctk1 is also implicated in rRNA synthesis. The results suggest that the RNA polymerase I transcription defect occurring in a ctk1 mutant strain causes rDNA contraction.

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          Most cited references 66

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          Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae.

          An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one-step PCR-mediated technique for deletion and modification of chromosomal genes. This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest. We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications. Using as selectable marker the S. cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5+ or Escherichia coli kan(r) gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), C- or N-terminal protein tagging [with GFP(S65T), GST, or the 3HA or 13Myc epitope], and partial N- or C-terminal deletions (with or without concomitant protein tagging). Because of the modular nature of the plasmids, they allow efficient and economical use of a small number of PCR primers for a wide variety of gene manipulations. Thus, these plasmids should further facilitate the rapid analysis of gene function in S. cerevisiae.
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            Elevated recombination rates in transcriptionally active DNA.

            We have examined the effect of RNA polymerase II-dependent transcription on recombination between directly repeated sequences of the GAL10 gene in S. cerevisiae. Direct repeat recombination leading either to plasmid loss or conversion was examined in isogenic strains containing null mutations in the positive activator, GAL4, or the repressor, GAL80. A 15-fold increase in the rate of plasmid loss is observed in cells constitutively expressing the construct compared with cells that are not. Conversion events that retain the integrated plasmid are not stimulated by expression of the repeats. Northern analysis of strains containing plasmid inserts with various promoter mutations suggests that the stimulation in recombination is mediated by events initiating within the integrated plasmid sequences.
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              A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae.

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                Author and article information

                Journal
                Nucleic Acids Res
                Nucleic Acids Research
                Nucleic Acids Research
                Oxford University Press
                0305-1048
                1362-4962
                October 2006
                October 2006
                19 August 2006
                : 34
                : 17
                : 4996-5006
                Affiliations
                simpleService de Biochimie et de Génétique Moléculaire, CEA/Saclay 91191 Gif/Yvette, France
                Author notes
                *To whom correspondence should be addressed. Tel: +33 1 4079 5123; Fax: +33 1 4079 5229; Email: valerie.goguel@ 123456espci.fr

                Present addresses: Céline Bouchoux, Chromosome Segregation Laboratory, Cancer Research UK, 44 Lincoln's Inn fields, London WC2A 3PX, UK

                Valérie Goguel, Gènes et Dynamique des Systèmes de Mémoire, CNRS UMR7637, ESPCI, 10 rue Vauquelin, 75005 Paris, France

                Article
                10.1093/nar/gkl493
                1635248
                16984969
                © 2006 The Author(s)

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

                Categories
                Molecular Biology

                Genetics

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