The Experts below are selected from a list of 288 Experts worldwide ranked by ideXlab platform
Yvonne Nygard - One of the best experts on this subject based on the ideXlab platform.
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synthetic control devices for gene regulation in penicillium chrysogenum
Microbial Cell Factories, 2019Co-Authors: Laszlo Mozsik, Zsofia Buttel, Roel A L Bovenberg, Arnold J M Driessen, Yvonne NygardAbstract:Orthogonal, synthetic control devices were developed for Penicillium chrysogenum, a model filamentous fungus and industrially relevant cell factory. In the synthetic transcription factor, the QF DNA-binding domain of the transcription factor of the quinic acid gene cluster of Neurospora crassa is fused to the VP16 activation domain. This synthetic transcription factor controls the expression of genes under a synthetic promoter containing quinic acid Upstream Activating Sequence (QUAS) elements, where it binds. A gene cluster may demand an expression tuned individually for each gene, which is a great advantage provided by this system. The control devices were characterized with respect to three of their main components: expression of the synthetic transcription factors, Upstream Activating Sequences, and the affinity of the DNA binding domain of the transcription factor to the Upstream Activating domain. This resulted in synthetic expression devices, with an expression ranging from hardly detectable to a level similar to that of highest expressed native genes. The versatility of the control device was demonstrated by fluorescent reporters and its application was confirmed by synthetically controlling the production of penicillin. The characterization of the control devices in microbioreactors, proved to give excellent indications for how the devices function in production strains and conditions. We anticipate that these well-characterized and robustly performing control devices can be widely applied for the production of secondary metabolites and other compounds in filamentous fungi.
Gary F. Merrill - One of the best experts on this subject based on the ideXlab platform.
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Thioredoxin Reductase-dependent Inhibition of MCB Cell Cycle Box Activity in Saccharomyces cerevisiae
Journal of Biological Chemistry, 1997Co-Authors: André K. Machado, Brian A Morgan, Gary F. MerrillAbstract:Abstract Mlu1 cell cycle box (MCB) elements are found near the start site of yeast genes expressed at G1/S. Basal promoters dependent on the elements for Upstream Activating Sequence activity are inactive in Δswi6 yeast. Yeast were screened for mutations that activated MCB reporter genes in the absence of Swi6. The mutations identified a single complementation group. Functional cloning revealed the mutations were alleles of theTRR1 gene encoding thioredoxin reductase. Although deletion of TRR1 activated MCB reporter genes, high copy expression did not suppress reporter gene activity. The trr1 mutations strongly (20-fold) stimulated MCB- and SCB (Swi4/Swi6 cell cycle box)-containing reporter genes, but also weakly (3-fold) stimulated reporter genes that lacked these elements. The trr1mutations did not affect the level or periodicity of three endogenous MCB gene mRNAs (TMP1, RNR1, andSWI4). Deletion of thioredoxin genes TRX1 andTRX2 recapitulated the stimulatory effect oftrr1 mutations on MCB reporter gene activity. Conditions expected to oxidize thioredoxin (exposure to H2O2) induced MCB gene expression, whereas conditions expected to conserve thioredoxin (exposure to hydroxyurea) inhibited MCB gene expression. The results suggest that thioredoxin oxidation contributes to MCB element activation and suggest a link between thioredoxin-oxidizing processes such as ribonucleotide reduction and cell cycle-specific gene transcription.
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Thioredoxin Reductase-dependent Inhibition of MCB Cell Cycle Box Activity in Saccharomyces cerevisiae
Journal of Biological Chemistry, 1997Co-Authors: André K. Machado, Brian A Morgan, Gary F. MerrillAbstract:Mlu1 cell cycle box (MCB) elements are found near the start site of yeast genes expressed at G1/S. Basal promoters dependent on the elements for Upstream Activating Sequence activity are inactive in Deltaswi6 yeast. Yeast were screened for mutations that activated MCB reporter genes in the absence of Swi6. The mutations identified a single complementation group. Functional cloning revealed the mutations were alleles of the TRR1 gene encoding thioredoxin reductase. Although deletion of TRR1 activated MCB reporter genes, high copy expression did not suppress reporter gene activity. The trr1 mutations strongly (20-fold) stimulated MCB- and SCB (Swi4/Swi6 cell cycle box)-containing reporter genes, but also weakly (3-fold) stimulated reporter genes that lacked these elements. The trr1 mutations did not affect the level or periodicity of three endogenous MCB gene mRNAs (TMP1, RNR1, and SWI4). Deletion of thioredoxin genes TRX1 and TRX2 recapitulated the stimulatory effect of trr1 mutations on MCB reporter gene activity. Conditions expected to oxidize thioredoxin (exposure to H2O2) induced MCB gene expression, whereas conditions expected to conserve thioredoxin (exposure to hydroxyurea) inhibited MCB gene expression. The results suggest that thioredoxin oxidation contributes to MCB element activation and suggest a link between thioredoxin-oxidizing processes such as ribonucleotide reduction and cell cycle-specific gene transcription.
George M. Carman - One of the best experts on this subject based on the ideXlab platform.
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Phosphatidate Phosphatase Plays Role in Zinc-mediated Regulation of Phospholipid Synthesis in Yeast
The Journal of biological chemistry, 2011Co-Authors: Aníbal Soto-cardalda, Florencia Pascual, Hyeon-son Choi, George M. CarmanAbstract:In the yeast Saccharomyces cerevisiae, the synthesis of phospholipids is coordinately regulated by mechanisms that control the homeostasis of the essential mineral zinc (Carman, G.M., and Han, G. S. (2007) Regulation of phospholipid synthesis in Saccharomyces cerevisiae by zinc depletion. Biochim. Biophys. Acta 1771, 322-330; Eide, D. J. (2009) Homeostatic and adaptive responses to zinc deficiency in Saccharomyces cerevisiae. J. Biol. Chem. 284, 18565-18569). The synthesis of phosphatidylcholine is balanced by the repression of CDP-diacylglycerol pathway enzymes and the induction of Kennedy pathway enzymes. PAH1-encoded phosphatidate phosphatase catalyzes the penultimate step in triacylglycerol synthesis, and the diacylglycerol generated in the reaction may also be used for phosphatidylcholine synthesis via the Kennedy pathway. In this work, we showed that the expression of PAH1-encoded phosphatidate phosphatase was induced by zinc deficiency through a mechanism that involved interaction of the Zap1p zinc-responsive transcription factor with putative Upstream Activating Sequence zinc-responsive elements in the PAH1 promoter. The pah1Δ mutation resulted in the derepression of the CHO1-encoded phosphatidylserine synthase (CDP-diacylglycerol pathway enzyme) and loss of the zinc-mediated regulation of the enzyme. Loss of phosphatidate phosphatase also resulted in the derepression of the CKI1-encoded choline kinase (Kennedy pathway enzyme) but decreased the synthesis of phosphatidylcholine when cells were deficient of zinc. This result confirmed the role phosphatidate phosphatase plays in phosphatidylcholine synthesis via the Kennedy pathway.
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Regulation of phospholipid synthesis in yeast by zinc.
Biochemical Society Transactions, 2005Co-Authors: George M. CarmanAbstract:The yeast Saccharomyces cerevisiae has the ability to cope with a variety of stress conditions (e.g. zinc deficiency) by regulating the expression of enzyme activities including those involved with phospholipid synthesis. Zinc is an essential mineral required for the growth and metabolism of S. cerevisiae . Depletion of zinc from the growth medium of wild-type cells results in alterations in phospholipid composition including an increase in PI (phosphatidylinositol) and a decrease in phosphatidylethanolamine. These changes can be attributed to an increase in PIS1 -encoded PI synthase activity and a decrease in the activities of several CDP-diacylglycerol pathway enzymes including the CHO1 -encoded PS (phosphatidylserine) synthase. The reduction in PS synthase in response to zinc depletion is due to a repression mechanism that involves the UAS INO (inositol Upstream Activating Sequence) element in the CHO1 promoter and the negative transcription factor Opi1p. These factors are also responsible for the inositol-mediated repression of CHO1 . This regulation may play an important role in allowing cells to adapt to zinc deficiency given the essential roles that phospholipids play in the structure and function of cellular membranes.
Roel A L Bovenberg - One of the best experts on this subject based on the ideXlab platform.
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synthetic control devices for gene regulation in penicillium chrysogenum
Microbial Cell Factories, 2019Co-Authors: Laszlo Mozsik, Zsofia Buttel, Roel A L Bovenberg, Arnold J M Driessen, Yvonne NygardAbstract:Orthogonal, synthetic control devices were developed for Penicillium chrysogenum, a model filamentous fungus and industrially relevant cell factory. In the synthetic transcription factor, the QF DNA-binding domain of the transcription factor of the quinic acid gene cluster of Neurospora crassa is fused to the VP16 activation domain. This synthetic transcription factor controls the expression of genes under a synthetic promoter containing quinic acid Upstream Activating Sequence (QUAS) elements, where it binds. A gene cluster may demand an expression tuned individually for each gene, which is a great advantage provided by this system. The control devices were characterized with respect to three of their main components: expression of the synthetic transcription factors, Upstream Activating Sequences, and the affinity of the DNA binding domain of the transcription factor to the Upstream Activating domain. This resulted in synthetic expression devices, with an expression ranging from hardly detectable to a level similar to that of highest expressed native genes. The versatility of the control device was demonstrated by fluorescent reporters and its application was confirmed by synthetically controlling the production of penicillin. The characterization of the control devices in microbioreactors, proved to give excellent indications for how the devices function in production strains and conditions. We anticipate that these well-characterized and robustly performing control devices can be widely applied for the production of secondary metabolites and other compounds in filamentous fungi.
Georges Rapoport - One of the best experts on this subject based on the ideXlab platform.
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two different mechanisms mediate catabolite repression of the bacillus subtilis levanase operon
Journal of Bacteriology, 1995Co-Authors: Isabelle Martinverstraete, Jorg Stulke, A Klier, Georges RapoportAbstract:There are two levels of control of the expression of the levanase operon in Bacillus subtilis: induction by fructose, which involves a positive regulator, LevR, and the fructose phosphotransferase system encoded by this operon (lev-PTS), and a global regulation, catabolite repression. The LevR activator interacts with its target, the Upstream Activating Sequence (UAS), to stimulate the transcription of the E sigma L complex bound at the "-12, -24" promoter. Levanase operon expression in the presence of glucose was tested in strains carrying a ccpA gene disruption or a ptsH1 mutation in which Ser-46 of HPr is replaced by Ala. In a levR+ inducible genetic background, the expression of the levanase operon was partially resistant to catabolite repression in both mutants, indicating that the CcpA repressor and the HPr-SerP protein are involved in the glucose control of this operon. In addition, a cis-acting catabolite-responsive element (CRE) of the levanase operon was identified and investigated by site-directed mutagenesis. The CRE Sequence TGAAAACGCTT(a)ACA is located between positions -50 and -36 from the transcriptional start site, between the UAS and the -12, -24 promoter. However, in a background constitutive for levanase, neither HPr, CcpA, nor CRE is involved in glucose repression, suggesting the existence of a different pathway of glucose regulation. Using truncated LevR proteins, we showed that this CcpA-independent pathway required the presence of the domain of LevR (amino acids 411 to 689) homologous to the BglG family of bacterial antiterminators.
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Interactions of Wild-type and Truncated LevR of Bacillus subtilis with the Upstream Activating Sequence of the Levanase Operon
Journal of molecular biology, 1994Co-Authors: Isabelle Martin-verstraete, Michel Débarbouilé, André Klier, Georges RapoportAbstract:Abstract Transcription of the levanase operon of Bacillus subtilis is controlled by LevR, an activator of the NifA/NtrC family of regulators. An Upstream Activating Sequence (UAS) located in a 16 bp palindromic structure has previously been characterized. LevR was overproduced in B. subtilis and interaction between the activator and the UAS was demonstrated by gel shift and footprint experiments. The LevR protein specifically binds to the two-halves of the palindromic structure centered at -125 bases Upstream from the transcriptional start site. In addition, footprint analysis suggests that LevR interacts with a third DNA region located at positions -90 to -80. To investigate the function of the different domains of the LevR activator, stop codons were introduced at various positions in the levR gene. The ability of the truncated LevR polypeptides to activate transcription, to respond to the inducer or to interact with the UAS was tested. The results obtained suggest that LevR is a multidomain protein. The amino-terminal part of the protein is required for DNA binding whereas the central domain allows the activation of transcription. The carboxy-terminal region is involved in the modulation of the LevR activity by the inducer.
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Mutagenesis of the Bacillus subtilis "-12, -24" promoter of the levanase operon and evidence for the existence of an Upstream Activating Sequence.
Journal of molecular biology, 1992Co-Authors: Isabelle Martin-verstraete, André Klier, Michel Débarbouillé, Georges RapoportAbstract:The levanase operon of Bacillus subtilis is controlled by RNA polymerase associated with sigma 54 factor and by the LevR activator that is homologous to the NifA/NtrC family of regulators. A "-12, -24" promoter is present at the appropriate distance from the transcription start site. The drastic down effect of base substitutions in the TGGCAC, TTGCA consensus Sequence on the expression of the levanase operon confirmed the involvement of the "-12, -24" region in promoter function. Deletion derivatives of the Upstream Sequence of the operon promoter were constructed using translational levD'-'lacZ fusions and were integrated as single copies at the amyE locus of the B. subtilis chromosome. A cis-acting DNA Sequence that is required for activation of the operon promoter by LevR was identified. This regulatory Sequence is about 50 base-pairs long and is centered 125 base-pairs Upstream from the transcription start site in a region containing a 16 base-pair palindromic structure. This region of dyad symmetry functions as a regulatory element when placed up to at least 600 base-pairs Upstream from the "-12, -24" promoter, although the efficacy of activation is lowered. Thus, in common with most sigma 54-dependent promoters, an Upstream Activating Sequence (UAS) is involved in the control of expression of the levanase operon. The isolation and characterization of eight mutations in the UAS region confirmed the importance of the palindromic structure in promoter activation. Moreover, the expression of the levanase operon was inhibited by placing the UAS in trans on a multicopy plasmid, probably through titration of the LevR polypeptide. In conclusion, the levanase promoter region can be divided into two regulatory Sequences: the "-12, -24" promoter recognized by the sigma 54 RNA polymerase holoenzyme and the UAS, an inverted repeat Sequence that is probably the LevR binding site.
