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5-Fluoroorotic Acid

The Experts below are selected from a list of 240 Experts worldwide ranked by ideXlab platform

Marlies P Rossmann – 1st expert on this subject based on the ideXlab platform

  • a common telomeric gene silencing assay is affected by nucleotide metabolism
    Molecular Cell, 2011
    Co-Authors: Marlies P Rossmann, Olga Tsaponina, Andrei Chabes, Bruce Stillman

    Abstract:

    Telomere-associated position-effect variegation (TPEV) in budding yeast has been used as a model for understanding epigenetic inheritance and gene silencing. A widely used assay to identify mutants with improper TPEV employs the URA3 gene at the telomere of chromosome VII-L that can be counterselected with 5-Fluoroorotic Acid (5-FOA). 5-FOA resistance has been inferred to represent lack of transcription of URA3 and therefore to represent heterochromatin-induced gene silencing. For two genes implicated in telomere silencing, POL30 and DOT1, we show that the URA3 telomere reporter assay does not reflect their role in heterochromatin formation. Rather, an imbalance in ribonucleotide reductase (RNR), which is induced by 5-FOA, and the specific promoter of URA3 fused to ADH4 at telomere VII-L are jointly responsible for the variegated phenotype. We conclude that metabolic changes caused by the drug employed and certain mutants being studied are incompatible with the use of certain prototrophic markers for TPEV.

Bruce Stillman – 2nd expert on this subject based on the ideXlab platform

  • a common telomeric gene silencing assay is affected by nucleotide metabolism
    Molecular Cell, 2011
    Co-Authors: Marlies P Rossmann, Olga Tsaponina, Andrei Chabes, Bruce Stillman

    Abstract:

    Telomere-associated position-effect variegation (TPEV) in budding yeast has been used as a model for understanding epigenetic inheritance and gene silencing. A widely used assay to identify mutants with improper TPEV employs the URA3 gene at the telomere of chromosome VII-L that can be counterselected with 5-Fluoroorotic Acid (5-FOA). 5-FOA resistance has been inferred to represent lack of transcription of URA3 and therefore to represent heterochromatin-induced gene silencing. For two genes implicated in telomere silencing, POL30 and DOT1, we show that the URA3 telomere reporter assay does not reflect their role in heterochromatin formation. Rather, an imbalance in ribonucleotide reductase (RNR), which is induced by 5-FOA, and the specific promoter of URA3 fused to ADH4 at telomere VII-L are jointly responsible for the variegated phenotype. We conclude that metabolic changes caused by the drug employed and certain mutants being studied are incompatible with the use of certain prototrophic markers for TPEV.

Vladimir Larionov – 3rd expert on this subject based on the ideXlab platform

  • A general cloning system to selectively isolate any eukaryotic or prokaryotic genomic region in yeast
    BMC Genomics, 2003
    Co-Authors: Vladimir N Noskov, Natalay Kouprina, Sun-hee Leem, Ilia Ouspenski, J Carl Barrett, Vladimir Larionov

    Abstract:

    Background Transformation-associated recombination (TAR) cloning in yeast is a unique method for selective isolation of large chromosomal fragments or entire genes from complex genomes. The technique involves homologous recombination, during yeast spheroplast transformation, between genomic DNA and a TAR vector that has short (~ 60 bp) 5′ and 3′ gene targeting sequences (hooks). Result TAR cloning requires that the cloned DNA fragment carry at least one autonomously replicating sequence ( ARS ) that can function as the origin of replication in yeast, which prevents wide application of the method. In this paper, we describe a novel TAR cloning system that allows isolation of genomic regions lacking yeast ARS -like sequences. ARS is inserted into the TAR vector along with URA3 as a counter-selectable marker. The hooks are placed between the TATA box and the transcription initiation site of URA3 . Insertion of any sequence between hooks results in inactivation of URA3 expression. That inactivation confers resistance to 5-Fluoroorotic Acid, allowing selection of TAR cloning events against background vector recircularization events. Conclusion The new system greatly expands the area of application of TAR cloning by allowing isolation of any chromosomal region from eukaryotic and prokaryotic genomes regardless of the presence of autonomously replicating sequences.

  • Integrity of human YACs during propagation in recombination-deficient yeast strains
    Genomics, 1999
    Co-Authors: N. Kouprina, Nana Nikolaishvili, Joan P. Graves, Maxim Koriabine, Michael A. Resnick, Vladimir Larionov

    Abstract:

    Abstract Several isogenic strains with defects in recombination/repair genes ( RAD1, RAD50, RAD51, RAD52, RAD54, and RAD55 ) were examined for their ability to propagate accurately a variety of linear and circular yeast artificial chromosomes (YACs) containing human DNA inserts. To assess YAC stability, the human DNA inserts were internally marked by an ADE2-pBR-URA3 cassette. Following selection for Ura − clones on 5-Fluoroorotic Acid containing medium, the following types of YAC deletions were identified: (i) those caused by homologous recombination with a telomeric pBR sequence; (ii) internal deletions, presumed to occur by recombination between commonly occurring DNA repeats such as Alu and LINE sequences; and (iii) deletions leading to loss of part of a YAC arm. rad52 host strains, but not other recombination-deficient strains, decreased the rate of all types of YAC deletions 25- to 400-fold. We have also developed and tested kar1 strains with a conditional RAD52 gene that allow transfer of a YAC from any host into a recombination-deficient background. These strains provide an efficient tool for stabilization of YACs and are useful for allowing additional recombinational modification of YACs.