Academic literature on the topic 'Gal4/gal80'

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

La plupart de nos cellules sont diploïdes possédant deux copies de chaque chromosome. Lors de la mitose, la formation d’un fuseau bipolaire avec un centrosome à chaque pôle permet la ségrégation correcte des chromosomes, essentiel au maintien de la stabilité génétique. Il existe néanmoins des variations du contenu chromosomique comme la polyploïdie, définit comme le doublement de l’ensemble des chromosomes et l’aneuploïdie, définie comme la perte ou le gain de chromosome entier. Bien qu’observées, la fréquence des cellules aneuploïdies dans les tissus d’un organisme sain reste controversée.De façon importante, la duplication du génome et l’aneuploïdie sont associées à des pathologies et sont considérées comme une caractéristique du cancer. En effet, un nombre anormal de chromosomes est souvent associé à une instabilité chromosomique. Toutefois le rôle et les implications de ces variations dans l’initiation et la progression de tumeur restent peu compris.J’ai d’abord étudié les conséquences de la polyploïdie sur la division des cellules. J’ai utilisé des approches in vivo et in vitro en induisant la polyploidisation par défaut de cytocinèse dans des cellules souches neurales de drosophile et des cellules cancéreuses humaines. L’analyse de leur mitose m’a permis de découvrir que la présence de chromosomes et de centrosomes en excès conduisait invariablement à la formation de fuseaux multipolaires. En modélisant les cellules polyploïdes, j’ai découvert qu’au-delà de la quantité, la conformation spatiale de l’ADN contribue à cette multipolarité. Des perturbations expérimentales au niveau de l’ADN et du fuseau m’ont permis de démontrer que la présence d’ADN en excès agit comme une barrière physique bloquant la coalescence des multiples pôles et par conséquent empêchant la bipolarité. De façon intéressante, j’ai réussi à restaurer la bipolarité en supprimant la « barrière d’ADN » par ablation avec laser ou en augmentant la longueur des microtubules pour contourner celle-ci. Alors que l’amplification centrosomale était considérée comme unique acteur, mes résultats identifient l’excès d’ADN comme contributeur clef de la multipolarité et de l’instabilité chromosomique typique des cellules polyploïdes.Puis, je me suis ensuite intéressée à l’aneuploïdie, dont la fréquence en contexte sain reste un sujet de débat intense. De plus, les outils développés jusqu’à présent pour évaluer le taux d’aneuploïdie manque d’une dimension temporelle. J’ai donc généré un outil génétique innovant de visualisation et de suivi des cellules aneuploïdes in vivo chez la drosophile. J’ai utilisé l’expression de la GFP comme gène rapporteur, contrôlée par le système GAL4/UAS et son inhibition par GAL80. Ainsi, la perte aléatoire du chromosome contenant la séquence du GAL80 entraine l’apparition d’un signal GFP dans les cellules aneuploïdes. Celles-ci peuvent donc être facilement détectées et suivies en temps-réel dans les tissus. Utilisant ce système, j’ai découvert que la perte de chromosome était un évènement très rare dans les tissus de la mouche. Cet outil combiné à d’autres marqueurs fluorescents et/ou utilisé dans divers contextes génétiques pourrait aider à la compréhension de la genèse et du devenir des cellules aneuploïdes in vivo.De plus, j’ai constaté que le cerveau de la larve présentait un nombre important de cellules GFP. De manière surprenante, ces cellules ne résultaient pas de la perte de chromosomes mais de la perte d’expression du gène GAL80. Ces résultats inattendus ont de fortes implications pour la communauté des drosophilistes car cela peut mener à des faux positifs dans les expériences de génération de clones. J’ai aussi découvert que les cellules souches neurales présentaient un mosaïsme dans l’expression des gènes, qui diffèrent d’autres organes et s’adaptent à des stimuli environnementaux. Ceci représente possiblement un niveau de plasticité dans le cerveau nécessaire à la diversité neuronale, l’adaptation et la survie
Most animal cells are diploid, containing two copies of each chromosome. Establishment of proper bipolar mitotic spindle containing two centrosomes, one at each pole contributes to accurate chromosome segregation. This is essential for the maintenance of genome stability, tissue and organism homeostasis. However, numerical deviations to the diploid set are observed in healthy tissues. Polyploidy is the doubling of the whole chromosome set and aneuploidy concerns the gain or loss of whole chromosomes. Importantly, whole genome duplications and aneuploidy have also been associated to pathological conditions. For example, variations to genome content are associated with chromosome instability and cancer development, however their exact contribution to cancer genome remains poorly understood.In the first part of my PhD project, I investigated the consequences of polyploidy during cell division. I found that the presence of extra DNA and extra centrosomes generated invariably multipolar spindles. Then I identified contributors to the multipolar status using in vivo approaches in Drosophila neural stem cells and in vitro culture of cancer cells. Further I combined DNA and spindle perturbations with computer modelling and found that in polyploid cells, the presence of excessive DNA acts as a physical barrier blocking spindle pole coalescence and bipolarity. Indeed, laser ablation to disrupt and increase in microtubule stability and length to bypass the DNA-barrier could rescue bipolar spindle formation. This discovery challenges the current view that suggested extra-centrosomes as only contributor to spindle multipolarity and provides a rational to understand chromosome instability typical of polyploid cells.The aim of the second part of my PhD project was to generate a novel tool to quantitively probe chromosome loss in vivo in Drosophila tissues. Aneuploidy has been observed in various physiological tissues, however the frequency of this error remained highly debatable. In addition, tools developed so far to assess aneuploidy lack a temporal dimension. To circumvent this, I used the expression of a GFP report gene driven by the GAL4/UAS system and its inhibition by GAL80. In principle, the random loss of the chromosome carrying the GAL80 sequence leads to GFP appearance in aneuploid cells that can therefore be followed in live tissues. I found that chromosome loss was extremely infrequent in most tissues of the wild type fly. This tool combined with fluorescent marker and/or tested in various genetic background, might help understanding mechanisms behind aneuploidy genesis and outcome in vivo.While developing this tool, I discovered that in the larval brain, GFP cells where not a by-product of chromosome loss but rather an unexpected mis-regulation in the expression of the GAL80 gene. These results have strong implications for the Drosophila community as it can result in false positive in clonal experiments. Further, I discovered a mosaicism and plasticity of the Drosophila brain in neural stem cells for gene expression which differs from other organs and that is influenced by environmental stimuli. This possibly reflects a certain level of plasticity in the brain necessary for neuronal diversity, adaptation and survival