The Experts below are selected from a list of 240 Experts worldwide ranked by ideXlab platform
Marlies P Rossmann - One of the best experts on this subject based on the ideXlab platform.
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a common telomeric gene silencing assay is affected by nucleotide metabolism
Molecular Cell, 2011Co-Authors: Marlies P Rossmann, Olga Tsaponina, Andrei Chabes, Bruce StillmanAbstract: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 - One of the best experts on this subject based on the ideXlab platform.
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a common telomeric gene silencing assay is affected by nucleotide metabolism
Molecular Cell, 2011Co-Authors: Marlies P Rossmann, Olga Tsaponina, Andrei Chabes, Bruce StillmanAbstract: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 - One of the best experts on this subject based on the ideXlab platform.
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A general cloning system to selectively isolate any eukaryotic or prokaryotic genomic region in yeast
BMC Genomics, 2003Co-Authors: Vladimir N Noskov, Natalay Kouprina, Sun-hee Leem, Ilia Ouspenski, J Carl Barrett, Vladimir LarionovAbstract: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.
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Integrity of human YACs during propagation in recombination-deficient yeast strains
Genomics, 1999Co-Authors: N. Kouprina, Nana Nikolaishvili, Joan P. Graves, Maxim Koriabine, Michael A. Resnick, Vladimir LarionovAbstract: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.
Robert W. Thornburg - One of the best experts on this subject based on the ideXlab platform.
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Fluoroorotic Acid-selected Nicotiana plumbaginifolia cell lines with a stable thymine starvation phenotype have lost the thymine-regulated transcriptional program.
Plant Physiology, 2000Co-Authors: Djoko Santoso, Robert W. ThornburgAbstract:We have selected 143 independent Nicotiana plumbaginifolia cell lines that survive in the presence of 5-Fluoroorotic Acid. These lines show several diverse phenotypes. The majority of these cell lines showed reduced levels of UMP synthase. However, one particular phenotype, which represents 14% of the total independent lines (20 cell lines), showed an unexpected, high level of UMP synthase and was therefore analyzed in detail. The selected cell lines showed no differences with wild-type cells with respect to uptake of orotic Acid, affinity of UMP synthase for its substrates, or UMP synthase gene-copy number. Alternative detoxification mechanisms were also excluded. The elevated enzyme activity was correlated with elevated UMP synthase protein levels as well as elevated UMP synthase mRNA levels. In contrast to wild-type cell lines, the fluoroorotic Acid-selected cell lines did not respond to thymine or to other biochemicals that affect thymine levels. In addition, there was also a concomitant up-regulation of aspartate transcarbamoylase, however, dihydroorotase and dihydroorotate dehydrogenase are not up-regulated in these cell lines.
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Uridine 5′-Monophosphate Synthase Is Transcriptionally Regulated by Pyrimidine Levels in Nicotiana plumbaginifolia
Plant Physiology, 1998Co-Authors: Djoko Santoso, Robert W. ThornburgAbstract:To understand the regulation and expression of pyrimidine biosynthesis in plants, we have examined the effect of the metabolic inhibitor 5-Fluoroorotic Acid (FOA) on uridine-5′-monophosphate synthase (UMPSase) expression in cell cultures of Nicotiana plumbaginifolia. UMPSase is the rate-limiting step of pyrimidine biosynthesis in plants. Addition of FOA causes an up-regulation of UMPSase enzyme activity in cell cultures after a lag phase of several days. Western-blot analysis demonstrated that the up-regulation in enzyme activity was caused by increased expression of the UMPSase protein. Northern-blot analysis demonstrated a higher level of UMPSase mRNA in the FOA-induced tissues than in control tissues. Run-on transcriptional assays showed that the UMPSase gene was transcriptionally activated after FOA treatment. The mechanism of toxicity of FOA is through thymine starvation. We found that addition of thymine abrogated the FOA-mediated up-regulation of UMPSase. In addition, methotrexate and aminopterin, which affect thymine levels by inhibiting dihydrofolate reductase, also up-regulate UMPSase in N. plumbaginifolia cells.
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Isolation and Characterization of UMP Synthase Mutants from Haploid Cell Suspensions of Nicotiana tabacum
Plant Physiology, 1992Co-Authors: Djoko Santoso, Robert W. ThornburgAbstract:Uridine 5′-monophosphate (UMP) synthase mutants of tobacco have been produced from haploid cell-suspension cultures of a transgenic Nicotiana tabacum line, Tr25. The mutants were induced by incubating the suspension-cultured cells with 1 mm N -nitroso- N -methylurea for either 5 or 12 hours. Twenty mutant calli were isolated on selection medium containing 20 milligrams per liter of 5-Fluoroorotic Acid. Of those tested, most had reduced regeneration capacity. Characterization of UMP synthase activities in the isolated calli showed that UMP synthase activity varied from 8 to nearly 100% of the wild-type activity. The growth of the calli on the media containing different levels of 5-Fluoroorotic Acid correlated with decreasing UMP synthase activity. Because the UMP synthase enzyme has two separate enzymic activities (orotate phosphoribosyl transferase and orotidine-5′-monophosphate decarboxylase), several mutants were further characterized to determine how the mutations affected each of the two enzymic activities. In each case, the enzymic activity affected was the orotate phosphoribosyl transferase and not the orotidine-5′-monophosphate decarboxylase. The wound-inducible phenotype of the Tr25 plants as measured by the activation of the pin 2-CAT gene remained unchanged by introduction of the UMP synthase mutations.
Charles K. Singleton - One of the best experts on this subject based on the ideXlab platform.
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identification and characterization of the thiamine transporter gene of saccharomyces cerevisiae
Gene, 1997Co-Authors: Charles K. SingletonAbstract:A positive selection scheme is described that selects for thiamine transporter clones. The scheme is based on the rescue of lethality, under non-permissive conditions, of Saccharomyces cerevisiae strains that are conditional for thiamine biosynthesis and are defective in thiamine transport. Transport defective strains were generated by selection for resistance to the lethal thiamine analog, pyrithiamine. Pyrithiamine resistance was shown to be a recessive, single gene trait that resulted from the mutation of the thiamine transporter gene, as suggested by previous work. Conditional thiamine biosynthesis was generated by cloning THI4, a thiamine biosynthetic gene, into a URA3 containing plasmid and transforming a strain disrupted in THI4. Thus, plating on 5-Fluoroorotic Acid causes the loss of thiamine synthesis ability. The gene for the yeast thiamine transporter, THI7, was cloned using this scheme. The predicted 598 amino Acid transporter is a member of the major facilitator superfamily of transporters and thus possesses 12 transmembrane spanning segments with amino and carboxy termini intracellularly located. Several alterations in the coding region were characterized that result in greatly reduced ability to transport thiamine. The level of transporter mRNA was found to be rapidly and dramatically reduced by the addition of thiamine to the growth medium.
