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Robert W. Thornburg - One of the best experts on this subject based on the ideXlab platform.

  • Fluoroorotic Acid-selected Nicotiana plumbaginifolia cell lines with a stable thymine starvation phenotype have lost the thymine-regulated transcriptional program.
    Plant Physiology, 2000
    Co-Authors: Djoko Santoso, Robert W. Thornburg
    Abstract:

    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.

  • Uridine 5′-Monophosphate Synthase Is Transcriptionally Regulated by Pyrimidine Levels in Nicotiana plumbaginifolia
    Plant Physiology, 1998
    Co-Authors: Djoko Santoso, Robert W. Thornburg
    Abstract:

    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.

  • uridine 5 monophosphate synthase is transcriptionally regulated by pyrimidine levels in nicotiana plumbaginifolia
    Plant Physiology, 1998
    Co-Authors: Djoko Santoso, Robert W. Thornburg
    Abstract:

    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.

  • Isolation and Characterization of UMP Synthase Mutants from Haploid Cell Suspensions of Nicotiana tabacum
    Plant Physiology, 1992
    Co-Authors: Djoko Santoso, Robert W. Thornburg
    Abstract:

    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.

Djoko Santoso - One of the best experts on this subject based on the ideXlab platform.

  • Fluoroorotic Acid-selected Nicotiana plumbaginifolia cell lines with a stable thymine starvation phenotype have lost the thymine-regulated transcriptional program.
    Plant Physiology, 2000
    Co-Authors: Djoko Santoso, Robert W. Thornburg
    Abstract:

    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.

  • Uridine 5′-Monophosphate Synthase Is Transcriptionally Regulated by Pyrimidine Levels in Nicotiana plumbaginifolia
    Plant Physiology, 1998
    Co-Authors: Djoko Santoso, Robert W. Thornburg
    Abstract:

    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.

  • uridine 5 monophosphate synthase is transcriptionally regulated by pyrimidine levels in nicotiana plumbaginifolia
    Plant Physiology, 1998
    Co-Authors: Djoko Santoso, Robert W. Thornburg
    Abstract:

    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.

  • Isolation and Characterization of UMP Synthase Mutants from Haploid Cell Suspensions of Nicotiana tabacum
    Plant Physiology, 1992
    Co-Authors: Djoko Santoso, Robert W. Thornburg
    Abstract:

    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.

Moshe Mevarech - One of the best experts on this subject based on the ideXlab platform.

  • development of a gene knockout system for the halophilic archaeon haloferax volcanii by use of the pyre gene
    Journal of Bacteriology, 2003
    Co-Authors: Gili Bitanbanin, Ron Ortenberg, Moshe Mevarech
    Abstract:

    So far, the extremely halophilic archaeon Haloferax volcanii has the best genetic tools among the archaea. However, the lack of an efficient gene knockout system for this organism has hampered further genetic studies. In this paper we describe the development of pyrE-based positive selection and counterselection systems to generate an efficient gene knockout system. The H. volacanii pyrE1 and pyrE2 genes were isolated, and the pyrE2 gene was shown to code for the physiological enzyme orotate phosphoribosyl transferase. A ΔpyrE2 strain was constructed and used to isolate deletion mutants by the following two steps: (i) integration of a nonreplicative plasmid carrying both the pyrE2 wild-type gene, as a selectable marker, and a cloned chromosomal DNA fragment containing a deletion in the desired gene; and (ii) excision of the integrated plasmid after selection with 5-Fluoroorotic Acid. Application of this gene knockout system is described.

Jef D Boeke - One of the best experts on this subject based on the ideXlab platform.

  • superloser a plasmid shuffling vector for saccharomyces cerevisiae with exceedingly low background
    G3: Genes Genomes Genetics, 2019
    Co-Authors: Max A B Haase, David M Truong, Jef D Boeke
    Abstract:

    Here we report a new plasmid shuffle vector for forcing budding yeast (Saccharomyces cerevisiae) to incorporate a new genetic pathway in place of a native pathway – even an essential one – while maintaining low false positive rates (less than 1 in 108 per cell). This plasmid, dubbed “Superloser,” was designed with reduced sequence similarity to commonly used yeast plasmids (i.e., pRS400 series) to limit recombination, a process that in our experience leads to retention of the yeast gene(s) instead of the desired gene(s). In addition, Superloser utilizes two orthogonal copies of the counter-selectable marker URA3 to reduce spontaneous 5-Fluoroorotic Acid resistance. Finally, the CEN/ARS sequence is fused to the GAL1-10 promoter, which disrupts plasmid segregation in the presence of the sugar galactose, causing Superloser to rapidly be removed from a population of cells. We show one proof-of-concept shuffling experiment: swapping yeast’s core histones out for their human counterparts. Superloser is especially useful for forcing yeast to use highly unfavorable genes, such as human histones, as it enables plating a large number of cells (1.4x109) on a single 10 cm petri dish while maintaining a very low background. Therefore, Superloser is a useful tool for yeast geneticists to effectively shuffle low viability genes and/or pathways in yeast that may arise in as few as 1 in 108 cells.

  • superloser a plasmid shuffling vector for saccharomyces cerevisiae with exceedingly low background
    bioRxiv, 2019
    Co-Authors: Max A B Haase, David M Truong, Jef D Boeke
    Abstract:

    Abstract Here we report a new plasmid shuffle vector for forcing budding yeast (Saccharomyces cerevisiae) to incorporate a new genetic pathway in place of a native pathway – even essential ones – while maintaining low false positive rates (less than 1 in 108 per cell). This plasmid, dubbed “Superloser”, was designed with reduced sequence similarity to commonly used yeast plasmids (i.e. pRS400 series) to limit recombination, a process that in our experience leads to retention of the yeast gene(s) instead of the desired gene(s). In addition, Superloser utilizes two orthogonal copies of the counter-selectable marker URA3 to reduce spontaneous 5-Fluoroorotic Acid resistance. Finally, the CEN/ARS sequence is fused to the GAL1-10 promoter, which disrupts plasmid segregation in the presence of the sugar galactose, causing Superloser to rapidly be removed from a population of cells. We show one proof of concept shuffling experiment: swapping yeast’s core histones out for their human counterparts. Superloser is especially useful for forcing yeast to use highly unfavorable genes, such as human histones, as it enables plating a large number of cells (1.4×109) on a single 10 cm petri dish while maintaining a very low background. Therefore, Superloser is a useful tool for yeast geneticists to effectively shuffle low viability genes and/or pathways in yeast that may arise in as low as 1 in 108 cells.

J. Christopher Fenno - One of the best experts on this subject based on the ideXlab platform.

  • Characterization of Treponema denticola pyrF encoding orotidine-5'-monophosphate decarboxylase.
    Fems Microbiology Letters, 2006
    Co-Authors: Ricardo Capone, Nora Pakulis, Turki Y. Alhazzazi, Yu Ning, J. Christopher Fenno
    Abstract:

    The Treponema denticola ATCC 35405 genome annotation contains most of the genes for de novo pyrimidine biosynthesis. To initiate characterization of pyrimidine synthesis in Treponema, we focused on TDE2110 (the putative pyrF, encoding orotidine-5′-monophosphate decarboxlyase). Unlike the parent strain, an isogenic pyrF mutant was resistant to 5-Fluoroorotic Acid. In complex medium, growth of the pyrF mutant was independent of added uracil, indicating activity of a uracil uptake/salvage pathway. Transcription of pyrF was greatly reduced in T. denticola grown in excess uracil, demonstrating that de novo pyrimidine synthesis is regulated and suggesting a feedback mechanism. Treponema denticola PyrF complemented uracil auxotrophy in an Escherichia coli pyrF mutant. This study provides biochemical confirmation of T. denticola genome predictions of de novo and salvage pyrimidine pathways and provides proof of concept that pyrF has potential as a selectable marker in T. denticola.