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The ability to independently control gene expression in two different tissues in the same animal is emerging as a major need, especially in the context of inter-organ communication studies. This type of study is made possible by technologies combining the GAL4/UAS and a second binary expression system such as the LexA system or QF system. Here, we describe a resource of reagents that facilitate combined use of the GAL4/UAS and a second binary system in various Drosophila tissues. Focusing on genes with well-characterized GAL4 expression patterns, we generated a set of more than 40 LexA-GAD and QF2 insertions by CRISPR knock-in and verified their tissue specificity in larvae. We also built constructs that encode QF2 and LexA-GAD transcription factors in a single vector. Following successful integration of this construct into the fly genome, FLP/FRT recombination is used to isolate fly lines that express only QF2 or LexA-GAD. Finally, using new compatible shRNA vectors, we evaluated both LexA and QF systems for in vivo gene knockdown and are generating a library of such RNAi fly lines as a community resource. Together, these LexA and QF system vectors and fly lines will provide a new set of tools for researchers who need to activate or repress two different genes in an orthogonal manner in the same animal.

Abstract One of the greatest strengths of Drosophila genetics is its easily observable and selectable phenotypic markers. The mini-white marker has been widely used as a transgenic marker for Drosophila transgenesis. Flies carrying a mini-white construct can exhibit various eye colors ranging from pale orange to intense red, depending on the insertion site and gene dosage. Because the two copies of the mini-white marker show a stronger orange color, this is often used for selecting progenies carrying two transgenes together in a single chromosome after chromosomal recombination. However, some GAL4 lines available in the fly community originally have very strong red eyes. Without employing another marker, such as GFP, generating a recombinant chromosome with the strong red-eyed GAL4 and a desired UAS-transgene construct may be difficult. Therefore, we decided to change the red eyes of GAL4 lines to orange color. To change the eye color of the fly, we tested the CRISPR/Cas9 method with a guide RNA targeting the white gene with OK371-GAL4 and elav-GAL4. After a simple screening, we have successfully obtained multiple lines of orange-eyed OK371-GAL4 and elav-GAL4 that still maintain their original expression patterns. All of these simple experiments were performed by undergraduate students, allowing them to learn about a variety of different genetic experiments and genome editing while contributing to the fly research community by creating fruit fly lines that will be used in real-world research.

One of the greatest strengths of Drosophila genetics is its easily observable and selectable phenotypic markers. The mini-white marker has been widely used as a transgenic marker for Drosophila transgenesis. Flies carrying a mini-white construct can exhibit various eye colors ranging from pale orange to intense red, depending on the insertion site and gene dosage. Because the two copies of the mini-white marker show a stronger orange color, this is often used for selecting progenies carrying two transgenes together in a single chromosome after chromosomal recombination. However, some GAL4 lines available in the fly community originally have very strong red eyes. Without employing another marker, such as GFP, generating a recombinant chromosome with the strong red-eyed GAL4 and a desired UAS-transgene construct may be difficult. Therefore, we decided to change the red eyes of GAL4 lines to orange color. To change the eye color of the fly, we tested the CRISPR/Cas9 method with a guide RNA targeting the white gene with OK371-GAL4 and elav-GAL4. After a simple screening, we have successfully obtained multiple lines of orange-eyed OK371-GAL4 and elav-GAL4 that still maintain their original expression patterns. All of these simple experiments were performed by undergraduate students, allowing them to learn about a variety of different genetic experiments and genome editing while contributing to the fly research community by creating fruit fly lines that will be used in real-world research. [ABSTRACT FROM AUTHOR]

The ability to independently control gene expression in two different tissues in the same animal is emerging as a major need, especially in the context of inter-organ communication studies. This type of study is made possible by technologies combining the GAL4/UAS and a second binary expression system such as the LexA system or QF system. Here, we describe a resource of reagents that facilitate combined use of the GAL4/UAS and a second binary system in various Drosophila tissues. Focusing on genes with well-characterized GAL4 expression patterns, we generated a set of more than 40 LexA-GAD and QF2 insertions by CRISPR knock-in and verified their tissue specificity in larvae. We also built constructs that encode QF2 and LexA-GAD transcription factors in a single vector. Following successful integration of this construct into the fly genome, FLP/FRT recombination is used to isolate fly lines that express only QF2 or LexA-GAD. Finally, using new compatible shRNA vectors, we evaluated both LexA and QF systems for in vivo gene knockdown and are generating a library of such RNAi fly lines as a community resource. Together, these LexA and QF system vectors and fly lines will provide a new set of tools for researchers who need to activate or repress two different genes in an orthogonal manner in the same animal.

Drosophila has been a powerful model system for biological studies due to the wide range of genetic tools established for it. Among these tools, Gal4 is the most abundant, offering unparalleled tissue and developmental stage specificity for gene manipulation. In comparison, other genetic reagents are far fewer in choices. Here we present a genetic toolkit for converting Gal4 strains into LexA and Flippase transgenes through simple genetic crosses and fluorescence screening. We demonstrate the proof-of-principle by converting ten Gal4 lines that exhibit diverse tissue specificities and examined the activity patterns of the converted LexA and Flippase lines. Gal4- to-LexA and Flp conversion is fast and convenient and should greatly expand the choices of LexA and Flp for binary expression and FRTbased mosaic analysis, respectively, in Drosophila. [ABSTRACT FROM AUTHOR]

Genetically-encoded combinatorial peptide libraries are convenient tools to identify peptides to be used as therapeutics, antimicrobials and functional synthetic biology modules. Here, we report the identification and characterization of a cyclic peptide, G4CP2, that interferes with the GAL4 protein, a transcription factor responsible for the activation of galactose catabolism in yeast and widely exploited in molecular biology. G4CP2 was identified by screening CYCLIC, a Yeast Two-Hybrid-based combinatorial library of cyclic peptides developed in our laboratory. G4CP2 interferes with GAL4-mediated activation of galactose metabolic enzymes both when expressed intracellularly, as a recombinant peptide, and when provided exogenously, as a chemically-synthesized cyclic peptide. Our results support the application of G4CP2 in microbial biotechnology and, additionally, demonstrate that CYCLIC can be used as a tool for the rapid identification of peptides, virtually without any limitations with respect to the target protein. The possible biotechnological applications of cyclic peptides are also discussed.

Precise, repeatable genetic access to specific neurons via GAL4/UAS and related methods is a key advantage of Drosophila neuroscience. Neuronal targeting is typically documented using light microscopy of full GAL4 expression patterns, which generally lack the single-cell resolution required for reliable cell type identification. Here, we use stochastic GAL4 labeling with the MultiColor FlpOut approach to generate cellular resolution confocal images at large scale. We are releasing aligned images of 74,000 such adult central nervous systems. An anticipated use of this resource is to bridge the gap between neurons identified by electron or light microscopy. Identifying individual neurons that make up each GAL4 expression pattern improves the prediction of split-GAL4 combinations targeting particular neurons. To this end, we have made the images searchable on the NeuronBridge website. We demonstrate the potential of NeuronBridge to rapidly and effectively identify neuron matches based on morphology across imaging modalities and datasets.

Previously, the Nine amino acid TransActivation Domain (9aaTAD) was identified in the Gal4 region 862–870 (DDVYNYLFD). Here, we identified 9aaTADs in the distal Gal4 orthologs by our prediction algorithm and found their conservation in the family. The 9aaTAD function as strong activators was demonstrated. We identified adjacent Gal4 region 871–811 (DEDTPPNPKKE) as a natural 9aaTAD inhibitory domain located at the extreme Gal4 terminus. Moreover, we identified conserved Gal4 region 172–185 (FDWSEEDDMSDGLP), which was capable to reverse the 9aaTAD inhibition. In conclusion, our results uncover the existence of the cryptic inhibitory domains, which need to be carefully implemented in all functional studies with transcription factors to avoid incorrect conclusions. • Gal4 9aaTAD could be inhibited by adjacent region 862–870 (DEDTPPNPKKE). • The inhibited Gal4 9aaTAD could be reactivated by region 172–185 (FDWSEEDDMSDGLP). • The 9aaTAD activation domains could be identified by our 9aaTAD online prediction algorithm. [ABSTRACT FROM AUTHOR]

Cdk8 of the RNA polymerase II mediator kinase complex regulates gene expression by phosphorylating sequence-specific transcription factors. This function is conserved amongst eukaryotes, but the signals and mechanisms regulating Cdk8 activity and phosphorylation of its substrates are unknown. Full induction of the GAL genes in yeast requires phosphorylation of the transcriptional activator Gal4 by Cdk8. We used a screen to identify regulators of the Cdk8-dependent phosphorylation on Gal4, from which we identified multiple mutants with defects in TORC1 signaling. One mutant, designated gal four throttle 1 (gft1) was identified as a recessive allele of hom3, encoding aspartokinase, and mutations in hom3 caused effects typical of inhibition of TORC1, including rapamycin sensitivity and enhanced nuclear localization of the TORC1-responsive transcription factor Gat1. Mutations in hom3 also inhibit phosphorylation of Gal4 in vivo at the Cdk8-dependent site on Gal4, as did mutations of tor1, but these mutations did not affect activity of Cdk8 assayed in vitro. Disruption of cdc55, encoding a regulatory subunit of the TORC1-regulated protein phosphatase PP2A, suppressed the effect of hom3 and tor1 mutations on GAL expression, and also restored phosphorylation of Gal4 at the Cdk8-dependent site in vivo. These observations demonstrate that TORC1 signaling regulates GAL induction through the activity of PP2A/Cdc55 and suggest that Cdk8-dependent phosphorylation of Gal4 is opposed by PP2A/Cdc55 dephosphorylation. These results provide insight into how induction of transcription by a specific inducer can be modulated by global nutritional signals through regulation of Cdk8-dependent phosphorylation. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are post-transcriptional regulators that target specific mRNAs for repression and thus play key roles in many biological processes, including insect wing morphogenesis. miR-2 is an invertebrate-specific miRNA family that has been predicted in the fruit fly, Drosophila melanogaster, to be involved in regulating the Notch signaling pathway. We show here that miR-2 plays a critical role in wing morphogenesis in the silkworm, Bombyx mori, a lepidopteran model insect. Transgenic over-expression of a miR-2 cluster using a Gal4/UAS system results in deformed adult wings, supporting the conclusion that miR-2 regulates functions essential for normal wing morphogenesis. Two genes, abnormal wing disc (awd) and fringe (fng), which are positive regulators in Notch signaling, are identified as miR-2 targets and validated by a dual-luciferase reporter assay. The relative abundance of both awd and fng expression products was reduced significantly in transgenic animals, implicating them in the abnormal wing phenotype. Furthermore, somatic mutagenesis analysis of awd and fng using the CRISPR/Cas9 system and knock-out mutants also resulted in deformed wings similar to those observed in the miR-2 overexpression transgenic animals. The critical role of miR-2 in Bombyx wing morphogenesis may provide a potential target in future lepidopteran pest control.

The Drosophila melanogaster larval neuromuscular system is extensively used by researchers to study neuronal cell biology, and Drosophila glutamatergic motor neurons have become a major model system. There are two main Types of glutamatergic motor neurons, Ib and Is, with different structural and physiological properties at synaptic level at the neuromuscular junction. To generate genetic tools to identify and manipulate motor neurons of each Type, we screened for GAL4 driver lines for this purpose. Here we describe GAL4 drivers specific for examples of neurons within each Type, Ib or Is. These drivers showed high expression levels and were expressed in only few motor neurons, making them amenable tools for specific studies of both axonal and synapse biology in identified Type I motor neurons.

Methylating chemicals are common in industry and agriculture and are often toxic, partly due to their propensity to methylate DNA. The Escherichia coli Ada protein detects methylating compounds by sensing aberrant methyl adducts on the phosphoester backbone of DNA. We characterize this system as a genetic sensor and engineer it to lower the detection threshold. By overexpressing Ada from a plasmid, we improve the sensor’s dynamic range to 350-fold induction and lower its detection threshold to 40 μM for methyl iodide. In eukaryotes, there is no known sensor of methyl adducts on the phosphoester backbone of DNA. By fusing the N-terminal domain of Ada to the Gal4 transcriptional activation domain, we built a functional sensor for methyl phosphotriester adducts in Saccharomyces cerevisiae. This sensor can be tuned to variable specifications by altering the expression level of the chimeric sensor and changing the number of Ada operators upstream of the Gal4-sensitive reporter promoter. These changes result in a detection threshold of 28 μM and 5.2-fold induction in response to methyl iodide. When the yeast sensor is exposed to different SN1 and SN2 alkylating compounds, its response profile is similar to that observed for the native Ada protein in E. coli, indicating that its native function is retained in yeast. Finally, we demonstrate that the specifications achieved for the yeast sensor are suitable for detecting methylating compounds at relevant concentrations in environmental samples. This work demonstrates the movement of a sensor from a prokaryotic to eukaryotic system and its rational tuning to achieve desired specifications.

The ability to independently control gene expression in two different tissues in the same animal is emerging as a major need, especially in the context of inter-organ communication studies. This type of study is made possible by technologies combining the GAL4/UAS and a second binary expression system such as the LexA system or QF system. Here, we describe a resource of reagents that facilitate combined use of the GAL4/UAS and a second binary system in various Drosophila tissues. Focusing on genes with well-characterized GAL4 expression patterns, we generated a set of more than 40 LexA-GAD and QF2 insertions by CRISPR knock-in and verified their tissue specificity in larvae. We also built constructs that encode QF2 and LexA-GAD transcription factors in a single vector. Following successful integration of this construct into the fly genome, FLP/FRT recombination is used to isolate fly lines that express only QF2 or LexA-GAD. Finally, using new compatible shRNA vectors, we evaluated both LexA and QF systems for in vivo gene knockdown and are generating a library of such RNAi fly lines as a community resource. Together, these LexA and QF system vectors and fly lines will provide a new set of tools for researchers who need to activate or repress two different genes in an orthogonal manner in the same animal., Competing Interests: JZ, BJ, RL, BE, JB, AR, CF, CV, YH, NP No competing interests declared, (© 2023, Zirin et al.)

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

The zebrafish model organism has been of exceptional utility for the study of vertebrate development and disease through the application of tissue-specific labelling and overexpression of genes carrying patient-derived mutations. However, there remains a need for a binary expression system that is both non-toxic and not silenced over animal generations by DNA methylation. The Q binary expression system derived from the fungus Neurospora crassa is ideal, because the consensus binding site for the QF transcription factor lacks CpG dinucleotides, precluding silencing by CpG-meditated methylation. To optimize this system for zebrafish, we systematically tested several variants of the QF transcription factor: QF full length; QF2, which lacks the middle domain; QF2w, which is an attenuated version of QF2; and chimeric QFGal4. We found that full length QF and QF2 were strongly toxic to zebrafish embryos, QF2w was mildly toxic, and QFGal4 was well tolerated, when injected as RNA or expressed ubiquitously from stable transgenes. In addition, QFGal4 robustly activated a Tg(QUAS:GFP NLS ) reporter transgene. To increase the utility of this system, we also modified the QF effector sequence termed QUAS, which consists of five copies of the QF binding site. Specifically, we decreased both the CpG dinucleotide content, as well as the repetitiveness of QUAS, to reduce the risk of transgene silencing via CpG methylation. Moreover, these modifications to QUAS removed leaky QF-independent neural expression that we detected in the original QUAS sequence. To demonstrate the utility of our QF optimizations, we show how the Q-system can be used for lineage tracing using a Cre-dependent Tg(ubi:QFGal4-switch) transgene. We also demonstrate that QFGal4 can be used in transient injections to tag and label endogenous genes by knocking in QFGal4 into sox2 and ubiquitin C genes. • We optimized the Q binary expression system for zebrafish and other vertebrates. • QFGal4 is a non-toxic transactivator that can be ubiquitously expressed in zebrafish. • We developed QF effector sequences with reduced CpG content to reduce risk silencing. • The Q system can be used for gene-tagging and lineage tracing applications. [ABSTRACT FROM AUTHOR]

The universal nine-amino-acid transactivation domains (9aaTADs) have been identified in numerous transcription activators. Here, we identified the conserved 9aaTAD motif in all nine members of the specificity protein (SP) family. Previously, the Sp1 transcription factor has been defined as a glutamine-rich activator. We showed by amino acid substitutions that the glutamine residues are completely dispensable for 9aaTAD function and are not conserved in the SP family. We described the origin and evolutionary history of 9aaTADs. The 9aaTADs of the ancestral Sp2 gene became inactivated in early chordates. We next discovered that an accumulation of valines in 9aaTADs inactivated their transactivation function and enabled their strict conservation during evolution. Subsequently, in chordates, Sp2 has duplicated and created new paralogs, Sp1, Sp3, and Sp4 (the SP1–4 clade). During chordate evolution, the dormancy of the Sp2 activation domain lasted over 100 million years. The dormant but still intact ancestral Sp2 activation domains allowed diversification of the SP1–4 clade into activators and repressors. By valine substitution in the 9aaTADs, Sp1 and Sp3 regained their original activator function found in ancestral lower metazoan sea sponges. Therefore, the vertebrate SP1–4 clade could include both repressors and activators. Furthermore, we identified secondary 9aaTADs in Sp2 introns present from fish to primates, including humans. In the gibbon genome, introns containing 9aaTADs were used as exons, which turned the Sp2 gene into an activator. Similarly, we identified introns containing 9aaTADs used conditionally as exons in the (SP family-unrelated) transcription factor SREBP1, suggesting that the intron-9aaTAD reservoir is a general phenomenon. [ABSTRACT FROM AUTHOR]

In some cases, gene transfer from euchromatin to constitutive heterochromatin as a result of chromosomal rearrangement is accompanied by epigenetic inactivation of this gene (cis-inactivation). In the case of trans-inactivation, transgenes in the normal chromosome are repressed by the cis-inactivation-causing rearranged homologous chromosome. Trans-inactivation is a result of the somatic pairing of homologs and the transfer of the normal chromosomal segment to the heterochromatic compartment of the nucleus. Previously, we have shown that the degree of trans-inactivation of the UAS-eGFP reporter gene in adult flies depends on its transcription level that can be regulated by temperature using the GAL4 transcription activator and its temperature-sensitive inhibitor GAL80ts. In this paper, we investigated the epigenetic inheritance of the active/repressed state of the trans-inactivated reporter gene at different expression levels by measuring eGFP fluorescence in the individual cells of Malpighian tubules in adult flies. High expression levels at the embryonic stage protected the eGFP gene from trans-inactivation in adult flies. The activated state was inherited over the entire period of development and differentiation, while the activating effect of GAL4 was turned off. [ABSTRACT FROM AUTHOR]

Abstract The traditional treatment for cancer is lack of specificity and efficacy. Modular synthetic regulatory RNAs, such as inhibitive RNA (iRNA) and active RNA (aRNA), may overcome these limitations. Here, we synthesize a new iRNA to bind the upstream activating sequence (UAS) of a minimal promoter that drives expression of artificial miRNAs (amiRNAs) targeting MYC, which represses the binding interaction between UAS and GAL4 fusion protein (GAL4-VP64) in GAL4/UAS system. The aRNA driven by a tumor-specific mutant human telomerase reverse transcriptase (hTERT) promoter is created to interact with iRNA to expose UAS again in bladder cancer. Without the aRNA, mRNA and protein levels of MYC, cell growth, cell apoptosis and cell migration were no significance in two bladder cancer cell lines, T24 and 5637, and human foreskin fibroblast (HFF) cells. The aRNA significantly inhibited the expression of MYC in mRNA and protein levels, as well as the proliferation and migration of the cancer cells, but not in HFF cells. These results indicated that regulatory RNAs selectively controlled the expression of amiRNAs and ultimately suppress the progression of bladder cancer cells without affecting normal cells. Synthetic regulatory RNAs might be a selective therapeutic approach for bladder cancer.

Eggshell patterning has been extensively studied in Drosophila melanogaster. However, the cis-regulatory modules (CRMs), which control spatiotemporal expression of these patterns, are vastly unexplored. The FlyLight collection contains >7000 intergenic and intronic DNA fragments that, if containing CRMs, can drive the transcription factor GAL4. We cross-listed the 84 genes known to be expressed during D. melanogaster oogenesis with the ∼1200 listed genes of the FlyLight collection, and found 22 common genes that are represented by 281 FlyLight fly lines. Of these lines, 54 show expression patterns during oogenesis when crossed to an UAS-GFP reporter. Of the 54 lines, 16 recapitulate the full or partial pattern of the associated gene pattern. Interestingly, while the average DNA fragment size is ∼3 kb in length, the vast majority of fragments show one type of spatiotemporal pattern in oogenesis. Mapping the distribution of all 54 lines, we found a significant enrichment of CRMs in the first intron of the associated genes’ model. In addition, we demonstrate the use of different anteriorly active FlyLight lines as tools to disrupt eggshell patterning in a targeted manner. Our screen provides further evidence that complex gene patterns are assembled combinatorially by different CRMs controlling the expression of genes in simple domains.

ABSTRACT Clustered regularly interspaced short palindromic repeat (CRISPR) methodology is not only an efficient tool in gene editing but also an attractive platform to facilitate DNA, RNA, and protein interactions. We describe here the implementation of a CRISPR-based system to regulate expression in the clinically important yeast Candida albicans. By fusing an allele of Streptococcus pyogenes Cas9 devoid of nuclease activity to a transcriptional repressor (Nrg1) or activator (Gal4), we were able to show specific repression or activation of the tester gene CAT1, encoding the cytosolic catalase. We generated strains where a 1.6-kbp upstream regulatory region of CAT1 controls the expression of the green fluorescent protein (GFP) and demonstrated the functionality of the constructs by quantitative PCR (qPCR), flow cytometry, and analysis of sensitivity/resistance to hydrogen peroxide. Activation and repression were strongly dependent on the position of the complex in this regulatory region. We also improved transcriptional activation using an RNA scaffolding strategy to allow interaction of inactive variants of Cas9 (dCas9) with the RNA binding protein MCP (monocyte chemoattractant protein) fused to the VP64 activator. The strategy shown here may facilitate the analysis of complex regulatory traits in this fungal pathogen. IMPORTANCE CRISPR technology is a new and efficient way to edit genomes, but it is also an appealing way to regulate gene expression. We have implemented CRISPR as a gene expression platform in Candida albicans using fusions between a Cas9 inactive enzyme and specific repressors or activators and demonstrated its functionality. This will allow future manipulation of complex virulence pathways in this important fungal pathogen.

Decoding the functional connectivity of the nervous system is facilitated by transgenic methods that express a genetically encoded reporter or effector in specific neurons; however, most transgenic lines show broad spatiotemporal and cell-type expression. Increased specificity can be achieved using intersectional genetic methods which restrict reporter expression to cells that co-express multiple drivers, such as Gal4 and Cre. To facilitate intersectional targeting in zebrafish, we have generated more than 50 new Cre lines, and co-registered brain expression images with the Zebrafish Brain Browser, a cellular resolution atlas of 264 transgenic lines. Lines labeling neurons of interest can be identified using a web-browser to perform a 3D spatial search (zbbrowser.com). This resource facilitates the design of intersectional genetic experiments and will advance a wide range of precision circuit-mapping studies.

The cold-tolerant yeast Saccharomyces cerevisiae is industrially useful for lager fermentation, high-quality wine, and frozen dough production. S. cerevisiae Cheongdo is a recent isolate from frozen peach samples which has a good fermentation performance at low temperatures and desirable flavor profiles. Here, phenotype microarray was used to investigate industrial potentials of S. cerevisiae Cheongdo using 192 carbon sources. Compared to commercial wine yeast S. cerevisiae EC1118, Cheongdo showed significantly different growth rates on 34 substrates. The principal component analysis of the results highlighted that the better growth of Cheongdo on galactose than on EC1118 was the most significant difference between the two strains. The intact GAL4 gene and the galactose fermentation performance at a low temperatures suggested that S. cerevisiae Cheongdo is a promising host for industrial fermentation rich in galactose, such as lactose and agarose.

As a DNA binding transcriptional activator, Gal4 promotes the expression of genes responsible for galactose metabolism. The Gal4 protein from Saccharomyces cerevisiae (baker’s yeast) has become a model for studying eukaryotic transcriptional activation in general because its regulatory properties mirror those of several eukaryotic organisms, including mammals. Given the availability of a crystallographic structure for Gal4, here we implement an in silico mutagenesis technique that makes use of a four-body knowledge-based energy function, in order to empirically quantify the structural impacts associated with single residue substitutions on the Gal4 protein. These results were used to examine the structure-function relationship in Gal4 based on a recently published experimental mutagenesis study, whereby functional changes to a uniformly distributed set of 1,068 single residue Gal4 variants were obtained by measuring their transcriptional activation levels relative to wild-type. A significant correlation was observed between computed (scalar) structural effect data and measured activity values for this collection of single residue Gal4 variants. Additionally, attribute vectors quantifying position-specific environmental impacts were generated for each of the Gal4 variants via computational mutagenesis, and we implemented supervised classification and regression statistical machine learning algorithms to train predictive models of variant Gal4 activity based on these structural changes. All models performed well under cross-validation testing, with balanced accuracy reaching 91% among the classification models, and with the actual and predicted activity values displaying a correlation as high as r = 0.80 for the regression models. Reliable predictions of transcriptional activation levels for Gal4 variants that have yet to be studied can be instantly generated by submitting their respective structure-based feature vectors to the trained models for testing. Such a computational pre-screening of Gal4 variants may potentially reduce costs associated with running large-scale mutagenesis experiments. [ABSTRACT FROM AUTHOR]

The Drosophila melanogaster larval neuromuscular system is extensively used by researchers to study neuronal cell biology, and Drosophila glutamatergic motor neurons have become a major model system. There are two main Types of glutamatergic motor neurons, Ib and Is, with different structural and physiological properties at synaptic level at the neuromuscular junction. To generate genetic tools to identify and manipulate motor neurons of each Type, we screened for GAL4 driver lines for this purpose. Here we describe GAL4 drivers specific for examples of neurons within each Type, Ib or Is. These drivers showed high expression levels and were expressed in only few motor neurons, making them amenable tools for specific studies of both axonal and synapse biology in identified Type I motor neurons. [ABSTRACT FROM AUTHOR]

As a DNA binding transcriptional activator, Gal4 promotes the expression of genes responsible for galactose metabolism. The Gal4 protein from Saccharomyces cerevisiae (baker’s yeast) has become a model for studying eukaryotic transcriptional activation in general because its regulatory properties mirror those of several eukaryotic organisms, including mammals. Given the availability of a crystallographic structure for Gal4, here we implement an in silico mutagenesis technique that makes use of a four-body knowledge-based energy function, in order to empirically quantify the structural impacts associated with single residue substitutions on the Gal4 protein. These results were used to examine the structure-function relationship in Gal4 based on a recently published experimental mutagenesis study, whereby functional changes to a uniformly distributed set of 1,068 single residue Gal4 variants were obtained by measuring their transcriptional activation levels relative to wild-type. A significant correlation was observed between computed (scalar) structural effect data and measured activity values for this collection of single residue Gal4 variants. Additionally, attribute vectors quantifying position-specific environmental impacts were generated for each of the Gal4 variants via computational mutagenesis, and we implemented supervised classification and regression statistical machine learning algorithms to train predictive models of variant Gal4 activity based on these structural changes. All models performed well under cross-validation testing, with balanced accuracy reaching 91% among the classification models, and with the actual and predicted activity values displaying a correlation as high as r = 0.80 for the regression models. Reliable predictions of transcriptional activation levels for Gal4 variants that have yet to be studied can be instantly generated by submitting their respective structure-based feature vectors to the trained models for testing. Such a computational pre-screening of Gal4 variants may potentially reduce costs associated with running large-scale mutagenesis experiments.

The commonly used expression systems in Saccharomyces cerevisiae typically rely on either constitutive or galactose-regulated promoters. The lack of inducible systems in S. cerevisiae limits the precise temporal regulation of protein function and yeast metabolism. We herein repurposed the galactose-regulated system to make it respond to cyanamide. By using a cyanamide-inducible DDI2 promoter to control Gal4 expression in CEN.PK2-1C with Δgal80, a tight and graded cyanamide-inducible GAL system with an enhanced signal output was constructed. Subsequently, we demonstrated that the cyanamide-inducible GAL system was capable of tightly regulating the pentafunctional Aro1 protein to achieve conditional shikimate pathway activity. Taken together, the cyanamide-inducible GAL system could be implemented for both fundamental research and applied biotechnology.

The central complex, a set of neuropils in the center of the insect brain, plays a crucial role in spatial aspects of sensory integration and motor control. Stereotyped neurons interconnect these neuropils with one another and with accessory structures. We screened over 5,000 Drosophila melanogaster GAL4 lines for expression in two neuropils, the noduli (NO) of the central complex and the asymmetrical body (AB), and used multicolor stochastic labeling to analyze the morphology, polarity, and organization of individual cells in a subset of the GAL4 lines that showed expression in these neuropils. We identified nine NO and three AB cell types and describe them here. The morphology of the NO neurons suggests that they receive input primarily in the lateral accessory lobe and send output to each of the six paired noduli. We demonstrate that the AB is a bilateral structure which exhibits asymmetry in size between the left and right bodies. We show that the AB neurons directly connect the AB to the central complex and accessory neuropils, that they target both the left and right ABs, and that one cell type preferentially innervates the right AB. We propose that the AB be considered a central complex neuropil in Drosophila. Finally, we present highly restricted GAL4 lines for most identified protocerebral bridge, NO, and AB cell types. These lines, generated using the split‐GAL4 method, will facilitate anatomical studies, behavioral assays, and physiological experiments. The authors use the multicolor flip‐out strategy in Drosophila to analyze neurons in two neuropils: the noduli of the central complex and the asymmetrical body. A set of split‐GAL4 lines is provided for previously published protocerebral bridge cell types as well as cell types presented here. [ABSTRACT FROM AUTHOR]

Position effect variegation (PEV) is a perturbation of genes expression resulting from the changes in their chromatin organization due to the abnormal juxtaposition with heterochromatin. The exact molecular mechanisms of PEV remain enigmatic in spite of the long history of PEV studies. Here, we developed a genetic model consisting of PEV-inducing chromosome rearrangement and a reporter gene under control of the UAS regulatory element. Expression of the reporter gene could be regulated by adjustment of the GAL4 transactivator activity. Two UAS-based systems of expression control were tested-with thermosensitive GAL4 repressor GAL80ts and GAL4-based artificial transactivator GeneSwitch. Both systems were able to regulate the expression of the UAS-controlled reporter gene over a wide range, but GAL80ts repressed the reporter gene more efficiently. Measurements of the heterochromatin-mediated repression of the reporter gene in the GAL4+GAL80ts system point to the existence of a threshold level of expression, above which no PEV is observed. [ABSTRACT FROM AUTHOR]

As a DNA binding transcriptional activator, Gal4 promotes the expression of genes responsible for galactose metabolism. The Gal4 protein from Saccharomyces cerevisiae (baker’s yeast) has become a model for studying eukaryotic transcriptional activation in general because its regulatory properties mirror those of several eukaryotic organisms, including mammals. Given the availability of a crystallographic structure for Gal4, here we implement an in silico mutagenesis technique that makes use of a four-body knowledge-based energy function, in order to empirically quantify the structural impacts associated with single residue substitutions on the Gal4 protein. These results were used to examine the structure-function relationship in Gal4 based on a recently published experimental mutagenesis study, whereby functional changes to a uniformly distributed set of 1,068 single residue Gal4 variants were obtained by measuring their transcriptional activation levels relative to wild-type. A significant correlation was observed between computed (scalar) structural effect data and measured activity values for this collection of single residue Gal4 variants. Additionally, attribute vectors quantifying position-specific environmental impacts were generated for each of the Gal4 variants via computational mutagenesis, and we implemented supervised classification and regression statistical machine learning algorithms to train predictive models of variant Gal4 activity based on these structural changes. All models performed well under cross-validation testing, with balanced accuracy reaching 91% among the classification models, and with the actual and predicted activity values displaying a correlation as high as r = 0.80 for the regression models. Reliable predictions of transcriptional activation levels for Gal4 variants that have yet to be studied can be instantly generated by submitting their respective structure-based feature vectors to the trained models for testing. Such a computational pre-screening of Gal4 variants may potentially reduce costs associated with running large-scale mutagenesis experiments. [ABSTRACT FROM AUTHOR]

Abstract: Conflict between cell growth and product accumulation is frequently encountered in biosynthesis of secondary metabolites. Herein, a temperature‐dependent dynamic control strategy was developed by modifying the GAL regulation system to facilitate two‐stage fermentation in yeast. A temperature‐sensitive Gal4 mutant Gal4M9 was created by directed evolution, and used as a protein switch in ΔGAL80 yeast. After EGFP‐reported validation of its temperature‐responsive induction capability, the sensitivity and stringency of this system in multi‐gene pathway regulation was tested, using lycopene as an example product. When Gal4M9 was used to control the expression of PGAL‐driven pathway genes, growth and production was successfully decoupled upon temperature shift during fermentation, accumulating 44% higher biomass and 177% more lycopene than the control strain with wild‐type Gal4. This is the first example of adopting temperature as an input signal for metabolic pathway regulation in yeast cell factories. [ABSTRACT FROM AUTHOR]

In Drosophila melanogaster larvae, the prime site of external taste reception is the terminal organ (TO). Though investigation on the TO's implications in taste perception has been expanding rapidly, the sensilla of the TO have been essentially unexplored. In this study, we performed a systematic anatomical and molecular analysis of the TO. We precisely define morphological types of TO sensilla taking advantage of volume electron microscopy and 3D image analysis. We corroborate the presence of five external types of sensilla: papilla, pit, spot, knob, and modified papilla. Detailed 3D analysis of their structural organization allowed a finer discrimination into subtypes. We classify three subtypes of papilla and pit sensilla, respectively, and two subtypes of knob sensilla. Further, we determine the repertoire of receptor genes for each sensillum by analyzing GAL4 driver lines of Ir, Gr, Ppk, and Trp receptor genes. We construct a map of the TO, in which the receptor genes are mapped to neurons of individual sensilla. While modified papillum and spot sensilla are not labeled by any GAL4 driver, neurons of the pit, papilla, and knob type are labeled by partially overlapping but different subsets of GAL4 driver lines of the Ir, Gr, and Ppk gene family. The results suggest that pit, papilla and knob sensilla act in contact chemosensation. However, they likely do these employing different stimulus transduction mechanisms to sense the diverse chemicals of their environment. [ABSTRACT FROM AUTHOR]

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

Eggshell patterning has been extensively studied in Drosophila melanogaster. However, the cis-regulatory modules (CRMs), which control spatiotemporal expression of these patterns, are vastly unexplored. The FlyLight collection contains >7000 intergenic and intronic DNA fragments that, if containing CRMs, can drive the transcription factor GAL4. We cross-listed the 84 genes known to be expressed during D. melanogaster oogenesis with the ~1200 listed genes of the FlyLight collection, and found 22 common genes that are represented by 281 FlyLight fly lines. Of these lines, 54 show expression patterns during oogenesis when crossed to an UAS-GFP reporter. Of the 54 lines, 16 recapitulate the full or partial pattern of the associated gene pattern. Interestingly, while the average DNA fragment size is ~3 kb in length, the vast majority of fragments show one type of spatiotemporal pattern in oogenesis. Mapping the distribution of all 54 lines, we found a significant enrichment of CRMs in the first intron of the associated genes' model. In addition, we demonstrate the use of different anteriorly active FlyLight lines as tools to disrupt eggshell patterning in a targeted manner. Our screen provides further evidence that complex gene patterns are assembled combinatorially by different CRMs controlling the expression of genes in simple domains. [ABSTRACT FROM AUTHOR]

Genetically-encoded combinatorial peptide libraries are convenient tools to identify peptides to be used as therapeutics, antimicrobials and functional synthetic biology modules. Here, we report the identification and characterization of a cyclic peptide, G4CP2, that interferes with the GAL4 protein, a transcription factor responsible for the activation of galactose catabolism in yeast and widely exploited in molecular biology. G4CP2 was identified by screening CYCLIC, a Yeast Two-Hybrid-based combinatorial library of cyclic peptides developed in our laboratory. G4CP2 interferes with GAL4-mediated activation of galactose metabolic enzymes both when expressed intracellularly, as a recombinant peptide, and when provided exogenously, as a chemically-synthesized cyclic peptide. Our results support the application of G4CP2 in microbial biotechnology and, additionally, demonstrate that CYCLIC can be used as a tool for the rapid identification of peptides, virtually without any limitations with respect to the target protein. The possible biotechnological applications of cyclic peptides are also discussed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Rosa, Tagliani, Bertaso, Tadini, Visentin, Gourlay, Pricl, Feni, Pellegrino, Pesaresi and Masiero.)

Transgenic methods enable the selective manipulation of neurons for functional mapping of neuronal circuits. Using confocal microscopy, we have imaged the cellular-level expression of 109 transgenic lines in live 6 day post fertilization larvae, including 80 Gal4 enhancer trap lines, 9 Cre enhancer trap lines and 20 transgenic lines that express fluorescent proteins in defined gene-specific patterns. Image stacks were acquired at single micron resolution, together with a broadly expressed neural marker, which we used to align enhancer trap reporter patterns into a common 3-dimensional reference space. To facilitate use of this resource, we have written software that enables searching for transgenic lines that label cells within a selectable 3-dimensional region of interest or neuroanatomical area. Many of the imaged enhancer trap lines show intrinsic brain-specific expression. However, to increase the utility of lines that also drive expression in non-neuronal tissue we have designed a novel UAS reporter, that suppresses expression in heart, muscle and skin through the incorporation of microRNA binding sites in a synthetic 3' untranslated region. Finally, we mapped the site of transgene integration, thus providing molecular identification of the expression pattern for most lines. Cumulatively, this library of enhancer trap lines provides genetic access to 70 % of the larval brain and is therefore a powerful and broadly accessible tool for the dissection of neural circuits in larval zebrafish.

Yeast hybrid systems have been widely used due to their convenience and low cost. Based on these systems, many methods have been developed to analyze protein-protein, protein-DNA and protein-RNA interactions. In this paper, we are reviewing these different yeast hybrid systems. According to the number of hybrid proteins, yeast hybrid systems can be divided into three categories, yeast one-hybrid, yeast two-hybrid and yeast three-hybrid systems. Alternatively, yeast hybrid systems can be categorized according to the subcellular localization of the protein interaction process in the cell into nuclear protein-protein interactions, cytosol protein-protein interactions and membrane protein-protein interactions. Throughout the review, we focus on the progress and limitations of each yeast hybrid system over the recent years. [ABSTRACT FROM AUTHOR]

Drosophila neurexin (DNRX) plays a critical role in proper architecture development and synaptic function in vivo. However, the temporal and spatial expression pattern of DNRX still remains unclear. For this study, we generated a novel Drosophila transgenic strain termed the DNRX-Gal4 transgenic line, with characteristic features in agreement with the endogenous DNRX expression pattern. DNRX expression was examined by driving the expression of a GFP reporter (nuclear-localized and membrane- localized GFP) using the DNRX-Gal4 promoter. We found that DNRX was expressed preferentially in central and motor neurons in embryos, larvae and adults, but not in glial cells. DNRX was expressed in pre- and post-synaptic areas in third instar larvae neuromuscular junctions (NMJs). Reporter expression was also observed in the salivary glands, guts, wings and legs of adult flies. In the adult brain, reporter expression was observed throughout several brain regions, including the mushroom body (MBs), antennal lobe (AL) and optic lobe neurons, which is consistent with endogenous DNRX expression via antibody staining. Interestingly, DNRX was also expressed in clock neurons. Meanwhile, we found that DNRX expression in the MBs was required for olfactory learning and memory. [ABSTRACT FROM AUTHOR]

The Drosophila gene fruitless expresses male and female specific transcription factors which are responsible for the generation of male specific neuronal circuitry for courtship behavior. Mutations in this gene may lead to bisexual behavior in males. Bisexual behavior in males also occurs in the absence of the neuropeptide SIFamide. We show here that the SIFamide neurons do not express fruitless. However, when fruitless neurons are made to express RNAi specific for the SIFamide receptor, male flies engage in bisexual behavior, showing that SIFamide acts on fruitless neurons. If neurons expressing a SIFaR- gal4 transgene are killed by the apoptotic protein reaper or when these neurons express SIFamide receptor RNAi, males also show male–male courtship behavior. We next used this transgene to localize neurons that express the SIFamide receptor. Such neurons are ubiquitously present in the central nervous and we also found two neurons in the uterus that project into the central nervous system. [ABSTRACT FROM AUTHOR]