GAL Genes Research Articles

Clade- and species-specific features of genome evolution in the Saccharomycetaceae.

Many aspects of the genomes of yeast species in the family Saccharomycetaceae have been well conserved during evolution. They have similar genome sizes, genome contents, and extensive collinearity of gene order along chromosomes. Gene functions can often be inferred reliably by using information from Saccharomyces cerevisiae. Beyond this conservative picture however, there are many instances where a species or a clade diverges substantially from the S. cerevisiae paradigm—for example, by the amplification of a gene family, or by the absence of a biochemical pathway or a protein complex. Here, we review clade-specific features, focusing on genomes sequenced in our laboratory from the post-WGD genera Naumovozyma, Kazachstania and Tetrapisispora, and from the non-WGD species Torulaspora delbrueckii. Examples include the loss of the pathway for histidine synthesis in the cockroach-associated species Tetrapisispora blattae; the presence of a large telomeric GAL gene cluster in To. delbrueckii; losses of the dynein and dynactin complexes in several independent yeast lineages; fragmentation of the MAT locus and loss of the HO gene in Kazachstania africana; and the patchy phylogenetic distribution of RNAi pathway components.

Effects of galactose adaptation in yeast for ethanol fermentation from red seaweed, Gracilaria verrucosa.

A total monosaccharide concentration of 39.6g/L, representing 74.0% conversion of 53.5g/L total carbohydrate from 80g dw/L (8% w/v) Gracilaria verrucosa slurry, was obtained by thermal acid hydrolysis and enzymatic saccharification. G. verrucosa hydrolysate was used as a substrate for ethanol production by 'separate hydrolysis and fermentation' (SHF). The ethanol production and yield (Y EtOH) from Saccharomyces cerevisiae KCCM 1129 with and without adaptation to high galactose concentrations were 18.3g/L with Y EtOH of 0.46 and 13.4g/L with Y EtOH of 0.34, respectively. Relationship between galactose adaptation effects and mRNA transcriptional levels were evaluated with GAL gene family, regulator genes of the GAL genetic switch and repressor genes in non-adapted and adapted S. cerevisiae. The development of galactose adaptation for ethanol fermentation of G. verrucosa hydrolysates allowed us to enhance the overall ethanol yields and obtain a comprehensive understanding of the gene expression levels and metabolic pathways involved.

Rrd1p, an RNA Polymerase II‐specific Prolyl Isomerase and Activator of Phosphoprotein Phosphatase, Promotes Transcription Independently of Rapamycin Response

Rrd1p (resistance to rapamycin deletion 1) has been previously implicated in controlling transcription of rapamycin-regulated genes in response to rapamycin treatment. Intriguingly, we show here that Rrd1p associates with the coding sequence of a galactose-inducible and rapamycin non-responsive GAL1 gene, and promotes the association of RNA polymerase II with GAL1 in the absence of rapamycin treatment following transcriptional induction. Consistently, nucleosomal disassembly at GAL1 is impaired in the absence of Rrd1p, and GAL1 transcription is reduced in the Δrrd1 strain. Likewise, Rrd1p associates with the coding sequences of other rapamycin non-responsive and inducible GAL genes to promote their transcription in the absence of rapamycin treatment. Similarly, inducible, but rapamycin-responsive, non-GAL genes such as CTT1, STL1 and CUP1 are also regulated by Rrd1p. However, transcription of these inducible GAL and non-GAL genes is not altered in the absence of Rrd1p when the steady-state is reached after long transcriptional induction. Consistently, transcription of the constitutively active genes is not changed in the Δrrd1 strain. Taken together, our results demonstrate a new function of Rrd1p in stimulation of initial rounds of transcription, but not steady-state/constitutive transcription, of both rapamycin-responsive and non-responsive genes independently of rapamycin treatment.

Evolutionary engineering of Saccharomyces cerevisiae for efficient conversion of red algal biosugars to bioethanol

The aim of this work was to apply the evolutionary engineering to construct a mutant Saccharomyces cerevisiae HJ7-14 resistant on 2-deoxy-d-glucose and with an enhanced ability of bioethanol production from galactose, a mono-sugar in red algae. In batch and repeated-batch fermentations, HJ7-14 metabolized 5-fold more galactose and produced ethanol 2.1-fold faster than the parental D452-2 strain. Transcriptional analysis of genes involved in the galactose metabolism revealed that moderate relief from the glucose-mediated repression of the transcription of the GAL genes might enable HJ7-14 to metabolize galactose rapidly. HJ7-14 produced 7.4g/L ethanol from hydrolysates of the red alga Gelidium amansii within 12h, which was 1.5-times faster than that observed with D452-2. We demonstrate conclusively that evolutionary engineering is a promising tool to manipulate the complex galactose metabolism in S. cerevisiae to produce bioethanol from red alga.

Galanin pathogenic mutations in temporal lobe epilepsy.

Temporal lobe epilepsy (TLE) is a common epilepsy syndrome with a complex etiology. Despite evidence for the participation of genetic factors, the genetic basis of TLE remains largely unknown. A role for the galanin neuropeptide in the regulation of epileptic seizures has been established in animal models more than two decades ago. However, until now there was no report of pathogenic mutations in GAL, the galanin-encoding gene, and therefore its role in human epilepsy was not established. Here, we studied a family with a pair of monozygotic twins affected by TLE and two unaffected siblings born to healthy parents. Exome sequencing revealed that both twins carried a novel de novo mutation (p.A39E) in the GAL gene. Functional analysis revealed that the p.A39E mutant showed antagonistic activity against galanin receptor 1 (GalR1)-mediated response, and decreased binding affinity and reduced agonist properties for GalR2. These findings suggest that the p.A39E mutant could impair galanin signaling in the hippocampus, leading to increased glutamatergic excitation and ultimately to TLE. In a cohort of 582 cases, we did not observe any pathogenic mutations indicating that mutations in GAL are a rare cause of TLE. The identification of a novel de novo mutation in a biologically-relevant candidate gene, coupled with functional evidence that the mutant protein disrupts galanin signaling, strongly supports GAL as the causal gene for the TLE in this family. Given the availability of galanin agonists which inhibit seizures, our findings could potentially have direct implications for the development of anti-epileptic treatment.

Dynamics of Gal80p in the Gal80p-Gal3p Complex Differ Significantly from the Dynamics in the Gal80p-Gal1p Complex: Implications for the Higher Specificity of Gal3p Towards Transcriptional Induction of Gal Genes

Expression of the GAL gene in Sacharomyces cerevisiae is regulated by three proteins; Gal3p/Gal1p, Gal80p and Gal4p. Both Gal3p and Gal1p act as transcriptional inducers, though Gal3p has a higher activity than Gal1p. The difference in activity may depend on the strength of the interaction and dynamical behavior of these proteins during complex formation with the repressor protein Gal80p. To address these queries we have modeled the binding interface of the Gal1p-Gal80p and Gal3p-Gal80p complexes. The comparison of the dynamics of these proteins in the complex and in the Apo protein was carried out. It was observed that the binding of Gal3p with Gal80p induces significant flexibility in Gal80p on a surface different from the one involved in binding with Gal3p. Several other differences at the interface between the Gal3p-Gal80p and the Gal1p-Gal80p complex were observed, which might permit Gal3p to act as a transcriptional inducer with higher activity. Further, on the basis of our finding we have proposed the dynamical event and plausible mechanism of complex formation of Gal3p and Gal1p with Gal80p at the molecular level.

Live-Cell Single-Molecule Imaging of Sense and Antisense Transcription of a Yeast Gene

The eukaryotic genome is pervasively transcribed, giving rise to various sorts of non-coding RNAs whose mechanisms of action are for the most part not understood. Recent technological advances now allow direct visualization of the synthesis of nascent transcripts from individual genes over time by decorating RNAs with fluorescent proteins. Using the orthogonal RNA-binding MS2 and PP7 bacteriophage coat proteins, we were recently able to tag two regions of the same RNA in two different colors [Coulon et al. 2014, eLife, in press]. Here, we used this technique to visualize simultaneously sense and antisense transcription from the GAL10 locus in yeast, during activation of the GAL pathway. Fluorescence fluctuations recorded in both channels at the transcription site reflect the kinetics of transcription on both strands as the GAL10 gene gets activated in response to galactose. We observe transient antisense transcription occurring almost exclusively prior to the appearance of sense transcription. Using cross-correlation analysis, we uncovered specific temporal windows relatively to sense activation where antisense transcription is enriched or depleted - likely reflecting the biochemical mechanisms underlying activation. Once transcription of the GAL10 gene starts, transcripts are produced in bursts separated by periods of inactivity, occasionally leaving the opportunity for antisense transcription to happen. We developed a method for applying fluctuation correlation analysis to non-stationary time traces. This allowed us to isolate the bursting kinetics even in the non-steadystate context of a transient response to galactose. By modeling the autocorrelation of a bursting gene, we were able to infer from our data how the elongation rate, burst size and burst frequency of the GAL10 gene are modulated by different doses of galactose. This work shows how in vivo single-molecule methods and fluctuation analysis can reveal unanticipated mechanisms of transcriptional regulation.

Observing Signal Transduction Directly at the Single-Molecule Level in Live Eukaryotic Cells

All cells dynamically sense their environment through signal transduction mechanisms, allowing them to respond to environmental changes at the genetic level. The glucose sensory mechanism in Sacchoromyces cerevisiae is a model system of signal transduction. It uses the multi-copy inhibitor of the GAL gene expression Mig1 protein to repress unwanted genes in the presence of elevated level of extracellular glucose. In wild-type cell strains, and in genetically and biochemically impaired signal transduction phenotypes, we have fluorescently labelled the Mig1 protein with the green fluorescent protein GFP via chromosomal integration at native levels of expression, in addition to the RNAP nuclear reporter protein Nrd1 with the red fluorescent protein mCherry. [1] Using millisecond dual-color fluorescence excitation with Slimfield microscopy, [2] we track the fate of single molecules of Mig1 in live yeast cells at millisecond timescales. We present data showing a consistent difference between confined, high stoichiometry assemblages of bound Mig1 in the nucleus and freely diffusing single Mig1 molecules across the entire cell as well as more complex behavior in Mig1 traversing the nuclear membrane. The mobility and stoichiometry distribution of Mig1 assemblages we observe is functionally dependent on the signal transduction pathway and the concentration of extracellular glucose, allowing us to directly observe gene regulatory events.1. Bendrioua, L. et al. Yeast AMP-activated Protein Kinase Monitors Glucose Concentration Changes and Absolute Glucose Levels. J. Biol. Chem. 289, 12863-75 (2014).2. Reyes-Lamothe, R., Sherratt, D. J. & Leake, M. C. Stoichiometry and architecture of active DNA replication machinery in Escherichia coli. Science 328, 498-501 (2010).

Millisecond single-molecule localization microscopy combined with convolution analysis and automated image segmentation to determine protein concentrations in complexly structured, functional cells, one cell at a time.

We present a single-molecule tool called the CoPro (concentration of proteins) method that uses millisecond imaging with convolution analysis, automated image segmentation and super-resolution localization microscopy to generate robust estimates for protein concentration in different compartments of single living cells, validated using realistic simulations of complex multiple compartment cell types. We demonstrate its utility experimentally on model Escherichia coli bacteria and Saccharomyces cerevisiae budding yeast cells, and use it to address the biological question of how signals are transduced in cells. Cells in all domains of life dynamically sense their environment through signal transduction mechanisms, many involving gene regulation. The glucose sensing mechanism of S. cerevisiae is a model system for studying gene regulatory signal transduction. It uses the multi-copy expression inhibitor of the GAL gene family, Mig1, to repress unwanted genes in the presence of elevated extracellular glucose concentrations. We fluorescently labelled Mig1 molecules with green fluorescent protein (GFP) via chromosomal integration at physiological expression levels in living S. cerevisiae cells, in addition to the RNA polymerase protein Nrd1 with the fluorescent protein reporter mCherry. Using CoPro we make quantitative estimates of Mig1 and Nrd1 protein concentrations in the cytoplasm and nucleus compartments on a cell-by-cell basis under physiological conditions. These estimates indicate a ∼4-fold shift towards higher values in the concentration of diffusive Mig1 in the nucleus if the external glucose concentration is raised, whereas equivalent levels in the cytoplasm shift to smaller values with a relative change an order of magnitude smaller. This compares with Nrd1 which is not involved directly in glucose sensing, and which is almost exclusively localized in the nucleus under high and low external glucose levels. CoPro facilitates time-resolved quantification of protein concentrations in single functional cells, and enables the distributions of concentrations across a cell population to be measured. This could be useful in investigating several cellular processes that are mediated by proteins, especially where changes in protein concentration in a single cell in response to changes in the extracellular chemical environment are subtle and rapid and may be smaller than the variability across a cell population.

Evolution of moonlighting proteins: insight from yeasts.

The present article addresses the possibilities offered by yeasts to study the problem of the evolution of moonlighting proteins. It focuses on data available on hexokinase from Saccharomyces cerevisiae that moonlights in catabolite repression and on galactokinase from Kluyveromyces lactis that moonlights controlling the induction of the GAL genes. Possible experimental approaches to studying the evolution of moonlighting hexose kinases are suggested.

Efficient generation of knock-in transgenic zebrafish carrying reporter/driver genes by CRISPR/Cas9-mediated genome engineering.

The type II bacterial CRISPR/Cas9 system is rapidly becoming popular for genome-engineering due to its simplicity, flexibility, and high efficiency. Recently, targeted knock-in of a long DNA fragment via homology-independent DNA repair has been achieved in zebrafish using CRISPR/Cas9 system. This raised the possibility that knock-in transgenic zebrafish could be efficiently generated using CRISPR/Cas9. However, how widely this method can be applied for the targeting integration of foreign genes into endogenous genomic loci is unclear. Here, we report efficient generation of knock-in transgenic zebrafish that have cell-type specific Gal4 or reporter gene expression. A donor plasmid containing a heat-shock promoter was co-injected with a short guide RNA (sgRNA) targeted for genome digestion, a sgRNA targeted for donor plasmid digestion, and Cas9 mRNA. We have succeeded in establishing stable knock-in transgenic fish with several different constructs for 4 genetic loci at a frequency being exceeding 25%. Due to its simplicity, design flexibility, and high efficiency, we propose that CRISPR/Cas9-mediated knock-in will become a standard method for the generation transgenic zebrafish.

Dynamics of Gal80p in the Gal80p-Gal3p complex differ significantly from the dynamics in the Gal80p-Gal1p complex: implications for the higher specificity of Gal3p.

The expression of the GAL gene in Sacharomyces cerevisiae is regulated by three proteins; Gal3p/Gal1p, Gal80p and Gal4p. Both Gal3p and Gal1p act as transcriptional inducers, though Gal3p has a higher activity than Gal1p. The difference in activity may depend on the strength of the interaction and dynamical behavior of these proteins during complex formation with the repressor protein Gal80p. To address these queries we have modeled the binding interface of the Gal1p-Gal80p and Gal3p-Gal80p complexes. The comparison of the dynamics of these proteins in the complex and in the Apo protein was carried out. It was observed that the binding of Gal3p with Gal80p induces significant flexibility in Gal80p on a surface different from the one involved in binding with Gal3p. Several other differences at the interface between the Gal3p-Gal80p and the Gal1p-Gal80p complex were observed, which might permit Gal3p to act as a transcriptional inducer with higher activity. Further, we have discussed the dynamical event and plausible mechanism of complex formation of Gal3p and Gal1p with Gal80p at the molecular level.

Regulation of glucose-dependent gene expression by the RNA helicase Dbp2 in Saccharomyces cerevisiae.

Cellular homeostasis requires a fine balance between energy uptake, utilization, and growth. Dbp2 is a member of the DEAD-box protein family in Saccharomyces cerevisiae with characterized ATPase and helicase activity in vitro. DEAD-box RNA helicases are a class of enzymes that utilize ATP hydrolysis to remodel RNA and/or RNA-protein (RNP) composition. Dbp2 has been proposed to utilize its helicase activity in vivo to promote RNA-protein complex assembly of both messenger (m)RNAs and long noncoding (lnc)RNAs. Previous work from our laboratory demonstrated that loss of DBP2 enhances the lncRNA-dependent transcriptional induction of the GAL genes by abolishing glucose-dependent repression. Herein, we report that either a carbon source switch or glucose deprivation results in rapid export of Dbp2 to the cytoplasm. Genome-wide RNA sequencing identified a new class of antisense hexose transporter transcripts that are specifically upregulated upon loss of DBP2. Further investigation revealed that both sense and antisense hexose transporter (HXT) transcripts are aberrantly expressed in DBP2-deficient cells and that this expression pathway can be partially mimicked in wild-type cells by glucose depletion. We also find that Dbp2 promotes ribosome biogenesis and represses alternative ATP-producing pathways, as loss of DBP2 alters the transcript levels of ribosome biosynthesis (snoRNAs and associated proteins) and respiration gene products. This suggests that Dbp2 is a key integrator of nutritional status and gene expression programs required for energy homeostasis.

Rrd1p, an RNA polymerase II-specific prolyl isomerase and activator of phosphoprotein phosphatase, promotes transcription independently of rapamycin response.

Rrd1p (resistance to rapamycin deletion 1) has been previously implicated in controlling transcription of rapamycin-regulated genes in response to rapamycin treatment. Intriguingly, we show here that Rrd1p associates with the coding sequence of a galactose-inducible and rapamycin non-responsive GAL1 gene, and promotes the association of RNA polymerase II with GAL1 in the absence of rapamycin treatment following transcriptional induction. Consistently, nucleosomal disassembly at GAL1 is impaired in the absence of Rrd1p, and GAL1 transcription is reduced in the Δrrd1 strain. Likewise, Rrd1p associates with the coding sequences of other rapamycin non-responsive and inducible GAL genes to promote their transcription in the absence of rapamycin treatment. Similarly, inducible, but rapamycin-responsive, non-GAL genes such as CTT1, STL1 and CUP1 are also regulated by Rrd1p. However, transcription of these inducible GAL and non-GAL genes is not altered in the absence of Rrd1p when the steady-state is reached after long transcriptional induction. Consistently, transcription of the constitutively active genes is not changed in the Δrrd1 strain. Taken together, our results demonstrate a new function of Rrd1p in stimulation of initial rounds of transcription, but not steady-state/constitutive transcription, of both rapamycin-responsive and non-responsive genes independently of rapamycin treatment.

PHD and TFIIS-Like domains of the Bye1 transcription factor determine its multivalent genomic distribution.

The BYpass of Ess1 (Bye1) protein is a putative S. cerevisiae transcription factor homologous to the human cancer-associated PHF3/DIDO family of proteins. Bye1 contains a Plant Homeodomain (PHD) and a TFIIS-like domain. The Bye1 PHD finger interacts with tri-methylated lysine 4 of histone H3 (H3K4me3) while the TFIIS-like domain binds to RNA polymerase (Pol) II. Here, we investigated the contribution of these structural features to Bye1 recruitment to chromatin as well as its function in transcriptional regulation. Genome-wide analysis of Bye1 distribution revealed at least two distinct modes of association with actively transcribed genes: within the core of Pol II- and Pol III-transcribed genes concomitant with the presence of the TFIIS transcription factor and, additionally, with promoters of a subset of Pol II-transcribed genes. Specific loss of H3K4me3 abolishes Bye1 association to gene promoters, but doesn’t affect its binding within gene bodies. Genetic interactions suggested an essential role of Bye1 in cell fitness under stress conditions compensating the absence of TFIIS. Furthermore, BYE1 deletion resulted in the attenuation of GAL genes expression upon galactose-mediated induction indicating its positive role in transcription regulation. Together, these findings point to a bimodal role of Bye1 in regulation of Pol II transcription. It is recruited via its PHD domain to H3K4 tri-methylated promoters at early steps of transcription. Once Pol II is engaged into elongation, Bye1 binds directly to the transcriptional machinery, modulating its progression along the gene.

Minimum length of direct repeat sequences required for efficient homologous recombination induced by zinc finger nuclease in yeast.

Zinc finger nuclease (ZFN) technology is a powerful molecular tool for targeted genome modifications and genetic engineering. However, screening for specific ZFs and validation of ZFN activity are labor intensive and time consuming. We previously designed a yeast-based ZFN screening and validation system by inserting a ZFN binding site flanked by a 164 bp direct repeat sequence into the middle of a Gal4 transcription factor, disrupting the open reading frame of the yeast Gal4 gene. Expression of the ZFN causes a double stranded break at its binding site, which promotes the cellular DNA repair system to restore expression of a functional Gal transcriptional factor via homologous recombination. Expression of Gal4 transcription factor leads to activation of three reporter genes in an AH109 yeast two-hybrid strain. However, the 164 bp direct repeat appears to generate spontaneous homologous recombination frequently, resulting in many false positive ZFNs. To overcome this, a series of DNA fragments of various lengths from 10 to 150 bp with 10 bp increase each and 164 bp direct repeats flanking the ZFN binding site were designed and constructed. The results demonstrated that the minimum length required for ZFN-induced homologous recombination was 30 bp, which almost eliminated spontaneous recombination. Using the 30 bp direct repeat sequence, ZFN could efficiently induce homologous recombination, while false positive ZFNs resulting from spontaneous homologous recombination were minimized. Thus, this study provided a simple, fast and sensitive ZFN screening and activity validation system in yeast.

Getting too sweet: galectin-1 dysregulation in gestational diabetes mellitus

Galectin-1 (gal-1) is a prototype carbohydrate-binding protein, whose dysregulation is associated with adverse pregnancy outcomes such as spontaneous abortion and pre-eclampsia. Furthermore, it is known that faulty gal-1 protein production or gene regulation can be caused by single-nucleotide polymorphisms in the LGALS1 gene. Gestational diabetes mellitus (GDM) is also an adverse pregnancy outcome and the most common metabolic disorder during gestation. However, gal-1 expression patterns during GDM remain largely unknown. Our aims were to define local and peripheral gal-1 expression patterns during pregnancy, and to investigate LGALS1 gene polymorphisms in GDM patients. Circulating gal-1 levels were determined by ELISA in GDM patients and normal pregnant controls, and LGALS1 gene polymorphisms were assessed for association with GDM. Placental tissues were collected from control and GDM term pregnancies to evaluate local gal-1 expression by immunofluorescence. Our results show that GDM is associated with a failure to increase circulating gal-1 levels during the second and third trimester, as well as overexpression of gal-1 in placental tissue. Additionally, the LGALS1 polymorphism rs4820294 was associated with the development of GDM. In pregnancies complicated by GDM, we observed gal-1 dysregulation both locally in the placenta and peripherally in the circulation. Furthermore, the association between the LGALS1 polymorphism and GDM may indicate a genetic contribution to this adverse pregnancy outcome.

Stochastic galactokinase expression underlies GAL gene induction in a GAL3 mutant of Saccharomyces cerevisiae.

GAL1 and GAL3 are paralogous signal transducers that functionally inactivate Gal80p to activate the Gal4p-dependent transcriptional activation of GAL genes in Saccharomycescerevisiae in response to galactose. Unlike a wild-type strain, the gal3∆ strain shows delayed growth kinetics as a result of the signaling function of GAL1. The mechanism ensuring that GAL1 is eventually expressed to turn on the GAL switch in the gal3∆ strain remains a paradox. Using galactose and histidine growth complementation assays, we demonstrate that 0.3% of the gal3∆ cell population responds to galactose. This is corroborated by flow cytometry and microscopic analysis. The galactose responders and nonresponders isolated from the galactose-adapted population attain the original bimodal state and this phenotype is found to be as hard wired as a genetic trait. Computational analysis suggests that the log-normal distribution in GAL4 synthesis can lead to bimodal expression of GAL80, resulting in the bimodal expression of GAL genes. Heterozygosity at the GAL80 but not at the GAL1, GAL2 or GAL4 locus alters the extent of bimodality of the gal3∆ cell population. We suggest that the asymmetric expression pattern between GAL1 and GAL3 results in the ability of S.cerevisiae to activate the GAL pathway by conferring nongenetic heterogeneity.

Deletion of the HXK2 gene in Saccharomyces cerevisiae enables mixed sugar fermentation of glucose and galactose in oxygen-limited conditions

Galactose is one of the major sugar components of red seaweeds along with glucose. In Saccharomyces cerevisiae, synthesis of galactose-metabolizing enzymes (the Leloir proteins) is under tight and complex regulation in the presence of glucose by a mechanism called catabolite repression. As a result, when both sugars are concurrently present in the medium under oxygen-limited conditions, the yeast cannot utilize galactose even after glucose consumption. Research efforts on mixed sugar fermentation of glucose/galactose, therefore, were made in the presence of oxygen, thereby resulting in sequential substrate consumption, and low ethanol yield and productivity. In this study, mixed sugar fermentation of glucose and galactose in oxygen-limited conditions was achieved by deleting the HXK2 gene, a moonlighting protein acting as hexokinase or repressor involved in catabolite repression of S. cerevisiae. Remarkably, the S. cerevisiae D452-2Δhxk2 strain utilized galactose after glucose depletion without a diauxic lag period, whereas the parental strain could not use galactose at all under oxygen-limited conditions. The Δhxk2 strain fermented galactose at the consumption rate of 3.02±0.10g/L/h to produce ethanol with a yield of 0.44±0.01g ethanol/g galactose. Complementation of the Δhxk2 strain with the plasmid-harbored HXK2 gene under the control of various promoters clearly showed that the galactose consumption rates were inversely related to the mRNA levels of the HXK2 gene. The transcription of the GAL genes was dramatically elevated by deleting the HXK2 gene, whereas the expressions of hexokinase genes were not significantly affected. We concluded that the HXK2-deleted strain is able to efficiently utilize both glucose and galactose in the oxygen-limited conditions by alleviating catabolite-repression.

Unmixing of fluorescence spectra to resolve quantitative time-series measurements of gene expression in plate readers

BackgroundTo connect gene expression with cellular physiology, we need to follow levels of proteins over time. Experiments typically use variants of Green Fluorescent Protein (GFP), and time-series measurements require specialist expertise if single cells are to be followed. Fluorescence plate readers, however, a standard in many laboratories, can in principle provide similar data, albeit at a mean, population level. Nevertheless, extracting the average fluorescence per cell is challenging because autofluorescence can be substantial.ResultsHere we propose a general method for correcting plate reader measurements of fluorescent proteins that uses spectral unmixing and determines both the fluorescence per cell and the errors on that fluorescence. Combined with strain collections, such as the GFP fusion collection for budding yeast, our methodology allows quantitative measurements of protein levels of up to hundreds of genes and therefore provides complementary data to high throughput studies of transcription. We illustrate the method by following the induction of the GAL genes in Saccharomyces cerevisiae for over 20 hours in different sugars and argue that the order of appearance of the Leloir enzymes may be to reduce build-up of the toxic intermediate galactose-1-phosphate. Further, we quantify protein levels of over 40 genes, again over 20 hours, after cells experience a change in carbon source (from glycerol to glucose).ConclusionsOur methodology is sensitive, scalable, and should be applicable to other organisms. By allowing quantitative measurements on a per cell basis over tens of hours and over hundreds of genes, it should increase our understanding of the dynamic changes that drive cellular behaviour.