Use of lacZ fusions to delimit regulatory elements of the inducible divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae.

Yocum, R. Rogers; Hanley, Seán; West, Robert W.; Ptashne, Mark

doi:10.1128/mcb.4.10.1985

Cited by 320 publications

(194 citation statements)

References 61 publications

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“…In support of the coregulation model, the intergenic region between the S. cerevisiae GAL1 and GAL10 genes contains shared regulatory binding sites (15,(46)(47)(48), and galactose-1-phosphate, one of the intermediate products of galactose metabolism, is toxic (49).…”

Section: Resultsmentioning

confidence: 99%

Multiple GAL pathway gene clusters evolved independently and by different mechanisms in fungi

Slot

Rokas

2010

Proc. Natl. Acad. Sci. U.S.A.

150

168

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A notable characteristic of fungal genomes is that genes involved in successive steps of a metabolic pathway are often physically linked or clustered. To investigate how such clusters of functionally related genes are assembled and maintained, we examined the evolution of gene sequences and order in the galactose utilization (GAL) pathway in whole-genome data from 80 diverse fungi. We found that GAL gene clusters originated independently and by different mechanisms in three unrelated yeast lineages. Specifically, the GAL cluster found in Saccharomyces and Candida yeasts originated through the relocation of native unclustered genes, whereas the GAL cluster of Schizosaccharomyces yeasts was acquired through horizontal gene transfer from a Candida yeast. In contrast, the GAL cluster of Cryptococcus yeasts was assembled independently from the Saccharomyces/Candida and Schizosaccharomyces GAL clusters and coexists in the Cryptococcus genome with unclustered GAL paralogs. These independently evolved GAL clusters represent a striking example of analogy at the genomic level. We also found that species with GAL clusters exhibited significantly higher rates of GAL pathway loss than species with unclustered GAL genes. These results suggest that clustering of metabolic genes might facilitate fungal adaptation to changing environments both through the acquisition and loss of metabolic capacities. F ungal genes involved in successive steps of a pathway are frequently physically clustered (1-12). Evolutionary analysis of several different fungal gene clusters has shown that they have originated through different mechanisms (13-16). For example, the DAL cluster for allantoin utilization in Saccharomyces cerevisiae and close relatives originated through adaptive gene relocation (13), whereas a nitrate assimilation cluster in Trichoderma originated through horizontal gene transfer (14). However, it is unclear how these mechanisms shape the assembly of gene clusters, or how the evolutionary trajectories differ between species containing clusters and species in which the genes in a pathway are scattered across the genome.To better understand the origins and maintenance of gene clusters over a large evolutionary timescale and a range of ecological conditions, we investigated the evolution of the Leloir galactose utilization (GAL) pathway in fungi. The detailed knowledge on GAL pathway function in S. cerevisiae (15, 16), and the abundance of genomic data from a wide diversity of fungal species (17), make it an excellent model pathway to address these questions. Furthermore, the relative galactose content varies substantially among different plant substrata (from hundreds of mg/g legume seeds and algal mats to less than 1 mg/g in some fruits) (18-21), suggesting that the natural substrates of fungi have likely provided ample opportunity for populations to evolve niche-dependent adaptations for galactose utilization.The protein products of three GAL genes (GAL1, GAL7, and GAL10) are involved in four enzymatic steps (22). In the first s...

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Section: Resultsmentioning

confidence: 99%

Multiple GAL pathway gene clusters evolved independently and by different mechanisms in fungi

Slot

Rokas

2010

Proc. Natl. Acad. Sci. U.S.A.

150

168

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“…Assays-␤-Galactosidase activity assays were performed with permeabilized yeast cells grown to mid-log phase as described previously (18). The mean activities are given in Miller units and are the averages of three to four assays of at least four independent yeast transformants.…”

Section: ␤-Galactosidasementioning

confidence: 99%

Glucose-mediated Phosphorylation Converts the Transcription Factor Rgt1 from a Repressor to an Activator

Mosley¹,

Lakshmanan²,

Aryal

et al. 2003

Journal of Biological Chemistry

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Glucose, the most abundant carbon and energy source, regulates the expression of genes required for its own efficient metabolism. In the yeast Saccharomyces cerevisiae, glucose induces the expression of the hexose transporter (HXT) genes by modulating the activity of the transcription factor Rgt1 that functions as a repressor when glucose is absent. However, in the presence of high concentrations of glucose, Rgt1 is converted from a repressor to an activator and is required for maximal induction of HXT1 gene expression. We report that Rgt1 binds to the HXT1 promoter only in the absence of glucose, suggesting that Rgt1 increases HXT1 gene expression at high levels of glucose by an indirect mechanism. It is likely that Rgt1 stimulates the expression of an activator of the HXT1 gene at high concentrations of glucose. In addition, we demonstrate that Rgt1 becomes hyperphosphorylated in response to high glucose levels and that this phosphorylation event is required for Rgt1 to activate transcription. Furthermore, Rgt1 lacks the glucose-mediated phosphorylation in the snf3 rgt2 and grr1 mutants, which are defective in glucose induction of HXT gene expression. In these mutants, Rgt1 behaves as a constitutive repressor independent of the carbon source. We conclude that phosphorylation of Rgt1 in response to glucose is required to abolish the Rgt1-mediated repression of the HXT genes and to convert Rgt1 from a transcriptional repressor to an activator.The yeast Saccharomyes cerevisiae uses glucose as its preferred carbon and energy source. Glucose not only represses the expression of genes that are required for the metabolism of alternate sugars but also induces the transcription of genes that are essential for its own efficient utilization (1-3). Among the genes that are induced by glucose are the members of the HXT gene family, which encode glucose transporters. Glucose induces the expression of the HXT1-HXT4 genes by 10 -300-fold (4, 5).Several components of the glucose induction pathway required for HXT gene expression have been identified, including the glucose sensors Snf3 and Rgt2, that are responsible for sensing extracellular glucose and generating the intracellular signal (6, 7). A strain mutated for both sensors (snf3 rgt2 double mutant) is completely defective in glucose induction of the HXT gene expression (7). Another component that is absolutely essential for glucose induction of the HXT gene expression is the ubiquitin ligase Grr1 (5, 8). Two homologous proteins, Std1 and Mth1, have been shown to negatively regulate HXT gene expression and to interact with the carboxyl-terminal tails of the Snf3 and Rgt2 sensors (9 -11). Repression of HXT gene expression in the absence of glucose is abolished in a std1 mth1 double mutant (9, 11).The target of the glucose induction signal is the Cys 6 DNAbinding protein Rgt1, which belongs to the family of the Gal4 transcription factors (12). In the absence of glucose, Rgt1 represses HXT gene expression, whereas at high concentrations of glucose Rgt1 is required for maxima...

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“…Repression by Migl deletions was determined by measuring (3-gal expressed from a GAL1-lacZ fusion (pRY181) integrated at the LEU2 locus as described previously (Yocum et al, 1984), except cell densities (OD600) and product formation (OD420) was quantified in microtiter plates on a Molecular Devices plate reader. Yeast were grown in minimal medium lacking uracil and containing 2% glucose (repressing conditions) or 5% glycerol (nonrepressing conditions) to midlogarithmic phase (OD600 of about 1.0).…”

Section: Measurement Of Glucose Repressionmentioning

confidence: 99%

Regulated nuclear translocation of the Mig1 glucose repressor.

Vít

Waddle

Johnston

1997

MBoC

319

182

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Glucose represses the transcription of many genes in bakers yeast (Saccharomyces cerevisiae). Mig1 is a Cys2-His2 zinc finger protein that mediates glucose repression of several genes by binding to their promoters and recruiting the general repression complex Ssn6-Tup1. We have found that the subcellular localization of Mig1 is regulated by glucose. Mig1 is imported into the nucleus within minutes after the addition of glucose and is just as rapidly transported back to the cytoplasm when glucose is removed. This regulated nuclear localization requires components of the glucose repression signal transduction pathway. An internal region of the protein separate from the DNA binding and repression domains is necessary and sufficient for glucose-regulated nuclear import and export. Changes in the phosphorylation status of Mig1 are coincident with the changes in its localization, suggesting a possible regulatory role for phosphorylation. Our results suggest that a glucose-regulated nuclear import and/or export mechanism controls the activity of Mig1.

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Use of lacZ fusions to delimit regulatory elements of the inducible divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae.

Cited by 320 publications

References 61 publications

Multiple GAL pathway gene clusters evolved independently and by different mechanisms in fungi

Multiple GAL pathway gene clusters evolved independently and by different mechanisms in fungi

Glucose-mediated Phosphorylation Converts the Transcription Factor Rgt1 from a Repressor to an Activator

Regulated nuclear translocation of the Mig1 glucose repressor.

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