Zeitschriftenartikel: „Gal4/gal80“

1

Jiang, Fenglei, Benjamin R. Frey, Margery L. Evans, Jordan C. Friel und James E. Hopper. „Gene Activation by Dissociation of an Inhibitor from a Transcriptional Activation Domain“. Molecular and Cellular Biology 29, Nr. 20 (03.08.2009): 5604–10. http://dx.doi.org/10.1128/mcb.00632-09.

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ABSTRACT Gal4 is a prototypical eukaryotic transcriptional activator whose recruitment function is inhibited in the absence of galactose by the Gal80 protein through masking of its transcriptional activation domain (AD). A long-standing nondissociation model posits that galactose-activated Gal3 interacts with Gal4-bound Gal80 at the promoter, yielding a tripartite Gal3-Gal80-Gal4 complex with altered Gal80-Gal4 conformation to enable Gal4 AD activity. Some recent data challenge this model, whereas other recent data support the model. To address this controversy, we imaged fluorescent-protein-tagged Gal80, Gal4, and Gal3 in live cells containing a novel GAL gene array. We find that Gal80 rapidly dissociates from Gal4 in response to galactose. Importantly, this dissociation is Gal3 dependent and concurrent with Gal4-activated GAL gene expression. When galactose-triggered dissociation is followed by galactose depletion, preexisting Gal80 reassociates with Gal4, indicating that sequestration of Gal80 by Gal3 contributes to the observed Gal80-Gal4 dissociation. Moreover, the ratio of nuclear Gal80 to cytoplasmic Gal80 decreases in response to Gal80-Gal3 interaction. Taken together, these and other results provide strong support for a GAL gene switch model wherein Gal80 rapidly dissociates from Gal4 through a mechanism that involves sequestration of Gal80 by galactose-activated Gal3.

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2

Lue, N. F., D. I. Chasman, A. R. Buchman und R. D. Kornberg. „Interaction of GAL4 and GAL80 gene regulatory proteins in vitro“. Molecular and Cellular Biology 7, Nr. 10 (Oktober 1987): 3446–51. http://dx.doi.org/10.1128/mcb.7.10.3446-3451.1987.

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The GAL80 protein of Saccharomyces cerevisiae, synthesized in vitro, bound tightly to GAL4 protein and to a GAL4 protein-upstream activation sequence DNA complex, as shown by (i) coimmunoprecipitation of GAL4 and GAL80 proteins with anti-GAL4 antiserum, (ii) an electrophoretic mobility shift of a GAL4 protein-upstream activation sequence DNA complex upon the addition of GAL80 protein, and (iii) GAL4-dependent binding of GAL80 protein to upstream activation sequence DNA immobilized on Sepharose beads. Anti-GAL4 antisera were raised against a GAL4-URA3 fusion protein, which could be purified to homogeneity in a single step with the use of an affinity chromatographic procedure for the URA3 gene product.

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3

Lue, N. F., D. I. Chasman, A. R. Buchman und R. D. Kornberg. „Interaction of GAL4 and GAL80 gene regulatory proteins in vitro.“ Molecular and Cellular Biology 7, Nr. 10 (Oktober 1987): 3446–51. http://dx.doi.org/10.1128/mcb.7.10.3446.

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The GAL80 protein of Saccharomyces cerevisiae, synthesized in vitro, bound tightly to GAL4 protein and to a GAL4 protein-upstream activation sequence DNA complex, as shown by (i) coimmunoprecipitation of GAL4 and GAL80 proteins with anti-GAL4 antiserum, (ii) an electrophoretic mobility shift of a GAL4 protein-upstream activation sequence DNA complex upon the addition of GAL80 protein, and (iii) GAL4-dependent binding of GAL80 protein to upstream activation sequence DNA immobilized on Sepharose beads. Anti-GAL4 antisera were raised against a GAL4-URA3 fusion protein, which could be purified to homogeneity in a single step with the use of an affinity chromatographic procedure for the URA3 gene product.

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4

Salmeron, J. M., S. D. Langdon und S. A. Johnston. „Interaction between transcriptional activator protein LAC9 and negative regulatory protein GAL80“. Molecular and Cellular Biology 9, Nr. 7 (Juli 1989): 2950–56. http://dx.doi.org/10.1128/mcb.9.7.2950-2956.1989.

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In Saccharomyces cerevisiae, transcriptional activation mediated by the GAL4 regulatory protein is repressed in the absence of galactose by the binding of the GAL80 protein, an interaction that requires the carboxy-terminal 28 amino acids of GAL4. The homolog of GAL4 from Kluyveromyces lactis, LAC9, activates transcription in S. cerevisiae and is highly similar to GAL4 in its carboxyl terminus but is not repressed by wild-type levels of GAL80 protein. Here we show that GAL80 does repress LAC9-activated transcription in S. cerevisiae if overproduced. We sought to determine the molecular basis for the difference in the responses of the LAC9 and GAL4 proteins to GAL80. Our results indicate that this difference is due primarily to the fact that under wild-type conditions, the level of LAC9 protein in S. cerevisiae is much higher than that of GAL4, which suggests that LAC9 escapes GAL80-mediated repression by titration of GAL80 protein in vivo. The difference in response to GAL80 is not due to amino acid sequence differences between the LAC9 and GAL4 carboxyl termini. We discuss the implications of these results for the mechanism of galactose metabolism regulation in S. cerevisiae and K. lactis.

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Salmeron, J. M., S. D. Langdon und S. A. Johnston. „Interaction between transcriptional activator protein LAC9 and negative regulatory protein GAL80.“ Molecular and Cellular Biology 9, Nr. 7 (Juli 1989): 2950–56. http://dx.doi.org/10.1128/mcb.9.7.2950.

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In Saccharomyces cerevisiae, transcriptional activation mediated by the GAL4 regulatory protein is repressed in the absence of galactose by the binding of the GAL80 protein, an interaction that requires the carboxy-terminal 28 amino acids of GAL4. The homolog of GAL4 from Kluyveromyces lactis, LAC9, activates transcription in S. cerevisiae and is highly similar to GAL4 in its carboxyl terminus but is not repressed by wild-type levels of GAL80 protein. Here we show that GAL80 does repress LAC9-activated transcription in S. cerevisiae if overproduced. We sought to determine the molecular basis for the difference in the responses of the LAC9 and GAL4 proteins to GAL80. Our results indicate that this difference is due primarily to the fact that under wild-type conditions, the level of LAC9 protein in S. cerevisiae is much higher than that of GAL4, which suggests that LAC9 escapes GAL80-mediated repression by titration of GAL80 protein in vivo. The difference in response to GAL80 is not due to amino acid sequence differences between the LAC9 and GAL4 carboxyl termini. We discuss the implications of these results for the mechanism of galactose metabolism regulation in S. cerevisiae and K. lactis.

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6

Bhat, P. J., und J. E. Hopper. „Overproduction of the GAL1 or GAL3 protein causes galactose-independent activation of the GAL4 protein: evidence for a new model of induction for the yeast GAL/MEL regulon“. Molecular and Cellular Biology 12, Nr. 6 (Juni 1992): 2701–7. http://dx.doi.org/10.1128/mcb.12.6.2701-2707.1992.

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The transcriptional activation function of the Saccharomyces cerevisiae GAL4 protein is modulated by the GAL80 and GAL3 proteins. In the absence of galactose, GAL80 inhibits the function of GAL4, presumably by direct binding to the GAL4 protein. The presence of galactose triggers the relief of the GAL80 block. The key to this relief is the GAL3 protein. How GAL3 and galactose activate GAL4 is not understood, but the long-standing notion has been that a galactose derivative formed by catalytic activity of GAL3 is the inducer that interacts with GAL80 or the GAL80-GAL4 complex. Here we report that overproduction of the GAL3 protein causes constitutive expression of GAL/MEL genes in the absence of exogenous galactose. Overproduction of the GAL1 protein (galactokinase) also causes constitutivity, consistent with the observations that GAL1 is strikingly similar in amino acid sequence to GAL3 and has GAL3-like induction activity. Cells lacking the GAL10-encoded UDP-galactose-UDP-glucose epimerase retained the constitutivity response to overproduction of GAL3, making it unlikely that constitutivity is due to endogenously produced galactose. A galactose-independent mechanism of constitutivity is further indicated by the inducing properties of two newly created galactokinaseless alleles of GAL1. On the basis of these data, we propose a new model for galactose-induced activation of the GAL4 protein. This model invokes galactose-activation of the GAL3 and GAL1 proteins which in turn elicit an alteration of the GAL80-GAL4 complex to activate GAL4. This model is consistent with all the known features of the system and has important implications for manipulating GAL4-dependent transcriptional activation in vitro.

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7

Bhat, P. J., und J. E. Hopper. „Overproduction of the GAL1 or GAL3 protein causes galactose-independent activation of the GAL4 protein: evidence for a new model of induction for the yeast GAL/MEL regulon.“ Molecular and Cellular Biology 12, Nr. 6 (Juni 1992): 2701–7. http://dx.doi.org/10.1128/mcb.12.6.2701.

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The transcriptional activation function of the Saccharomyces cerevisiae GAL4 protein is modulated by the GAL80 and GAL3 proteins. In the absence of galactose, GAL80 inhibits the function of GAL4, presumably by direct binding to the GAL4 protein. The presence of galactose triggers the relief of the GAL80 block. The key to this relief is the GAL3 protein. How GAL3 and galactose activate GAL4 is not understood, but the long-standing notion has been that a galactose derivative formed by catalytic activity of GAL3 is the inducer that interacts with GAL80 or the GAL80-GAL4 complex. Here we report that overproduction of the GAL3 protein causes constitutive expression of GAL/MEL genes in the absence of exogenous galactose. Overproduction of the GAL1 protein (galactokinase) also causes constitutivity, consistent with the observations that GAL1 is strikingly similar in amino acid sequence to GAL3 and has GAL3-like induction activity. Cells lacking the GAL10-encoded UDP-galactose-UDP-glucose epimerase retained the constitutivity response to overproduction of GAL3, making it unlikely that constitutivity is due to endogenously produced galactose. A galactose-independent mechanism of constitutivity is further indicated by the inducing properties of two newly created galactokinaseless alleles of GAL1. On the basis of these data, we propose a new model for galactose-induced activation of the GAL4 protein. This model invokes galactose-activation of the GAL3 and GAL1 proteins which in turn elicit an alteration of the GAL80-GAL4 complex to activate GAL4. This model is consistent with all the known features of the system and has important implications for manipulating GAL4-dependent transcriptional activation in vitro.

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8

Parthun, M. R., und J. A. Jaehning. „A transcriptionally active form of GAL4 is phosphorylated and associated with GAL80“. Molecular and Cellular Biology 12, Nr. 11 (November 1992): 4981–87. http://dx.doi.org/10.1128/mcb.12.11.4981-4987.1992.

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The GAL4 activator and GAL80 repressor proteins regulate the expression of yeast genes in response to galactose. A complex of the two proteins isolated from glucose-grown cells is inactive in an in vitro transcription reaction but binds DNA and blocks activation by the GAL4-VP16 chimeric activator. The complex purified from galactose-grown cells contains a mixture of phosphorylated and unphosphorylated forms of GAL4. The galactose-induced form of GAL4 activates in vitro transcription to levels similar to those seen with GAL4-VP16. The induced GAL4 complex is indistinguishable in size and apparent shape from the uninduced complex, consistent with a continued association with GAL80. These results confirm in vivo analyses that correlate GAL4 phosphorylation with galactose induction and support a model of transcriptional activation that does not require GAL80 dissociation.

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9

Parthun, M. R., und J. A. Jaehning. „A transcriptionally active form of GAL4 is phosphorylated and associated with GAL80.“ Molecular and Cellular Biology 12, Nr. 11 (November 1992): 4981–87. http://dx.doi.org/10.1128/mcb.12.11.4981.

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The GAL4 activator and GAL80 repressor proteins regulate the expression of yeast genes in response to galactose. A complex of the two proteins isolated from glucose-grown cells is inactive in an in vitro transcription reaction but binds DNA and blocks activation by the GAL4-VP16 chimeric activator. The complex purified from galactose-grown cells contains a mixture of phosphorylated and unphosphorylated forms of GAL4. The galactose-induced form of GAL4 activates in vitro transcription to levels similar to those seen with GAL4-VP16. The induced GAL4 complex is indistinguishable in size and apparent shape from the uninduced complex, consistent with a continued association with GAL80. These results confirm in vivo analyses that correlate GAL4 phosphorylation with galactose induction and support a model of transcriptional activation that does not require GAL80 dissociation.

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10

Salmeron, J. M., K. K. Leuther und S. A. Johnston. „GAL4 mutations that separate the transcriptional activation and GAL80-interactive functions of the yeast GAL4 protein.“ Genetics 125, Nr. 1 (01.05.1990): 21–27. http://dx.doi.org/10.1093/genetics/125.1.21.

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Abstract The carboxy-terminal 28 amino acids of the Saccharomyces cerevisiae transcriptional activator protein GAL4 execute two functions--transcriptional activation and interaction with the negative regulatory protein, GAL80. Here we demonstrate that these two functions are separable by single amino acid changes within this region. We determined the sequences of four GAL4C-mutations, and characterized the abilities of the encoded GAL4C proteins to activate transcription of the galactose/melibiose regulon in the presence of GAL80 and superrepressible GAL80S alleles. One of the GAL4C mutations can be compensated by a specific GAL80S mutation, resulting in a wild-type phenotype. These results support the idea that while the GAL4 activation function tolerates at least minor alterations in the GAL4 carboxyl terminus, the GAL80-interactive function is highly sequence-specific and sensitive even to single amino acid alterations. They also argue that the GAL80S mutations affect the affinity of GAL80 for GAL4, and not the ability of GAL80 to bind inducer.

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Barwell, Taylor, Brian DeVeale, Luc Poirier, Jie Zheng, Frederique Seroude und Laurent Seroude. „Regulating the UAS/GAL4 system in adultDrosophilawith Tet-off GAL80 transgenes“. PeerJ 5 (14.12.2017): e4167. http://dx.doi.org/10.7717/peerj.4167.

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The UAS/GAL4 system is the most used method inDrosophila melanogasterfor directing the expression of a gene of interest to a specific tissue. However, the ability to control the temporal activity of GAL4 with this system is very limited. This study constructed and characterized Tet-off GAL80 transgenes designed to allow temporal control of GAL4 activity in aging adult muscles. By placing GAL80 under the control of a Tet-off promoter, GAL4 activity is regulated by the presence or absence of tetracycline in the diet. Almost complete inhibition of the expression of UAS transgenes during the pre-adult stages of the life cycle is obtained by using four copies and two types of Tet-off GAL80 transgenes. Upon treatment of newly emerged adults with tetracycline, induction of GAL4 activity is observed but the level of induction is influenced by the concentration of the inducer, the age, the sex and the anatomical location of the expression. The inhibition of GAL4 activity and the maintenance of induced expression are altered in old animals. This study reveals that the repressive ability of GAL80 is affected by the age and sex of the animal which is a major limitation to regulate gene expression with GAL80 in agedDrosophila.

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12

Zenke, F. T., W. Zachariae, A. Lunkes und K. D. Breunig. „Gal80 proteins of Kluyveromyces lactis and Saccharomyces cerevisiae are highly conserved but contribute differently to glucose repression of the galactose regulon“. Molecular and Cellular Biology 13, Nr. 12 (Dezember 1993): 7566–76. http://dx.doi.org/10.1128/mcb.13.12.7566-7576.1993.

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We cloned the GAL80 gene encoding the negative regulator of the transcriptional activator Gal4 (Lac9) from the yeast Kluyveromyces lactis. The deduced amino acid sequence of K. lactis GAL80 revealed a strong structural conservation between K. lactis Gal80 and the homologous Saccharomyces cerevisiae protein, with an overall identity of 60% and two conserved blocks with over 80% identical residues. K. lactis gal80 disruption mutants show constitutive expression of the lactose/galactose metabolic genes, confirming that K. lactis Gal80 functions in essentially in the same way as does S. cerevisiae Gal80, blocking activation by the transcriptional activator Lac9 (K. lactis Gal4) in the absence of an inducing sugar. However, in contrast to S. cerevisiae, in which Gal4-dependent activation is strongly inhibited by glucose even in a gal80 mutant, glucose repressibility is almost completely lost in gal80 mutants of K. lactis. Indirect evidence suggests that this difference in phenotype is due to a higher activator concentration in K. lactis which is able to overcome glucose repression. Expression of the K. lactis GAL80 gene is controlled by Lac9. Two high-affinity binding sites in the GAL80 promoter mediate a 70-fold induction by galactose and hence negative autoregulation by Gal80. Gal80 in turn not only controls Lac9 activity but also has a moderate influence on its rate of synthesis. Thus, a feedback control mechanism exists between the positive and negative regulators. By mutating the Lac9 binding sites of the GAL80 promoter, we could show that induction of GAL80 is required to prevent activation of the lactose/galactose regulon in glycerol or glucose plus galactose, whereas the noninduced level of Gal80 is sufficient to completely block Lac9 function in glucose.

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Zenke, F. T., W. Zachariae, A. Lunkes und K. D. Breunig. „Gal80 proteins of Kluyveromyces lactis and Saccharomyces cerevisiae are highly conserved but contribute differently to glucose repression of the galactose regulon.“ Molecular and Cellular Biology 13, Nr. 12 (Dezember 1993): 7566–76. http://dx.doi.org/10.1128/mcb.13.12.7566.

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We cloned the GAL80 gene encoding the negative regulator of the transcriptional activator Gal4 (Lac9) from the yeast Kluyveromyces lactis. The deduced amino acid sequence of K. lactis GAL80 revealed a strong structural conservation between K. lactis Gal80 and the homologous Saccharomyces cerevisiae protein, with an overall identity of 60% and two conserved blocks with over 80% identical residues. K. lactis gal80 disruption mutants show constitutive expression of the lactose/galactose metabolic genes, confirming that K. lactis Gal80 functions in essentially in the same way as does S. cerevisiae Gal80, blocking activation by the transcriptional activator Lac9 (K. lactis Gal4) in the absence of an inducing sugar. However, in contrast to S. cerevisiae, in which Gal4-dependent activation is strongly inhibited by glucose even in a gal80 mutant, glucose repressibility is almost completely lost in gal80 mutants of K. lactis. Indirect evidence suggests that this difference in phenotype is due to a higher activator concentration in K. lactis which is able to overcome glucose repression. Expression of the K. lactis GAL80 gene is controlled by Lac9. Two high-affinity binding sites in the GAL80 promoter mediate a 70-fold induction by galactose and hence negative autoregulation by Gal80. Gal80 in turn not only controls Lac9 activity but also has a moderate influence on its rate of synthesis. Thus, a feedback control mechanism exists between the positive and negative regulators. By mutating the Lac9 binding sites of the GAL80 promoter, we could show that induction of GAL80 is required to prevent activation of the lactose/galactose regulon in glycerol or glucose plus galactose, whereas the noninduced level of Gal80 is sufficient to completely block Lac9 function in glucose.

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Webster, Sophia H., Michael R. Vella und Maxwell J. Scott. „Development and testing of a novel killer–rescue self-limiting gene drive system in Drosophila melanogaster“. Proceedings of the Royal Society B: Biological Sciences 287, Nr. 1925 (15.04.2020): 20192994. http://dx.doi.org/10.1098/rspb.2019.2994.

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Here we report the development and testing of a novel self-limiting gene drive system, Killer–Rescue (K–R), in Drosophila melanogaster . This system is composed of an autoregulated Gal4 Killer (K) and a Gal4-activated Gal80 Rescue (R). Overexpression of Gal4 is lethal, but in the presence of R activation of Gal80 leads to much lower levels of Gal4 and rescue of lethality. We demonstrate that with a single 2 : 1 engineered to wild-type release, K drives R through the population and after nine generations, more than 98% of the population carry R and less than 2% of the population are wild-type flies. We discuss how this simple K–R gene drive system may be readily adapted for population replacement in a human health pest, Aedes aegypti , or for population suppression in an agricultural pest, Drosophila suzukii .

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Chasman, D. I., und R. D. Kornberg. „GAL4 protein: purification, association with GAL80 protein, and conserved domain structure“. Molecular and Cellular Biology 10, Nr. 6 (Juni 1990): 2916–23. http://dx.doi.org/10.1128/mcb.10.6.2916-2923.1990.

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Expression of the yeast Saccharomyces cerevisiae GAL4 protein under its own (galactose-inducible) control gave 5 to 10 times the level of protein observed when the GAL4 gene was on a high-copy plasmid. Purification of GAL4 by a procedure including affinity chromatography on a GAL4-binding DNA column yielded not only GAL4 but also a second protein, shown to be GAL80 by its reaction with an antipeptide antibody. Sequence comparisons of GAL4 and other members of a family of proteins sharing homologous cysteine finger motifs identified an additional region of homology in the middle of these proteins shown by genetic analysis to be important for GAL4 function. GAL4 could be cleaved proteolytically at the boundary of the conserved region, defining internal and carboxy-terminal folded domains.

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Chasman, D. I., und R. D. Kornberg. „GAL4 protein: purification, association with GAL80 protein, and conserved domain structure.“ Molecular and Cellular Biology 10, Nr. 6 (Juni 1990): 2916–23. http://dx.doi.org/10.1128/mcb.10.6.2916.

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Expression of the yeast Saccharomyces cerevisiae GAL4 protein under its own (galactose-inducible) control gave 5 to 10 times the level of protein observed when the GAL4 gene was on a high-copy plasmid. Purification of GAL4 by a procedure including affinity chromatography on a GAL4-binding DNA column yielded not only GAL4 but also a second protein, shown to be GAL80 by its reaction with an antipeptide antibody. Sequence comparisons of GAL4 and other members of a family of proteins sharing homologous cysteine finger motifs identified an additional region of homology in the middle of these proteins shown by genetic analysis to be important for GAL4 function. GAL4 could be cleaved proteolytically at the boundary of the conserved region, defining internal and carboxy-terminal folded domains.

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Blüher, Doreen, Annekathrin Reinhardt-Tews, Martin Hey, Hauke Lilie, Ralph Golbik, Karin D. Breunig und Alexander Anders. „An ancient oxidoreductase making differential use of its cofactors“. Biological Chemistry 395, Nr. 7-8 (01.07.2014): 855–69. http://dx.doi.org/10.1515/hsz-2014-0152.

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Abstract Many transcription factors contribute to cellular homeostasis by integrating multiple signals. Signaling via the yeast Gal80 protein, a negative regulator of the prototypic transcription activator Gal4, is primarily regulated by galactose. ScGal80 from Saccharomyces cerevisiae has been reported to bind NAD(P). Here, we show that the ability to bind these ligands is conserved in KlGal80, a Gal80 homolog from the distantly related yeast Kluyveromyces lactis. However, the homologs apparently have diverged with respect to response to the dinucleotide. Strikingly, ScGal80 binds NAD(P) and NAD(P)H with more than 50-fold higher affinity than KlGal80. In contrast to ScGal80, where NAD is neutral, NAD and NADP have a negative effect in KlGal80 on its interaction with a KlGal4-peptide in vitro. Swapping a loop in the NAD(P) binding Rossmann-fold of ScGal80 into KlGal80 increases the affinity for NAD(P) and has a significant impact on KlGal4 regulation in vivo. Apparently, dinucleotide binding allows coupling of the metabolic state of the cell to regulation of the GAL/LAC genes. The particular sequences involved in binding determine how exactly the metabolic state is sensed and integrated by Gal80 to regulate Gal4.

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Lettow, Julia, Rasha Aref und Hans-Joachim Schüller. „Transcriptional repressor Gal80 recruits corepressor complex Cyc8–Tup1 to structural genes of the Saccharomyces cerevisiae GAL regulon“. Current Genetics 68, Nr. 1 (07.10.2021): 115–24. http://dx.doi.org/10.1007/s00294-021-01215-x.

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AbstractUnder non-inducing conditions (absence of galactose), yeast structural genes of the GAL regulon are repressed by Gal80, preventing interaction of Gal4 bound to UASGAL promoter motifs with general factors of the transcriptional machinery. In this work, we show that Gal80 is also able to interact with histone deacetylase-recruiting corepressor proteins Cyc8 and Tup1, indicating an additional mechanism of gene repression. This is supported by our demonstration that a lexA–Gal80 fusion efficiently mediates repression of a reporter gene with an upstream lexA operator sequence. Corepressor interaction and in vivo gene repression could be mapped to a Gal80 minimal domain of 65 amino acids (aa 81-145). Site-directed mutagenesis of selected residues within this domain showed that a cluster of aromatic-hydrophobic amino acids (YLFV, aa 118-121) is important, although not solely responsible, for gene repression. Using chromatin immunoprecipitation, Cyc8 and Tup1 were shown to be present at the GAL1 promoter in a wild-type strain but not in a gal80 mutant strain under non-inducing (derepressing) growth conditions. Expression of a GAL1–lacZ fusion was elevated in a tup1 mutant (but not in a cyc8 mutant) grown in derepressing medium, indicating that Tup1 may be mainly responsible for this second mechanism of Gal80-dependent gene repression.

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Weaver, Lesley N., Tianlu Ma und Daniela Drummond-Barbosa. „Analysis of Gal4 Expression Patterns in Adult Drosophila Females“. G3: Genes|Genomes|Genetics 10, Nr. 11 (11.09.2020): 4147–58. http://dx.doi.org/10.1534/g3.120.401676.

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Precise genetic manipulation of specific cell types or tissues to pinpoint gene function requirement is a critical step in studies aimed at unraveling the intricacies of organismal physiology. Drosophila researchers heavily rely on the UAS/Gal4/Gal80 system for tissue-specific manipulations; however, it is often unclear whether the reported Gal4 expression patterns are indeed specific to the tissue of interest such that experimental results are not confounded by secondary sites of Gal4 expression. Here, we surveyed the expression patterns of commonly used Gal4 drivers in adult Drosophila female tissues under optimal conditions and found that multiple drivers have unreported secondary sites of expression beyond their published cell type/tissue expression pattern. These results underscore the importance of thoroughly characterizing Gal4 tools as part of a rigorous experimental design that avoids potential misinterpretation of results as we strive for understanding how the function of a specific gene/pathway in one tissue contributes to whole-body physiology.

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Leuther, K., und S. Johnston. „Nondissociation of GAL4 and GAL80 in vivo after galactose induction“. Science 256, Nr. 5061 (29.05.1992): 1333–35. http://dx.doi.org/10.1126/science.1598579.

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Selleck, S. B., und J. E. Majors. „In vivo DNA-binding properties of a yeast transcription activator protein“. Molecular and Cellular Biology 7, Nr. 9 (September 1987): 3260–67. http://dx.doi.org/10.1128/mcb.7.9.3260-3267.1987.

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UV light can serve as a molecular probe to identify DNA-protein interactions at nucleotide level resolution from intact yeast cells. We have used the photofootprinting technique to determine during which of three regulated states (uninduced, induced, and catabolite repressed) the transcriptional activator protein encoded by GAL4 binds to its recognition sites within the GAL1-GAL10 upstream activating sequence (UASG). GAL4 protein is bound to at least four, and probably five, related sequence blocks within UASG under both induced and uninduced states. GAL4-dependent photofootprints are lost under conditions of catabolite repression. We observed no footprint patterns unique to catabolite-repressed cells, which suggests that binding of a repressor to the UASG is not involved in this process. Photofootprints of the GAL10 TATA element are strictly correlated with transcription: uninduced, catabolite-repressed, and delta gal4 cells exhibit footprints characteristic of the inactive promoter; induced and delta gal80 cells, which express GAL10 constitutively, display footprints unique to the actively transcribed gene.

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Selleck, S. B., und J. E. Majors. „In vivo DNA-binding properties of a yeast transcription activator protein.“ Molecular and Cellular Biology 7, Nr. 9 (September 1987): 3260–67. http://dx.doi.org/10.1128/mcb.7.9.3260.

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UV light can serve as a molecular probe to identify DNA-protein interactions at nucleotide level resolution from intact yeast cells. We have used the photofootprinting technique to determine during which of three regulated states (uninduced, induced, and catabolite repressed) the transcriptional activator protein encoded by GAL4 binds to its recognition sites within the GAL1-GAL10 upstream activating sequence (UASG). GAL4 protein is bound to at least four, and probably five, related sequence blocks within UASG under both induced and uninduced states. GAL4-dependent photofootprints are lost under conditions of catabolite repression. We observed no footprint patterns unique to catabolite-repressed cells, which suggests that binding of a repressor to the UASG is not involved in this process. Photofootprints of the GAL10 TATA element are strictly correlated with transcription: uninduced, catabolite-repressed, and delta gal4 cells exhibit footprints characteristic of the inactive promoter; induced and delta gal80 cells, which express GAL10 constitutively, display footprints unique to the actively transcribed gene.

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Uemura, H., und Y. Jigami. „Role of GCR2 in transcriptional activation of yeast glycolytic genes“. Molecular and Cellular Biology 12, Nr. 9 (September 1992): 3834–42. http://dx.doi.org/10.1128/mcb.12.9.3834-3842.1992.

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The Saccharomyces cerevisiae GCR2 gene affects expression of most of the glycolytic genes. We report the nucleotide sequence of GCR2, which can potentially encode a 58,061-Da protein. There is a small cluster of asparagines near the center and a C-terminal region that would be highly charged but overall neutral. Fairly homologous regions were found between Gcr2 and Gcr1 proteins. To test potential interactions, the genetic method of S. Fields and O. Song (Nature [London] 340:245-246, 1989), which uses protein fusions of candidate gene products with, respectively, the N-terminal DNA-binding domain of Gal4 and the C-terminal activation domain II, assessing restoration of Gal4 function, was used. In a delta gal4 delta gal80 strain, double transformation by plasmids containing, respectively, a Gal4 (transcription-activating region)/Gcr1 fusion and a Gal4 (DNA-binding domain)/Gcr2 fusion activated lacZ expression from an integrated GAL1/lacZ fusion, indicating reconstitution of functional Gal4 through the interaction of Gcr1 and Gcr2 proteins. The Gal4 (transcription-activating region)/Gcr1 fusion protein alone complemented the defects of both gcr1 and gcr2 strains. Furthermore, a Rap1/Gcr2 fusion protein partially complemented the defects of gcr1 strains. These results suggest that Gcr2 has transcriptional activation activity and that the GCR1 and GCR2 gene products function together.

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Uemura, H., und Y. Jigami. „Role of GCR2 in transcriptional activation of yeast glycolytic genes.“ Molecular and Cellular Biology 12, Nr. 9 (September 1992): 3834–42. http://dx.doi.org/10.1128/mcb.12.9.3834.

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The Saccharomyces cerevisiae GCR2 gene affects expression of most of the glycolytic genes. We report the nucleotide sequence of GCR2, which can potentially encode a 58,061-Da protein. There is a small cluster of asparagines near the center and a C-terminal region that would be highly charged but overall neutral. Fairly homologous regions were found between Gcr2 and Gcr1 proteins. To test potential interactions, the genetic method of S. Fields and O. Song (Nature [London] 340:245-246, 1989), which uses protein fusions of candidate gene products with, respectively, the N-terminal DNA-binding domain of Gal4 and the C-terminal activation domain II, assessing restoration of Gal4 function, was used. In a delta gal4 delta gal80 strain, double transformation by plasmids containing, respectively, a Gal4 (transcription-activating region)/Gcr1 fusion and a Gal4 (DNA-binding domain)/Gcr2 fusion activated lacZ expression from an integrated GAL1/lacZ fusion, indicating reconstitution of functional Gal4 through the interaction of Gcr1 and Gcr2 proteins. The Gal4 (transcription-activating region)/Gcr1 fusion protein alone complemented the defects of both gcr1 and gcr2 strains. Furthermore, a Rap1/Gcr2 fusion protein partially complemented the defects of gcr1 strains. These results suggest that Gcr2 has transcriptional activation activity and that the GCR1 and GCR2 gene products function together.

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Suster, Maximiliano L., Laurent Seugnet, Michael Bate und Marla B. Sokolowski. „Refining GAL4-driven transgene expression inDrosophila with a GAL80 enhancer-trap“. genesis 39, Nr. 4 (2004): 240–45. http://dx.doi.org/10.1002/gene.20051.

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26

Zhao, Xianguo, Xingzhuo Yang, Pengfei Lv, Yuetong Xu, Xiangfeng Wang, Zhangwu Zhao und Juan Du. „Polycombregulates circadian rhythms inDrosophilain clock neurons“. Life Science Alliance 7, Nr. 1 (01.11.2023): e202302140. http://dx.doi.org/10.26508/lsa.202302140.

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Circadian rhythms are essential physiological feature for most living organisms. Previous studies have shown that epigenetic regulation plays a crucial role. There is a knowledge gap in the chromatin state of some key clock neuron clusters. In this study, we show that circadian rhythm is affected by the epigenetic regulatorPolycomb(Pc) within theDrosophilaclock neurons. To investigate the molecular mechanisms underlying the roles ofPcin these clock neuron clusters, we use targeted DamID (TaDa) to identify genes significantly bound by Pc in the neurons marked byC929-Gal4(including l-LNvs cluster),R6-Gal4(including s-LNvs cluster),R18H11-Gal4(including DN1 cluster), andDVpdf-Gal4,pdf-Gal80(including LNds cluster). It shows that Pc binds to the genes involved in the circadian rhythm pathways, arguing a direct role forPcin regulating circadian rhythms through specific clock genes. This study shows the identification of Pc targets in the clock neuron clusters, providing potential resource for understanding the regulatory mechanisms of circadian rhythms by the PcG complex. Thus, this study provided an example for epigenetic regulation of adult behavior.

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Eliason, Jessica, Ali Afify, Christopher Potter und lchiro Matsumura. „A GAL80 Collection To Inhibit GAL4 Transgenes in Drosophila Olfactory Sensory Neurons“. G3: Genes|Genomes|Genetics 8, Nr. 11 (27.09.2018): 3661–68. http://dx.doi.org/10.1534/g3.118.200569.

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Smith, Brittany N., Arash M. Ghazanfari, Rudolf A. Bohm, William P. Welch, Bing Zhang und John P. Masly. „A Flippase-Mediated GAL80/GAL4 Intersectional Resource for Dissecting Appendage Development inDrosophila“. G3: Genes|Genomes|Genetics 5, Nr. 10 (13.08.2015): 2105–12. http://dx.doi.org/10.1534/g3.115.019810.

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Faucherre, Adèle, und Hernán López-Schier. „Delaying Gal4-Driven Gene Expression in the Zebrafish with Morpholinos and Gal80“. PLoS ONE 6, Nr. 1 (26.01.2011): e16587. http://dx.doi.org/10.1371/journal.pone.0016587.

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Ma, Jun, und Mark Ptashne. „The carboxy-terminal 30 amino acids of GAL4 are recognized by GAL80“. Cell 50, Nr. 1 (Juli 1987): 137–42. http://dx.doi.org/10.1016/0092-8674(87)90670-2.

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Melcher, K., und S. A. Johnston. „GAL4 interacts with TATA-binding protein and coactivators.“ Molecular and Cellular Biology 15, Nr. 5 (Mai 1995): 2839–48. http://dx.doi.org/10.1128/mcb.15.5.2839.

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A major goal in understanding eukaryotic gene regulation is to identify the target(s) of transcriptional activators. Efforts to date have pointed to various candidates. Here we show that a 34-amino-acid peptide from the carboxy terminus of GAL4 is a strong activation domain (AD) and retains at least four proteins from a crude extract: the negative regulator GAL80, the TATA-binding protein (TBP), and the putative coactivators SUG1 and ADA2. TFIIB was not retained. Concentrating on TBP, we demonstrate in in vitro binding assays that its interaction with the AD is specific, direct, and salt stable up to at least 1.6 M NaCl. The effects of mutations in the GAL4 AD on transcriptional activation in vivo correlate with their affinities to TBP. A point mutation (L114K) in yeast TBP, which has been shown to compromise the mutant protein in both binding to the VP16 AD domain and activated transcription in vitro, reduces the affinity to the GAL4 AD to the same degree as to the VP16 AD. This suggests that these two prototypic activators make similar contacts with TBP.

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Casas-Tintó, Sergio, Mercedes Arnés und Alberto Ferrús. „Drosophila enhancer-Gal4 lines show ectopic expression during development“. Royal Society Open Science 4, Nr. 3 (März 2017): 170039. http://dx.doi.org/10.1098/rsos.170039.

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In Drosophila melanogaster the most widely used technique to drive gene expression is the binary UAS/Gal4 system. We show here that a set of nervous system specific enhancers ( elav , D42/ Toll-6 , OK6/ RapGAP1 ) display ectopic activity in epithelial tissues during development, which is seldom considered in experimental studies. This ectopic activity is variable, unstable and influenced by the primary sequence of the enhancer and the insertion site in the chromosome. In addition, the ectopic activity is independent of the protein expressed, Gal4, as it is reproduced also with the expression of Gal80. Another enhancer, LN2 from the sex lethal ( Sxl ) gene, shows sex-dependent features in its ectopic expression. Feminization of LN2 expressing males does not alter the male specific pattern indicating that the sexual dimorphism of LN2 expression is an intrinsic feature of this enhancer. Other X chromosome enhancers corresponding to genes not related to sex determination do not show sexual dimorphism in their ectopic expressions. Although variable and unstable, the ectopic activation of enhancer-Gal4 lines seems to be regulated in terms of tissue and intensity. To characterize the full domain of expression of enhancer-Gal4 constructs is relevant for the design of transgenic animal models and biotechnology tools, as well as for the correct interpretation of developmental and behavioural studies in which Gal4 lines are used.

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Oh, D., und J. E. Hopper. „Transcription of a yeast phosphoglucomutase isozyme gene is galactose inducible and glucose repressible“. Molecular and Cellular Biology 10, Nr. 4 (April 1990): 1415–22. http://dx.doi.org/10.1128/mcb.10.4.1415-1422.1990.

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The Saccharomyces cerevisiae GAL5 (PGM2) gene was isolated and shown to encode the major isozyme of phosphoglucomutase. Northern (RNA) blot hybridization revealed that the GAL5 transcript level increased three- to fourfold in response to galactose and was severely repressed in response to glucose. Total cellular phosphoglucomutase activity was likewise responsive to galactose and to glucose, and this responsiveness was found to be due primarily to variation in the activity of the major isozyme of phosphoglucomutase. These results imply that the major and minor isozymes of phosphoglucomutase have distinct roles in yeast cells. The galactose inducibility of GAL5 was found to be under the control of the GAL4, GAL80, and GAL3 genes. In striking contrast to other galactose-inducible genes, the GAL5 gene exhibited an unusually high GAL4-independent basal level of expression. These results have implications for metabolic trafficking.

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Oh, D., und J. E. Hopper. „Transcription of a yeast phosphoglucomutase isozyme gene is galactose inducible and glucose repressible.“ Molecular and Cellular Biology 10, Nr. 4 (April 1990): 1415–22. http://dx.doi.org/10.1128/mcb.10.4.1415.

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The Saccharomyces cerevisiae GAL5 (PGM2) gene was isolated and shown to encode the major isozyme of phosphoglucomutase. Northern (RNA) blot hybridization revealed that the GAL5 transcript level increased three- to fourfold in response to galactose and was severely repressed in response to glucose. Total cellular phosphoglucomutase activity was likewise responsive to galactose and to glucose, and this responsiveness was found to be due primarily to variation in the activity of the major isozyme of phosphoglucomutase. These results imply that the major and minor isozymes of phosphoglucomutase have distinct roles in yeast cells. The galactose inducibility of GAL5 was found to be under the control of the GAL4, GAL80, and GAL3 genes. In striking contrast to other galactose-inducible genes, the GAL5 gene exhibited an unusually high GAL4-independent basal level of expression. These results have implications for metabolic trafficking.

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Landis, Jessie E., Kevin Sungu, Hannah Sipe und Jeffrey M. Copeland. „RNAi of Complex I and V of the electron transport chain in glutamate neurons extends life span, increases sleep, and decreases locomotor activity in Drosophila melanogaster“. PLOS ONE 18, Nr. 6 (15.06.2023): e0286828. http://dx.doi.org/10.1371/journal.pone.0286828.

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RNAi targeting the electron transport chain has been proven to prolong life span in many different species, and experiments specifically with Drosophila melanogaster and Caenorhabditis elegans have shown a distinct role for neurons. To determine which subset of neurons is implicated in this life span extension, we used the GAL4/UAS system to activate RNAi against genes of Complex I and Complex V. We found life span extension of 18–24% with two glutamate neuron (D42 and VGlut) GAL4 lines. We used the GAL80 system to determine if the overlapping set of glutamate neurons in these two GAL4 lines imparts the life span extension. Limiting GAL4 activity to non-VGlut glutamate neurons in the D42 background failed to extend life span, suggesting that glutamate neurons have an important role in aging. Interestingly, RNAi of the electron transport chain in D42 glutamate neurons also caused an increase in daytime and nighttime sleep and a decrease in nighttime locomotor activity. Changes to sleep patterns and prolonged life span were not accompanied by any changes in female fertility or response to starvation. Our findings demonstrate that a small subset of neurons can control life span, and further studies can look into the contributions made by glutamate neurons.

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Lohr, D., und J. Lopez. „GAL4/GAL80-dependent Nucleosome Disruption/Deposition on the Upstream Regions of the YeastGAL1-10andGAL80Genes“. Journal of Biological Chemistry 270, Nr. 46 (17.11.1995): 27671–78. http://dx.doi.org/10.1074/jbc.270.46.27671.

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Anders, Alexander, Hauke Lilie, Kathlen Franke, Lutz Kapp, Jörg Stelling, Ernst D. Gilles und Karin D. Breunig. „The Galactose Switch inKluyveromyces lactisDepends on Nuclear Competition between Gal4 and Gal1 for Gal80 Binding“. Journal of Biological Chemistry 281, Nr. 39 (25.07.2006): 29337–48. http://dx.doi.org/10.1074/jbc.m604271200.

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Zachariae, W., und K. D. Breunig. „Expression of the transcriptional activator LAC9 (KlGAL4) in Kluyveromyces lactis is controlled by autoregulation“. Molecular and Cellular Biology 13, Nr. 5 (Mai 1993): 3058–66. http://dx.doi.org/10.1128/mcb.13.5.3058-3066.1993.

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The concentration of the transcriptional activator LAC9 (KlGAL4) of Kluyveromyces lactis is moderately regulated by the carbon source as is the case for GAL4, its homolog in Saccharomyces cerevisiae. Expression of the LAC9 gene is induced about twofold in galactose. This induction is due to autoregulation. The LAC9 gene product binds to a low-affinity binding site in the LAC9 promoter and moderately activates transcription in response to galactose above a basal level. As for the LAC9-controlled metabolic genes, induction of LAC9 is inhibited in the presence of glucose. This inhibition of induction is a prerequisite for glucose repression of the lactose-galactose metabolic pathway. On the other hand, induced LAC9 levels are required for optimal growth on galactose, since mutating the LAC9 binding site in the LAC9 promoter resulted in poor growth and reduced expression of LAC9-controlled genes. Thus, in addition to the GAL80-dependent regulation by protein-protein interaction, the regulation of LAC9 gene expression is an important parameter in determining carbon source control of the LAC-GAL regulon. Although the mode of control is different, the pattern of LAC9 gene regulation resembles that of the S. cerevisiae GAL4 gene, being lower in glucose and glucose-galactose than in galactose.

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Zachariae, W., und K. D. Breunig. „Expression of the transcriptional activator LAC9 (KlGAL4) in Kluyveromyces lactis is controlled by autoregulation.“ Molecular and Cellular Biology 13, Nr. 5 (Mai 1993): 3058–66. http://dx.doi.org/10.1128/mcb.13.5.3058.

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The concentration of the transcriptional activator LAC9 (KlGAL4) of Kluyveromyces lactis is moderately regulated by the carbon source as is the case for GAL4, its homolog in Saccharomyces cerevisiae. Expression of the LAC9 gene is induced about twofold in galactose. This induction is due to autoregulation. The LAC9 gene product binds to a low-affinity binding site in the LAC9 promoter and moderately activates transcription in response to galactose above a basal level. As for the LAC9-controlled metabolic genes, induction of LAC9 is inhibited in the presence of glucose. This inhibition of induction is a prerequisite for glucose repression of the lactose-galactose metabolic pathway. On the other hand, induced LAC9 levels are required for optimal growth on galactose, since mutating the LAC9 binding site in the LAC9 promoter resulted in poor growth and reduced expression of LAC9-controlled genes. Thus, in addition to the GAL80-dependent regulation by protein-protein interaction, the regulation of LAC9 gene expression is an important parameter in determining carbon source control of the LAC-GAL regulon. Although the mode of control is different, the pattern of LAC9 gene regulation resembles that of the S. cerevisiae GAL4 gene, being lower in glucose and glucose-galactose than in galactose.

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Lellek, Heinrich, Sybille Welker, Ines Diehl, Romy Kirsten und Jobst Greeve. „Reconstitution of mRNA Editing in Yeast Using a Gal4-ApoB-Gal80 Fusion Transcript as the Selectable Marker“. Journal of Biological Chemistry 277, Nr. 26 (25.04.2002): 23638–44. http://dx.doi.org/10.1074/jbc.m203517200.

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Suzuki, Y., Y. Nogi, A. Abe und T. Fukasawa. „GAL11 protein, an auxiliary transcription activator for genes encoding galactose-metabolizing enzymes in Saccharomyces cerevisiae“. Molecular and Cellular Biology 8, Nr. 11 (November 1988): 4991–99. http://dx.doi.org/10.1128/mcb.8.11.4991-4999.1988.

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Normal function of the GAL11 gene is required for maximum production of the enzymes encoded by GAL1, GAL7, and GAL10 (collectively termed GAL1,7,10) in Saccharomyces cerevisiae. Strains bearing a gal11 mutation synthesize these enzymes at 10 to 30% of the wild-type level in the induced state. In a DNA-RNA hybridization experiment, the gal11 effect was shown to be exerted at the transcription level. Yeast cells bearing the gal11 mutation were shown to grow on glycerol plus lactate more slowly than the wild type. We isolated recombinant plasmids carrying the GAL11 gene by complementation of the gal11 mutation. When the GAL11 locus was disrupted by insertion of the URA3 gene, the resulting yeast cells (gal11::URA3) exhibited phenotypes almost identical to those of the gal11 strains, with respect to both galactose utilization and growth on nonfermentable carbon sources. Deficiency of Gal4, the major transcription activator for GAL1,7,10, was epistatic over the gal11 defect. The Gal11 deficiency lowered the expression of GAL2 but not that of MEL1 or GAL80; expression of these genes is also known to be dependent on GAL4 function. We determined the nucleotide sequence of GAL11, which is predicted to encode a 107-kilodalton protein with stretches of polyglutamine and poly(glutamine-alanine). An alpha-helix-beta-turn-alpha-helix structure was found in a distal part of the predicted amino acid sequence. A possible role of the GAL11 product in the regulation of galactose-inducible genes is discussed.

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Suzuki, Y., Y. Nogi, A. Abe und T. Fukasawa. „GAL11 protein, an auxiliary transcription activator for genes encoding galactose-metabolizing enzymes in Saccharomyces cerevisiae.“ Molecular and Cellular Biology 8, Nr. 11 (November 1988): 4991–99. http://dx.doi.org/10.1128/mcb.8.11.4991.

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Normal function of the GAL11 gene is required for maximum production of the enzymes encoded by GAL1, GAL7, and GAL10 (collectively termed GAL1,7,10) in Saccharomyces cerevisiae. Strains bearing a gal11 mutation synthesize these enzymes at 10 to 30% of the wild-type level in the induced state. In a DNA-RNA hybridization experiment, the gal11 effect was shown to be exerted at the transcription level. Yeast cells bearing the gal11 mutation were shown to grow on glycerol plus lactate more slowly than the wild type. We isolated recombinant plasmids carrying the GAL11 gene by complementation of the gal11 mutation. When the GAL11 locus was disrupted by insertion of the URA3 gene, the resulting yeast cells (gal11::URA3) exhibited phenotypes almost identical to those of the gal11 strains, with respect to both galactose utilization and growth on nonfermentable carbon sources. Deficiency of Gal4, the major transcription activator for GAL1,7,10, was epistatic over the gal11 defect. The Gal11 deficiency lowered the expression of GAL2 but not that of MEL1 or GAL80; expression of these genes is also known to be dependent on GAL4 function. We determined the nucleotide sequence of GAL11, which is predicted to encode a 107-kilodalton protein with stretches of polyglutamine and poly(glutamine-alanine). An alpha-helix-beta-turn-alpha-helix structure was found in a distal part of the predicted amino acid sequence. A possible role of the GAL11 product in the regulation of galactose-inducible genes is discussed.

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Nevado, Julián, und Claudio F. Heredia. „Galactose induces in Saccharomyces cerevisiae sensitivity of the utilization of hexoses to inhibition by D-glucosamine“. Canadian Journal of Microbiology 42, Nr. 1 (01.01.1996): 6–11. http://dx.doi.org/10.1139/m96-002.

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Inhibition by glucosamine of the utilization of hexoses by Saccharomyces cerevisiae is induced by growing the cells in media with galactose as carbon source. The intensity of inhibition parallels the induction of the galactose pathway. These findings contrast with the fact that glucosamine is a substrate of the constitutive glucose but not of the inducible galactose transport and phosphorylation systems. The inhibition by glucosamine is pH dependent; the extent seems to be related with the phosphorylation of the hexosamine, as shown by its greater effect with substrates or with conditions that less interfere with the phosphorylation of the inhibitor. Inhibition is not a consequence of ATP depletion of the cell. Intracellular accumulated glucosamine derivatives impair the transport of glucose and mannose in yeast cells grown in galactose-supplemented media but not those grown with glucose or ethanol supplements (i.e., under conditions in which the utilization of these sugars is inhibited). However, impairment of the transport is not enough to explain the characteristics of the observed inhibition. The changes induced by growing the yeast in galactose that render the cells sensitive to glucosamine are under the control of the gal80 and gal4 genes.Key words: yeast, glycolysis inhibition, glucosamine, Saccharomyces cerevisiae.

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Torchia, Timothy E., und James E. Hopper. „GENETIC AND MOLECULAR ANALYSIS OF THE GAL3 GENE IN THE EXPRESSION OF THE GALACTOSE/MELIBIOSE REGULON OF SACCHAROMYCES CEREVISIAE“. Genetics 113, Nr. 2 (01.06.1986): 229–46. http://dx.doi.org/10.1093/genetics/113.2.229.

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ABSTRACT During the galactose adaptation period of a Saccharomyces cerevisiae strain bearing a naturally occurring gal3 allele, we found a longer induction lag and slower rate of accumulation of GAL10 and MEL1 RNAs compared to wild-type strains. A strain of genotype gal3 gal1 gal7 is noninducible for MEL1 gene expression, but this expression block is bypassed by overexpression of the GAL4 gene or by deletion of the GAL80 gene, either of which causes a constitutive phenotype. An otherwise wild-type strain that bears a chromosomal gal3 gene disruption mutation does not produce wild-type GAL3 RNA and exhibits induction comparable to a strain bearing the naturally occurring gal3. Based on this array of results, we conclude that the GAL3 gene product executes its function at a point before GAL4 mediated transcription of the GAL1-10-7 and MEL1 genes. Thus, the data are consistent with the previously advanced hypothesis that the GAL3 gene product functions to synthesize the inducer or coinducer molecule. In experiments in which the presence of either the plasmid-carried cloned GAL3 gene or the plasmid-carried cloned GAL1-10-7 genes allows MEL1 induction of a gal3 gal1 gal7 cell, we find that loss of the plasmid results in the shutoff of MEL1 expression even when galactose is continuously present. Either GAL3 function or GAL1-10-7 functions are therefore required for both the initiation and the maintenance of the induced state. Since the strains bearing either the naturally occurring gal3 allele or the gal3 disruption (null) allele do induce, the plasmid loss experiments indicate the existence of two completely independent induction initiation-maintenance pathways, one requiring GAL3 function, the other requiring GAL1-10-7 function. Finally, Northern blot analysis reveals two major GAL3 transcripts that differ in size by approximately 500 nucleotides.

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Egriboz, O., S. Goswami, X. Tao, K. Dotts, C. Schaeffer, V. Pilauri und J. E. Hopper. „Self-Association of the Gal4 Inhibitor Protein Gal80 Is Impaired by Gal3: Evidence for a New Mechanism in the GAL Gene Switch“. Molecular and Cellular Biology 33, Nr. 18 (15.07.2013): 3667–74. http://dx.doi.org/10.1128/mcb.00646-12.

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Li, Yan, Guanjun Chen und Weifeng Liu. „Alterations in the Interaction Between GAL4 and GAL80 Effect Regulation of the Yeast GAL Regulon Mediated by the F box Protein Dsg1“. Current Microbiology 61, Nr. 3 (05.02.2010): 210–16. http://dx.doi.org/10.1007/s00284-010-9598-1.

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Donelson, Nathan C., Richa Dixit, Israel Pichardo-Casas, Eva Y. Chiu, Robert T. Ohman, Justin B. Slawson, Mason Klein, Tudor A. Fulga, David Van Vactor und Leslie C. Griffith. „MicroRNAs Regulate Multiple Aspects of Locomotor Behavior in Drosophila“. G3: Genes|Genomes|Genetics 10, Nr. 1 (06.11.2019): 43–55. http://dx.doi.org/10.1534/g3.119.400793.

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Locomotion is an ancient and fundamental output of the nervous system required for animals to perform many other complex behaviors. Although the formation of motor circuits is known to be under developmental control of transcriptional mechanisms that define the fates and connectivity of the many neurons, glia and muscle constituents of these circuits, relatively little is known about the role of post-transcriptional regulation of locomotor behavior. MicroRNAs have emerged as a potentially rich source of modulators for neural development and function. In order to define the microRNAs required for normal locomotion in Drosophila melanogaster, we utilized a set of transgenic Gal4-dependent competitive inhibitors (microRNA sponges, or miR-SPs) to functionally assess ca. 140 high-confidence Drosophila microRNAs using automated quantitative movement tracking systems followed by multiparametric analysis. Using ubiquitous expression of miR-SP constructs, we identified a large number of microRNAs that modulate aspects of normal baseline adult locomotion. Addition of temperature-dependent Gal80 to identify microRNAs that act during adulthood revealed that the majority of these microRNAs play developmental roles. Comparison of ubiquitous and neural-specific miR-SP expression suggests that most of these microRNAs function within the nervous system. Parallel analyses of spontaneous locomotion in adults and in larvae also reveal that very few of the microRNAs required in the adult overlap with those that control the behavior of larval motor circuits. These screens suggest that a rich regulatory landscape underlies the formation and function of motor circuits and that many of these mechanisms are stage and/or parameter-specific.

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Sil, Alok Kumar, Samina Alam, Ping Xin, Ly Ma, Melissa Morgan, Colleen M. Lebo, Michael P. Woods und James E. Hopper. „The Gal3p-Gal80p-Gal4p Transcription Switch of Yeast: Gal3p Destabilizes the Gal80p-Gal4p Complex in Response to Galactose and ATP“. Molecular and Cellular Biology 19, Nr. 11 (01.11.1999): 7828–40. http://dx.doi.org/10.1128/mcb.19.11.7828.

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ABSTRACT The Gal3, Gal80, and Gal4 proteins of Saccharomyces cerevisiae comprise a signal transducer that governs the galactose-inducible Gal4p-mediated transcription activation ofGAL regulon genes. In the absence of galactose, Gal80p binds to Gal4p and prohibits Gal4p from activating transcription, whereas in the presence of galactose, Gal3p binds to Gal80p and relieves its inhibition of Gal4p. We have found that immunoprecipitation of full-length Gal4p from yeast extracts coprecipitates less Gal80p in the presence than in the absence of Gal3p, galactose, and ATP. We have also found that retention of Gal80p by GSTG4AD (amino acids [aa] 768 to 881) is markedly reduced in the presence compared to the absence of Gal3p, galactose, and ATP. Consistent with these in vitro results, an in vivo two-hybrid genetic interaction between Gal80p and Gal4p (aa 768 to 881) was shown to be weaker in the presence than in the absence of Gal3p and galactose. These compiled results indicate that the binding of Gal3p to Gal80p results in destabilization of a Gal80p-Gal4p complex. The destabilization was markedly higher for complexes consisting of G4AD (aa 768 to 881) than for full-length Gal4p, suggesting that Gal80p relocated to a second site on full-length Gal4p. Congruent with the idea of a second site, we discovered a two-hybrid genetic interaction involving Gal80p and the region of Gal4p encompassing aa 225 to 797, a region of Gal4p linearly remote from the previously recognized Gal80p binding peptide within Gal4p aa 768 to 881.

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Bro, Christoffer, Steen Knudsen, Birgitte Regenberg, Lisbeth Olsson und Jens Nielsen. „Improvement of Galactose Uptake in Saccharomyces cerevisiae through Overexpression of Phosphoglucomutase: Example of Transcript Analysis as a Tool in Inverse Metabolic Engineering“. Applied and Environmental Microbiology 71, Nr. 11 (November 2005): 6465–72. http://dx.doi.org/10.1128/aem.71.11.6465-6472.2005.

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ABSTRACT Through genome-wide transcript analysis of a reference strain and two recombinant Saccharomyces cerevisiae strains with different rates of galactose uptake, we obtained information about the global transcriptional response to metabolic engineering of the GAL gene regulatory network. One of the recombinant strains overexpressed the gene encoding the transcriptional activator Gal4, and in the other strain the genes encoding Gal80, Gal6, and Mig1, which are negative regulators of the GAL system, were deleted. Even though the galactose uptake rates were significantly different in the three strains, we surprisingly did not find any significant changes in the expression of the genes encoding the enzymes catalyzing the first steps of the pathway (i.e., the genes encoding Gal2, Gal1, Gal7, and Gal10). We did, however, find that PGM2, encoding the major isoenzyme of phosphoglucomutase, was slightly up-regulated in the two recombinant strains with higher galactose uptake rates. This indicated that PGM2 is a target for overexpression in terms of increasing the flux through the Leloir pathway, and through overexpression of PGM2 the galactose uptake rate could be increased by 70% compared to that of the reference strain. Based on our findings, we concluded that phosphoglucomutase plays a key role in controlling the flux through the Leloir pathway, probably due to increased conversion of glucose-1-phosphate to glucose-6-phosphate. This conclusion was supported by measurements of sugar phosphates, which showed that there were increased concentrations of glucose-6-phosphate, galactose-6-phosphate, and fructose-6-phosphate in the strain construct overexpressing PGM2.

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Bajwa, W., T. E. Torchia und J. E. Hopper. „Yeast regulatory gene GAL3: carbon regulation; UASGal elements in common with GAL1, GAL2, GAL7, GAL10, GAL80, and MEL1; encoded protein strikingly similar to yeast and Escherichia coli galactokinases“. Molecular and Cellular Biology 8, Nr. 8 (August 1988): 3439–47. http://dx.doi.org/10.1128/mcb.8.8.3439-3447.1988.

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GAL3 gene expression is required for rapid GAL4-mediated galactose induction of the galactose-melibiose regulon genes in Saccharomyces cerevisiae. Here we show by Northern (RNA) blot analysis that GAL3 gene expression is itself galactose inducible. Like the GAL1, GAL7, GAL10, and MEL1 genes, the GAL3 gene is severely glucose repressed. Like the MEL1 gene, but in contrast to the GAL1, GAL7, and GAL10 genes, GAL3 is expressed at readily detectable basal levels in cells grown in noninducing, nonrepressing media. We determined the sequence of the S. cerevisiae GAL3 gene and its 5'-noncoding region. Within the 5'-noncoding region of the GAL3 gene, we found two sequences similar to the UASGal elements of the other galactose-melibiose regulon genes. Deletion analysis indicated that only the most ATG proximal of these sequences is required for GAL3 expression. The coding region of GAL3 consists of a 1,275-base-pair open reading frame in the direction of transcription. A comparison of the deduced 425-amino-acid sequence with the protein data bank revealed three regions of striking similarity between the GAL3 protein and the GAL1-specified galactokinase of Saccharomyces carlsbergensis. One of these regions also showed striking similarity to sequences within the galactokinase protein of Escherichia coli. On the basis of these protein sequence similarities, we propose that the GAL3 protein binds a molecule identical to or structurally related to one of the substrates or products of the galactokinase-catalyzed reaction.

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