Gal4 Research Articles

Strain variation in Candida albicans glycolytic gene regulation.

Central carbon metabolism is vital for the proliferation of Candida albicans, a fungus that is prominent as a commensal and pathogen. Glycolytic genes are activated by overlapping activities of the transcription factors Tye7 and Gal4, as shown by studies in the SC5314 genetic background. However, regulatory relationships can vary among C. albicans isolates. Here, we analyzed Tye7- and Gal4-related phenotypes in five diverse clinical isolates of C. albicans. We tested growth properties and gene expression impact through Nanostring profiling and, for the two strains SC5314 and P87, RNA sequencing. Our results lead to three main conclusions. First, the functional redundancy of Tye7 and Gal4 for glycolytic gene activation is preserved among all strains tested. Second, at the gene expression level, strain P87 is an outlier with regard to tye7Δ/Δ impact, and strain SC5314 is an outlier with regard to gal4Δ/Δ impact. Third, while Gal4 is well known to be dispensable for induction of the GAL1, GAL7, and GAL10 galactose-specific metabolic genes, we find that gal4Δ/Δ mutants of several strains have a mild galactose fermentation defect, as assayed by growth on galactose with the respiration inhibitor antimycin A. Our findings indicate that even a central metabolic regulatory network is subject to strain variation and illustrates an unexpected genotype-phenotype relationship.The fungal commensal and pathogen Candida albicans rely upon metabolic flexibility to colonize and infect host niches. Central carbon metabolism is governed by two regulators, Tye7 and Gal4, as defined in the reference strain SC5314. Here, we have explored the impact of Tye7 and Gal4 on carbon utilization and gene expression across five diverse C. albicans clinical isolates. Novel aspects of this study are the finding that even a central metabolic regulatory network is subject to strain variation and the observation of an unexpected mutant phenotype.

Single-Neuron Labeling in Drosophila Using Multicolor FLP-Out.

Neurons exhibit some of the most striking examples of morphological diversity of any cell type. Thus, when studying neurons, the morphology of each neuron must be considered individually. However, neurons densely populate the central nervous system (CNS), making it difficult to ascertain fine morphological features due to a lack of spatial resolution. In Drosophila, this problem can be partially resolved by using driver lines that express the yeast transcription factor GAL4 in subsets of neurons. GAL4 can activate the expression of other introduced genetic elements such as genes for fluorescent proteins or other markers under the control of the GAL4 upstream activation sequences (UAS effectors). However, even highly specific GAL4 lines often label sets of potentially morphologically heterogeneous neurons. Here, we describe a protocol for using the multicolor flip-out (MCFO) technique in Drosophila melanogaster to stochastically label individual neurons within a GAL4 expression pattern. MCFO relies on the binary GAL4/UAS expression system in Drosophila but adds additional control for how densely the neurons within a GAL4 expression pattern are labeled via user-controlled heat shock. Specifically, three discrete UAS effector elements containing the sequences for unique epitope tags (FLAG, HA, and V5) linked to a gene for nonfluorescent GFP can be independently expressed under the control of GAL4 only when a transcriptional stop sequence in the UAS promoter sequence has been removed by heat shock-induced recombination. This effectively labels multiple individual neurons with either one or a combination of epitope tags that can be spectrally resolved with immunofluorescence. The MCFO technique is ideal for researchers who want to determine morphological features of CNS neurons in wild-type or mutant backgrounds.

A Gaussia luciferase reporter assay for the evaluation of coronavirus Nsp5/3CLpro activity

Human coronaviruses (hCoVs) infect millions of people every year. Among these, MERS, SARS-CoV-1, and SARS-CoV-2 caused significant morbidity and mortality and their emergence highlights the risk of possible future coronavirus outbreaks. Therefore, broadly-active anti-coronavirus drugs are needed. Pharmacological inhibition of the hCoV protease Nsp5 (3CLpro) is clinically beneficial as shown by the wide and effective use of Paxlovid (nirmatrelvir, ritonavir). However, further treatment options are required due to the risk of drug resistance. To facilitate the assessment of coronavirus protease function and its pharmacological inhibition, we developed an assay allowing rapid and reliable quantification of Nsp5 activity under biosafety level 1 conditions. It is based on an ACE2-Gal4 transcription factor fusion protein separated by a Nsp5 recognition site. Cleavage by Nsp5 releases the Gal4 transcription factor, which then induces the expression of Gaussia luciferase. Our assay is compatible with Nsp5 proteases from all hCoVs and allows simultaneous measurement of inhibitory and cytotoxic effects of the tested compounds. Proof-of-concept measurements confirmed that nirmatrelvir, GC376 and lopinavir inhibit SARS-CoV-2 Nsp5 function. Furthermore, the assay accurately predicted the impact of Nsp5 mutations on catalytic activity and inhibitor sensitivity. Overall, the reporter assay is suitable for evaluating viral protease activity.

Conditional Inhibition of Eip75B Eliminates the Effects of Mating and Mifepristone on Lifespan in Female Drosophila.

Mating in female Drosophila melanogaster causes midgut hypertrophy and reduced lifespan, and these effects are blocked by the drug mifepristone. Eip75B is a transcription factor previously reported to have pleiotropic effects on Drosophila lifespan. Because Eip75B null mutations are lethal, conditional systems and/or partial knock-down are needed to study Eip75B effects in adults. Previous studies showed that Eip75B is required for adult midgut cell proliferation in response to mating. To test the possible role of Eip75B in mediating the lifespan effects of mating and mifepristone, a tripartite FLP-recombinase-based conditional system was employed that provides controls for genetic background. Expression of a Hsp70-FLP transgene was induced in third instar larvae by a brief heat pulse. The FLP recombinase catalyzed the recombination and activation of an Actin5C-GAL4 transgene. The GAL4 transcription factor in turn activated expression of a UAS-Eip75B-RNAi transgene. Inhibition of Eip75B activity was confirmed by loss of midgut hypertrophy upon mating, and the lifespan effects of both mating and mifepristone were eliminated. In addition, the negative effects of mifepristone on egg production were eliminated. The data indicate that Eip75B mediates the effects of mating and mifepristone on female midgut hypertrophy, egg production, and lifespan.

Transcriptome analysis reveals the effect of cold storage time on the expression of genes related to oxidative metabolism in Chinese black truffle.

Chinese black truffle (Tuber indicum) is a hypogenous fungus of great value due to its distinctive aroma. In this study, both transcriptome and physicochemical analyses were performed to investigate the changes of nutrients and gene expression in truffle fruiting bodies during cold storage. The results of physicochemical analysis revealed the active metabolism of fruiting bodies in cold storage, showing the decreased contents of protein and soluble sugar, the variations in both polyphenol oxidase activity and total phenol content, and the detrimental effect of reactive oxygen species production caused by heavy metals (cadmium and lead) in truffles. Transcriptome analysis identified a total of 139,489 unigenes. Down-regulated expression of genes encoding the catalase-like domain-containing protein (katE), glutaredoxin protein (GRX), a copper/zinc superoxide dismutase (Sod_Cu), and aspartate aminotransferase (AAT) affected the degradation metabolism of intracellular oxides. Ribulose-5-phosphate-3-epimerase (RPE) was a key enzyme in response to oxidative stress in truffle cells through the pentose phosphate pathway (PPP). A total of 51,612 simple sequence repeats were identified, providing valuable resources for further genetic diversity analysis, molecular breeding, and genetic map-ping in T. indicum. Transcription factors GAL4 and SUF4-like protein were involved in glucose metabolism and histone methylation processes, respectively. Our study provided a fundamental characterization of the physicochemical and molecular variations in T. indicum during the cold storage at 4°C, providing strong experimental evidence to support the improvement of storage quality of T. indicum.

How transcription factor binding controls transcriptional bursting dynamics: A single-molecule view

In this issue, Pomp etal.1 simultaneously tracked transcriptional bursts of yeast gene GAL10 and transient binding of transcription factor Gal4 at the gene using novel methods. Dynamic exchange and infrequent long binding of Gal4 together enable prolonged transcriptional bursts of GAL10.

Protein-Protein Interaction via Two-Hybrid Assay in Yeast.

The yeast two-hybrid assay enables detecting interactions between proteins, which makes this tool of particular interest for plant-virus interaction studies. Basically, the reporter gene expression (HIS3) is activated by the binding of a transcription factor GAL4, which, like eukaryotic transcription factors, is modular in nature and consists of two structurally independent domains: DNA-binding (DB) and activation (AD) domains. The two proteins under investigation are expressed separately, one fused to the BD domain and the other to the AD domain. In the yeast strain AH109, activation of the reporter gene occurs only in cells that contain interacting proteins, reconstituting the transcription factor GAL4 which then binds to the responsive promoter and results in yeast colony growth.

Seeing red: a modified RUBY reporter assay for visualizing inplanta protein-protein interactions.

Seeing red: a modified RUBY reporter assay for visualizing inplanta protein-protein interactions.

Dynamic epistasis analysis reveals how chromatin remodeling regulates transcriptional bursting

Transcriptional bursting has been linked to the stochastic positioning of nucleosomes. However, how bursting is regulated by the remodeling of promoter nucleosomes is unknown. Here, we use single-molecule live-cell imaging of GAL10 transcription in Saccharomyces cerevisiae to measure how bursting changes upon combined perturbations of chromatin remodelers, the transcription factor Gal4 and preinitiation complex components. Using dynamic epistasis analysis, we reveal how the remodeling of different nucleosomes regulates transcriptional bursting parameters. At the nucleosome covering the Gal4 binding sites, RSC and Gal4 binding synergistically facilitate each burst. Conversely, nucleosome remodeling at the TATA box controls only the first burst upon galactose induction. At canonical TATA boxes, the nucleosomes are displaced by TBP binding to allow for transcription activation even in the absence of remodelers. Overall, our results reveal how promoter nucleosome remodeling together with Gal4 and preinitiation complex binding regulates transcriptional bursting.

DNA supercoiling restricts the transcriptional bursting of neighboring eukaryotic genes

DNA supercoiling has emerged as a major contributor to gene regulation in bacteria, but how DNA supercoiling impacts transcription dynamics in eukaryotes is unclear. Here, using single-molecule dual-color nascent transcription imaging in budding yeast, we show that transcriptional bursting of divergent and tandem GAL genes is coupled. Temporal coupling of neighboring genes requires rapid release of DNA supercoils by topoisomerases. When DNA supercoils accumulate, transcription of one gene inhibits transcription at itsadjacent genes. Transcription inhibition of the GAL genes results from destabilized binding of the transcription factor Gal4. Moreover, wild-type yeast minimizes supercoiling-mediated inhibition by maintaining sufficient levels of topoisomerases. Overall, we discover fundamental differences in transcriptional control by DNA supercoiling between bacteria and yeast and show that rapid supercoiling release in eukaryotes ensures proper gene expression of neighboring genes.

Enhancement of Vivid-based photo-activatable Gal4 transcription factor in mammalian cells.

The Gal4/UAS system is a versatile tool to manipulate exogenous gene expression of cells spatially and temporally in many model organisms. Many variations of light-controllable Gal4/UAS system are now available, following the development of photo-activatable (PA) molecular switches and integration of these tools. However, many PA-Gal4 transcription factors have undesired background transcription activities even in dark conditions, and this severely attenuates reliable light-controlled gene expression. Therefore, it is important to develop reliable PA-Gal4 transcription factors with robust light-induced gene expression and limited background activity. By optimization of synthetic PA-Gal4 transcription factors, we have validated configurations of Gal4 DNA biding domain, transcription activation domain and blue light-dependent dimer formation molecule Vivid (VVD), and applied types of transcription activation domains to develop a new PA-Gal4 transcription factor we have named eGAV (enhanced Gal4-VVD transcription factor). Background activity of eGAV in dark conditions was significantly lower than that of hGAVPO, a commonly used PA-Gal4 transcription factor, and maximum light-induced gene expression levels were also improved. Light-controlled gene expression was verified in cultured HEK293T cells with plasmid-transient transfections, and in mouse EpH4 cells with lentivirus vector-mediated transduction. Furthermore, light-controlled eGAV-mediated transcription was confirmed in transfected neural stem cells and progenitors in developing and adult mouse brain and chick spinal cord, and in adult mouse hepatocytes, demonstrating that eGAV can be applied to a wide range of experimental systems and model organisms.Key words: optogenetics, Gal4/UAS system, transcription, gene expression, Vivid.

Functional identification of the zebrafish Interleukin-1 receptor in an embryonic model of Il-1β-induced systemic inflammation.

Interleukin-1β (IL-1β) is a potent proinflammatory cytokine that plays a vital role in the innate immune system. To observe the innate immune response in vivo, several transgenic zebrafish lines have been developed to model IL-1β-induced inflammation and to visualize immune cell migration and proliferation in real time. However, our understanding of the IL-1β response in zebrafish is limited due to an incomplete genome annotation and a lack of functional data for the cytokine receptors involved in the inflammatory process. Here, we use a combination of database mining, genetic analyses, and functional assays to identify zebrafish Interleukin-1 receptor, type 1 (Il1r1). We identified putative zebrafish il1r1 candidate genes that encode proteins with predicted structures similar to human IL1R1. To examine functionality of these candidates, we designed highly effective morpholinos to disrupt gene expression in a zebrafish model of embryonic Il-1β-induced systemic inflammation. In this double transgenic model, ubb:Gal4-EcR, uas:il1βmat , the zebrafish ubiquitin b (ubb) promoter drives expression of the modified Gal4 transcription factor fused to the ecdysone receptor (EcR), which in turn drives the tightly-regulated expression and secretion of mature Il-1β only in the presence of the ecdysone analog tebufenozide (Teb). Application of Teb to ubb:Gal4-EcR, uas:il1βmat embryos causes premature death, fin degradation, substantial neutrophil expansion, and generation of reactive oxygen species (ROS). To rescue these deleterious phenotypes, we injected ubb:Gal4-EcR, uas:il1βmat embryos with putative il1r1 morpholinos and found that knockdown of only one candidate gene prevented the adverse effects caused by Il-1β. Mosaic knockout of il1r1 using the CRISPR/Cas9 system phenocopied these results. Taken together, our study identifies the functional zebrafish Il1r1 utilizing a genetic model of Il-1β-induced inflammation and provides valuable new insights to study inflammatory conditions specifically driven by Il-1β or related to Il1r1 function in zebrafish.

Yeast 2-hybrid assay for investigating the interaction between the centrosome proteins PLK4 and STIL.

Centrosomes act as a major microtubule-organizing center in many animal cells. This non-membranous cell organelle is involved in a wide range of cell functions like division, migration, and signaling. Centrosome houses hundreds of proteins that interact in a regulated manner to carry out their tasks. Most of these centrosome proteins have either the coiled-coil or disordered regions, making them challenging to purify for functional analysis. Yeast 2-hybrid (Y2H) is an in vivo based method for studying protein-protein interactions by exploiting the modular nature of yeast's GAL4 transcription factor. This method provides an alternative strategy for investigating bi-molecular interactions, especially when dealing with otherwise difficult proteins to purify by conventional biochemical approaches. We present a detailed protocol for showing the interaction between the two core centrosome proteins, i.e., PLK4 and STIL, using the Y2H assay.

Abstract 2478: Elevating SOX2 inhibits the proliferation of tumor cells independently of DNA binding

Abstract Purpose: The purpose of these studies was to understand how elevated SOX2 affects tumor cell proliferation. The stem cell transcription factor SOX2 has been implicated in ~20 different tumor types. Importantly, several studies have demonstrated that the effects of SOX2 during development and malignancy are dependent on its dosage. High levels of SOX2 in both contexts are associated with a quiescent, stem-like phenotype. SOX2high quiescent tumor cells present a significant clinical challenge as these cells are largely resistant to conventional chemotherapies and, thus, provide a reservoir to drive disease recurrence. Therefore, understanding the mechanisms through which elevated SOX2 inhibits tumor cell proliferation will provide insights into the biology of drug-resistant, quiescent tumor cells. Methods: Tumor cell lines representative of medulloblastoma (MB) and colorectal (CRC) tumor cells were engineered for the doxycycline (Dox)-inducible expression of mutant forms of SOX2 that are unable to bind to DNA. One SOX2 mutant was created by substituting a proline for a glycine in the DNA binding domain of SOX2 [SOX2(G76P)]. A second SOX2 mutant replaces the DNA binding domain of SOX2 with the DNA binding domain of the yeast transcription factor Gal4 (Gal4-SOX2). The effects of elevating mutant forms of SOX2 on cell proliferation and the cell cycle distribution of the cells were compared to the effects of elevating wild-type SOX2. Results: Our studies demonstrate that elevating SOX2, SOX2(G76P) or Gal4-SOX2 decreases the proliferation of MB and CRC cells to similar extents. In contrast, inducible expression of the DNA binding domain of Gal4 had no effect on cell proliferation. We previously reported that while SOX2 elevation inhibits the proliferation of a diverse set of tumor cell lines, it does not significantly alter their cell cycle distribution. Similarly, elevating SOX2(G76P) or Gal4-SOX2 did not significantly alter cell cycle distribution. To extend these findings, we are engineering additional SOX2 mutants to map the location of the domain of SOX2 required for growth inhibition. We recently engineered MB and CRC cells to express a mutant form of SOX2 that lacks the c-terminal R1 transactivation domain [Gal4-SOX2(ΔR1)]. Significantly, removing the R1 transactivation domain eliminated the growth inhibitory effects of Gal4-SOX2 when elevated. Conclusion: Our findings demonstrate that elevating two mutant forms of SOX2 that cannot bind to DNA inhibit the proliferation of tumor cell lines that represent two different forms of cancer, similar to the effects of wild-type SOX2. Equally important, our studies indicate that the c-terminal R1 transactivation domain of SOX2 is necessary, but no sufficient, to inhibit proliferation when SOX2 levels are elevated. Collectively, these findings and our previously published work indicate that elevated levels of SOX2 inhibit cell proliferation independently of DNA binding. Citation Format: Ethan Patrick Metz, Phillip J. Wilder, Angie Rizzino. Elevating SOX2 inhibits the proliferation of tumor cells independently of DNA binding [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2478.

Combinatorial Use of Bexarotene Plus ATRA for the Treatment of Acute Myeloid Leukemia

Aim: All-trans retinoic acid (ATRA) results in high response rates in acute promyelocytic leukemia (APL), but has not had a significant impact on non-APL forms of acute myeloid leukemia (AML), even though retinoic acid receptors have been implicated in hematopoietic maturation and self-renewal. RXRs bind as heterodimers with RARs, and are expressed at higher levels in AML cells than are RARs. We sought to determine whether natural RXR ligands exist in primary leukemia cells, whether RXR ligands might augment or inhibit leukemic cell growth, and whether RXRs might be rational targets for AML therapeutics.Background: Retinoid receptors are nuclear hormone receptors, induced during terminal myeloid maturation with highest expressed in M4 and M5 AML. Retinoids serve as intracellular messengers and are activating ligands for the retinoic acid receptors (RAR) and retinoid X receptors (RXR).Methods: We developed an in vivo reporter assay that measures intracellular, activating nuclear receptor ligands in mouse bone marrow cells. We generated transgenic mice with GFP regulated by a UAS promoter. The yeast Gal4 transcription factor recognizes UAS promoter sequences. When a fusion of the Gal4-DNA binding domain (DBD) and a retinoid receptor ligand-binding domain (LBD) is expressed in UAS-GFP bone marrow cells, and an activating ligand is present, the cells express GFP. Leukemia was generated in bone marrow cells from these mice and from Mx1-Cre x Rxrαfl/fl x Rxrβfl/fl mice using MSCV-MLL-AF9 retrovirus.Results: We found that intermediate levels of RXRA ligands are present in mouse MLL-AF9 leukemia cells in vivo. To determine if activation of RXRA is required for MLL-AF9 cell growth, we transduced MSCV-MLL-AF9 retrovirus into bone marrow cells from Mx1-Cre+ x Rxrαfl/fl x Rxrβfl/fl mice and transplanted these into recipient mice. Mx1-Cre is “leaky.” Without external activation of Mx1-Cre (i.e. without pIpC treatment) we found that all subsequent leukemias were associated with deleted (“floxed out”) Rxra and Rxrb alleles, suggesting that the deleted allele was positively selected for. In secondary transplants, we found that MLL-AF9 leukemias with deleted Rxrα and Rxrβ were associated with shorter survival when compared with MLL-AF9 leukemias with wild type Rxrα and Rxrβ, suggesting that naturally occurring RXR ligands suppressed leukemic growth. To determine if pharmacologic activation of retinoid receptors could inhibit MLL-AF9 cell growth, we treated MLL-AF9 cells ex vivo with ATRA and bexarotene. We found that ATRA modestly inhibited growth, and this effect reached a plateau at 62 nM (this may partially explain the limited clinical efficacy of ATRA in non-APL AML) (see Figure). In contrast, the EC50 of bexarotene was 125 nM and the EC50 decreased to 54 nM when bexarotene was combined with 62 nM ATRA. Higher doses of ATRA decreased the bexarotene EC50 further to 43 nM and resulted in near complete culture collapse. Finally, to determine whether combination bexarotene and ATRA inhibited cell growth by inducing differentiation or apoptosis, we examined cytomorphology, cell division by Celltrace Violet staining (similar to CFSE), and Annexin V. Unexpectedly, we observed no evidence of differentiation, but rather increased Annexin V associated with ATRA and bexarotene-induced growth arrest.Conclusion: RXRs act as tumor suppressors in MLL-AF9 murine leukemia cells in vivo, and the activation of RXR/RAR heterodimers with bexarotene and all-trans retinoic acid leads to synergistic apoptosis and growth arrest. Additional experiments are now being performed to test the efficacy of this dug combination in vivo and assess their potential use in elderly relapsed/refractory AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Using the GAL4-UAS System for Functional Genetics in Anopheles gambiae.

The bipartite GAL4-UAS system is a versatile and powerful tool for functional genetic analysis. The essence of the system is to cross transgenic 'driver' lines that express the yeast transcription factor GAL4 in a tissue specific manner, with transgenic 'responder' lines carrying a candidate gene/RNA interference construct whose expression is controlled by Upstream Activation Sequences (UAS) that bind GAL4. In the ensuing progeny, the gene or silencing construct is thus expressed in a prescribed spatiotemporal manner, enabling the resultant phenotypes to be assayed and gene function inferred. The binary system enables flexibility in experimental approaches to screen phenotypes generated by transgene expression in multiple tissue-specific patterns, even if severe fitness costs are induced. We have adapted this system for Anopheles gambiae, the principal malaria vector in Africa. In this article, we provide some of the common procedures used during GAL4-UAS analysis. We describe the An. gambiae GAL4-UAS lines already generated, as well as the cloning of new responder constructs for upregulation and RNAi knockdown. We specify a step by step guide for sexing of mosquito pupae to establish genetic crosses, that also includes screening progeny to follow inheritance of fluorescent gene markers that tag the driver and responder insertions. We also present a protocol for clearing An. gambiae embryos to study embryonic development. Finally, we introduce potential adaptions of the method to generate driver lines through CRISPR/Cas9 insertion of GAL4 downstream of target genes.

Mediator subunit Med15 dictates the conserved \u201cfuzzy\u201d binding mechanism of yeast transcription activators Gal4 and Gcn4

The acidic activation domain (AD) of yeast transcription factor Gal4 plays a dual role in transcription repression and activation through binding to Gal80 repressor and Mediator subunit Med15. The activation function of Gal4 arises from two hydrophobic regions within the 40-residue AD. We show by NMR that each AD region binds the Mediator subunit Med15 using a “fuzzy” protein interface. Remarkably, comparison of chemical shift perturbations shows that Gal4 and Gcn4, two intrinsically disordered ADs of different sequence, interact nearly identically with Med15. The finding that two ADs of different sequence use an identical fuzzy binding mechanism shows a common sequence-independent mechanism for AD-Mediator binding, similar to interactions within a hydrophobic cloud. In contrast, the same region of Gal4 AD interacts strongly with Gal80 via a distinct structured complex, implying that the structured binding partner of an intrinsically disordered protein dictates the type of protein–protein interaction.

Giant GAL gene clusters for the melibiose-galactose pathway in Torulaspora.

In many yeast species, the three genes at the centre of the galactose catabolism pathway, GAL1, GAL10 and GAL7, are neighbours in the genome and form a metabolic gene cluster. We report here that some yeast strains in the genus Torulaspora have much larger GAL clusters that include genes for melibiase (MEL1), galactose permease (GAL2), glucose transporter (HGT1), phosphoglucomutase (PGM1) and the transcription factor GAL4, in addition to GAL1, GAL10, and GAL7. Together, these eight genes encode almost all the steps in the pathway for catabolism of extracellular melibiose (a disaccharide of galactose and glucose). We show that a progenitor 5‐gene cluster containing GAL 7‐1‐10‐4‐2 was likely present in the common ancestor of Torulaspora and Zygotorulaspora. It added PGM1 and MEL1 in the ancestor of most Torulaspora species. It underwent further expansion in the T. pretoriensis clade, involving the fusion of three progenitor clusters in tandem and the gain of HGT1. These giant GAL clusters are highly polymorphic in structure, and subject to horizontal transfers, pseudogenization and gene losses. We identify recent horizontal transfers of complete GAL clusters from T. franciscae into one strain of T. delbrueckii, and from a relative of T. maleeae into one strain of T. globosa. The variability and dynamic evolution of GAL clusters in Torulaspora indicates that there is strong natural selection on the GAL pathway in this genus.

Directed evolution of the transcription factor Gal4 for development of an improved transcriptional regulation system in Saccharomyces cerevisiae

As a well-characterized eukaryotic DNA binding transcription factor, Gal4 has been extensively employed to construct controllable gene expression systems in Saccharomyces cerevisiae. The problem of insufficient inducibility arises in the constructs with multiples genes under control of GAL promoters due to the low expression level and activity of the native Gal4. In order to obtain improved transcriptional regulation systems for multi-gene pathways, Gal4 mutants with improved activity were created by directed evolution. During the preliminary screening, five positive Gal4 variants were isolated based on the lycopene-indicated high-throughput screening method. Analysis of the mutation sites revealed that the majority of positive mutations are localized in the middle homology region with unspecified function, suggesting an important role of this domain in transactivation of Gal4. Through combinatorial site-directed mutagenesis, the best variant Gal4T406A/V413A was obtained, which successfully increased the transcription of PGAL-driven lycopene pathway genes and led to 48 % higher lycopene accumulation relative to the wild-type Gal4. This study demonstrates the viability of modifying Gal4 activity by directed evolution for elevated expression of PGAL-driven genes and therefore enhanced production of the target metabolite.

A Study of Transcriptional Activation by the Transcription Factor GAL4 in Saccharomyces Cerevisiae by 3D Orbital Tracking and In Vivo RNA Labelling

Understanding what is going on at the molecular level within individual cells is challenging. But deciphering stochastic biomolecular processes is crucial for our understanding of gene transcription and the intricacies of cellular metabolism. With 3D orbital tracking, we are able to visualize and monitor pre-mRNA and transcription factors in real-time using fluorescent tagging within yeast cells at a high sampling rate. Our study demonstrates that we can track these molecules of interest, fluorescent-labeled GFP (pre-mRNA) and JF 646 dye (transcription factors), during the process of transcribing a gene that codes for metabolizing galactose.