Yeast Transcriptional Activator GAL4 Research Articles

A Bifunctional Amino Acid Enables Both Covalent Chemical Capture and Isolation of in Vivo Protein-Protein Interactions.

In vivo covalent chemical capture by using photoactivatable unnatural amino acids (UAAs) is a powerful tool for the identification of transient protein-protein interactions (PPIs) in their native environment. However, the isolation and characterization of the crosslinked complexes can be challenging. Here, we report the first in vivo incorporation of the bifunctional UAA BPKyne for the capture and direct labeling of crosslinked protein complexes through post-crosslinking functionalization of a bioorthogonal alkyne handle. Using the prototypical yeast transcriptional activator Gal4, we demonstrate that BPKyne is incorporated at the same level as the commonly used photoactivatable UAA pBpa and effectively captures the Gal4-Gal80 transcriptional complex. Post-crosslinking, the Gal4-Gal80 adduct was directly labeled by treatment of the alkyne handle with a biotin-azide probe; this enabled facile isolation and visualization of the crosslinked adduct from whole-cell lysate. This bifunctional amino acid extends the utility of the benzophenone crosslinker and expands our toolbox of chemical probes for mapping PPIs in their native cellular environment.

MicroRNA-8 targets the Wingless signaling pathway in the female mosquito fat body to regulate reproductive processes

Female mosquitoes require a blood meal for reproduction, and this blood meal provides the underlying mechanism for the spread of many important vector-borne diseases in humans. A deeper understanding of the molecular mechanisms linked to mosquito blood meal processes and reproductive events is of particular importance for devising innovative vector control strategies. We found that the conserved microRNA miR-8 is an essential regulator of mosquito reproductive events. Two strategies to inhibit miR-8 function in vivo were used for functional characterization: systemic antagomir depletion and spatiotemporal inhibition using the miRNA sponge transgenic method in combination with the yeast transcriptional activator gal4 protein/upstream activating sequence system. Depletion of miR-8 in the female mosquito results in defects related to egg development and deposition. We used a multialgorithm approach for miRNA target prediction in mosquito 3' UTRs and experimentally verified secreted wingless-interacting molecule (swim) as an authentic target of miR-8. Our findings demonstrate that miR-8 controls the activity of the long-range Wingless (Wg) signaling by regulating Swim expression in the female fat body. We discovered that the miR-8/Wg axis is critical for the proper secretion of lipophorin and vitellogenin by the fat body and subsequent accumulation of these yolk protein precursors by developing oocytes.

Solvent-exposed serines in the Gal4 DNA-binding domain are required for promoter occupancy and transcriptional activationin vivo

The yeast transcriptional activator Gal4 has long been the prototype for studies of eukaryotic transcription. Gal4 is phosphorylated in the DNA-binding domain (DBD); however, the molecular details and functional significance of this remain unknown. We mutagenized seven potential phosphoserines that lie on the solvent-exposed face of the DBD structure and assessed them for transcriptional activity and DNA binding in vivo. Serine to alanine mutants at positions 22, 47, and 85 show the greatest reduction in promoter occupancy and transcriptional activity at the MEL1 promoter containing a single UASGAL . Substitutions with the phosphomimetic aspartate restored DNA-binding and transcriptional activity at serines 22 and 85, suggesting that they are potential sites of Gal4 phosphorylation in vivo. In contrast, the serine to alanine mutants, except serine 22, were fully proficient for binding to the GAL1-10 promoter, containing multiple UASGAL sites, although they had a reduced ability to activate transcription. Collectively, these data show that at the GAL1-10 promoter, functions of the DBD in transcriptional activation can be uncoupled from roles in promoter binding. We suggest that the serines in the DBD mediate protein-protein contacts with the transcription machinery, leading to stabilization of Gal4 at promoters.

Rapid GAL Gene Switch of Saccharomyces cerevisiae Depends on Nuclear Gal3, Not Nucleocytoplasmic Trafficking of Gal3 and Gal80

The yeast transcriptional activator Gal4 localizes to UAS(GAL) sites even in the absence of galactose but cannot activate transcription due to an association with the Gal80 protein. By 4 min after galactose addition, Gal4-activated gene transcription ensues. It is well established that this rapid induction arises through a galactose-triggered association between the Gal80 and Gal3 proteins that decreases the association of Gal80 and Gal4. How this happens mechanistically remains unclear. Strikingly different hypotheses prevail concerning the possible roles of nucleocytoplasmic distribution and trafficking of Gal3 and Gal80 and where in the cell the initial Gal3-Gal80 association occurs. Here we tested two conflicting hypotheses by evaluating the subcellular distribution and dynamics of Gal3 and Gal80 with reference to induction kinetics. We determined that the rates of nucleocytoplasmic trafficking for both Gal80 and Gal3 are slow relative to the rate of induction. We find that depletion of the nuclear pool of Gal3 slows the induction kinetics. Thus, nuclear Gal3 is critical for rapid induction. Fluorescence-recovery-after-photobleaching experiments provided data suggesting that the Gal80-Gal4 complex exhibits kinetic stability in the absence of galactose. Finally, we detect Gal3 at the UAS(GAL) only if Gal80 is covalently linked to the DNA-binding domain. Taken altogether, these new findings lead us to propose that a transient interaction of Gal3 with Gal4-associated Gal80 could explain the rapid response of this system. This notion could also explain earlier observations.

Naturally occurring and artificially induced tumor-like formations in marine invertebrates: A search for permanent cell lines

Many problems of modern virology, biotechnology and ecology cannot be approached without the use of permanent cell lines from marine invertebrates. Tissues with high growth potential are useful for establishment of such cell lines. We estimated the prevalence of naturally occurring tumor-like lesions and neoplasms in bivalve molluscs from the Sea of Japan with the focus on cell mitotic activity and tried to induce similar formations in sea urchins in the laboratory. Inspecting nearly 3000 bivalves of eight species, we have detected the tumor-like lesions occurring in 0–15% of the total number of molluscs that were studied. Many of the molluscan tumor-like lesions had a low mitotic index (MI, 0.01–0.3%). Neoplasms with the MI 3–12% were met only in Mytilus and Pecten. Besides a search for naturally occurring tumor-like lesions and neoplasms in molluscs, we used modulation of gene expression by genetically engineered constructs bearing foreign genes, the yeast transcriptional activator gal4 gene or the agrobacterial rol oncogenes, in sea urchin embryos and sea urchin embryonic cells. We failed to develop a potential permanent cell line; however, the results obtained allow us to assert that some tumor-like formations can be useful objects for establishment of permanent cell lines. Only cells of malignant mussel hemocytes and developmental anomalies in sea urchin embryos transformed by the yeast gal4 gene were involved in active proliferation. Our findings suggest that the transformed by the yeast gal4 gene embryonic cells of sea urchins may be as a suitable model for future studies.

Construction, expression and characterization of a chimeric multi-domain protein mediating specific DNA transfer

The delivery of plasmid DNA to target cells using a simple, defined, non-viral system is an area of intense research in gene therapy. Here, we describe a novel DNA carrier protein termed TG, consisting of the DNA-binding domain of the yeast transcriptional activator GAL4 and human immunodeficiency virus type 1 Tat protein, which can transfer modified naked plasmid DNA into target cells to express foreign genes of interest. The TG protein was expressed in Escherichia coli ( E. coli), refolded and purified on an immobilized Ni 2+ affinity chromatography column. SDS–PAGE and Western blotting revealed that the fusion protein was highly expressed with a yield of approximately 275 mg/L. We also constructed the pIRES-UAS-EGFP DNA vector, consisting of upstream activating sequences (UASs) for the specific binding of the DNA-binding protein and the enhanced green fluorescent protein (EGFP) gene. The TG protein could bind specifically to pIRES-UAS-EGFP, forming a complex which could efficiently transfect target cells and result in detectable EGFP protein expression. Thus, these results provide a basis for development of efficient non-viral DNA transfer vectors for further improvements of gene therapy strategies.

A novel two-step transcriptional activation system for gene therapy directed toward epithelial cells

The two-step transcriptional activation (TSTA) mechanism in gene therapy amplifies cell type-specific promoter activity, allowing for increased levels of gene expression in target tissues. In this system, the specific promoter drives expression of a strong transcriptional activator that binds to DNA target sequences located upstream from a second promoter controlling the expression of the therapeutic gene. The majority of previous studies have exploited a fusion between the DNA binding domain of the yeast transcriptional activator Gal4 fused to the VP16 activation domain of herpes simplex virus 1 as the transcriptional activator. In this report, an alternative to this system is described based on a fusion protein containing the DNA binding domain of the bovine papillomavirus 1 transcriptional activator E2 fused to VP16 that induces target gene expression following binding to a minimal bovine papillomavirus 4 promoter containing upstream E2 binding sites and only 3 bp of promoter sequence upstream from the TATA box. VP16-E2 is superior to Gal4-VP16 as the transcriptional activator in a TSTA system driven by either of the two potentially cancer-specific promoters telomerase RNA and telomerase reverse transcriptase in several cell lines. Results also suggest that this new system has an advantage in epithelial cells and is therefore ideal for potential targeting of carcinomas. By incorporating the TRAIL gene as a transgene in the VP16-E2 TSTA system, selective killing of telomerase-positive cells occurs. We propose that our new system should be considered in future TSTA, particularly when targeting epithelial-derived cells.

Nicastrin Is Dispensable for γ-Secretase Protease Activity in the Presence of Specific Presenilin Mutations

γ-Secretase is a multisubunit membrane protein complex consisting of presenilin (PS1), nicastrin (NCT), anterior pharynx-1, and presenilin enhancer 2. To analyze the activity of familial Alzheimer disease mutants and to understand the roles of the subunits, we established a yeast transcriptional activator Gal4p system with artificial γ-secretase substrates containing amyloid precursor protein or Notch fragments. The γ-secretase activities were evaluated by transcriptional activation of reporter genes upon Gal4p release from the membrane-bound substrates, i.e. growth of yeast on histidine and adenine, or β-galactosidase assay. We screened and evaluated γ-secretase mutants using this reconstitution system in yeast, which does not possess endogenous γ-secretase activity. When we introduced familial Alzheimer mutants of PS1 in this system, their activities were shown to be loss of function. Although the protease activity of wild type PS1 depends on the other three subunits introduced, we obtained 15 new PS1 mutants, which are active in the absence of NCT. They possessed a S438P mutation at the ninth transmembrane domain (TM9) together with one missense mutation distributed through transmembrane and loop regions. These mutations were not related to familial Alzheimer mutations of PS1 as identified so far. The S438P mutant was partially active but required other mutations for full activation. Results of the β-galactosidase assay suggested that they have wild type protease activities, which were further confirmed by the endoproteolysis of PS1, amyloid β peptides, and Notch intracellular domain production in mammalian cells. These results suggest that NCT is dispensable for the protease activity of γ-secretase.

A Drosophila Protein Family Implicated in Pheromone Perception Is Related to Tay-Sachs GM2-Activator Protein

Low volatility, lipid-like cuticular hydrocarbon pheromones produced by Drosophila melanogaster females play an essential role in triggering and modulating mating behavior, but the chemosensory mechanisms involved remain poorly understood. Recently, we showed that the CheB42a protein, which is expressed in only 10 pheromone-sensing taste hairs on the front legs of males, modulates progression to late stages of male courtship behavior in response to female-specific cuticular hydrocarbons. Here we report that expression of all 12 genes in the CheB gene family is predominantly or exclusively gustatory-specific, and occurs in many different, often non-overlapping patterns. Only the Gr family of gustatory receptor genes displays a comparable variety of gustatory-specific expression patterns. Unlike Grs, however, expression of all but one CheB gene is sexually dimorphic. Like CheB42a, other CheBs may therefore function specifically in gustatory perception of pheromones. We also show that CheBs belong to the ML superfamily of lipid-binding proteins, and are most similar to human GM2-activator protein (GM2-AP). In particular, GM2-AP residues involved in ligand binding are conserved in CheBs but not in other ML proteins. Finally, CheB42a is specifically secreted into the inner lumen of pheromone-sensing taste hairs, where pheromones interact with membrane-bound receptors. We propose that CheB proteins interact directly with lipid-like Drosophila pheromones and modulate their detection by the gustatory signal transduction machinery. Furthermore, as loss of GM2-AP in Tay-Sachs disease prevents degradation of GM2 gangliosides and results in neurodegeneration, the function of CheBs in pheromone response may involve biochemical mechanisms critical for lipid metabolism in human neurons.

Mitochondrial Transcription Factor B2 Is Essential for Metabolic Function in Drosophila melanogaster Development

Characterization of the basal transcription machinery of mitochondrial DNA (mtDNA) is critical to understand mitochondrial pathophysiology. In mammalian in vitro systems, mtDNA transcription requires mtRNA polymerase, transcription factor A (TFAM), and either transcription factor B1 (TFB1M) or B2 (TFB2M). We have silenced the expression of TFB2M by RNA interference in Drosophila melanogaster. RNA interference knockdown of TF2BM causes lethality by arrest of larval development. Molecular analysis demonstrates that TF2BM is essential for mtDNA transcription during Drosophila development and is not redundant with TFB1M. The impairment of mtDNA transcription causes a dramatic decrease in oxidative phosphorylation and mitochondrial ATP synthesis in the long-lived larvae, and a metabolic shift to glycolysis, which partially restores ATP levels and elicits a compensatory response at the nuclear level that increases mitochondrial mass. At the cellular level, the mitochondrial dysfunction induced by TFB2M knockdown causes a severe reduction in cell proliferation without affecting cell growth, and increases the level of apoptosis. In contrast, cell differentiation and morphogenesis are largely unaffected. Our data demonstrate the essential role of TFB2M in mtDNA transcription in a multicellular organism, and reveal the complex cellular, biochemical, and molecular responses induced by impairment of oxidative phosphorylation during Drosophila development.

Aubergine Gene Overexpression in Somatic Tissues of auberginesting Mutants Interferes With the RNAi Pathway of a yellow Hairpin dsRNA in Drosophila melanogaster

AUBERGINE (AUB) is a member of the PPD family of proteins. These proteins are implicated in RNA interference. In this article we demonstrate that the expression of the aub gene and protein increase in aub(sting) mutants. We used a genetic method to test whether aub(sting) overexpression could interfere with proper functioning of the process of RNA interference in somatic tissues of Drosophila melanogaster. This method is based on a transgenic line bearing a construct in which a fragment of the yellow (y) gene is cloned to form an inverted repeat (y-IR) under the control of the upstream activation sequence (UAS) of the yeast transcriptional activator GAL4. The UAS-y-IR transgene and the Act5C-GAL4 driver were brought together on chromosome 3 via recombination. In the resulting strain (Act5C-y-IR), transcriptional activation by GAL4 constitutively produces a dsRNA hairpin bearing cognate sequences to the yellow gene causing continuing degradation of y mRNA resulting in yellow(1) (y(1)) phenocopies. In this genetic background, the mutation of any factor involved in RNAi should repress degradation of y mRNA, restoring the wild-type phenotype. We employed this genetic approach to show that an increased amount of AUBERGINE interferes with the regular functioning of the somatic RNAi pathway.

Enhancement of Exogenous Gene Expression by the Artificial Transcription Factor in Chinese Hamster Ovary Cells

Using the amino acids 1-147 of the yeast transcriptional activator GAL4 as the DNA-binding domain and four tandem repeats of the 12-aa peptide (DALDDFDLDMLG) of the herpesvirus VP16 as the activation domain, an artificial transcription factor, GVP4 was constructed via the linkage of the nuclear localization signal sequence of SV40. And then, GVP4 was cloned into expression vector pcDNA3.1/Hygro(+). Various amounts of targeting sites of artificial transcription factor were linked to the upstream of promoter CMV in exogenous gene expression vector pcDNA3.1(+) that separately harbored EGFP cDNA and t-PA cDNA. The CHO cells were co-transfected with GVP4 expression vector and EGFP or t-PA expression vector. The effect of GVP4 on exogenous gene expression was evaluated by measuring the fluorescence intensity of EGFP in CHO cells and the concentration of t-PA in the supernatant. GVP4 showed positive effect on enhancement of exogenous gene expression in CHO cells integrated with targeting sites of artificial transcription factor. And, CHO cells integrated with 10 targeting sites of GVP4 was more favorable to foreign gene expression, which resulted in 2-3-fold increase in both EGFP and t-PA expressions. These results indicated that artificial transcription factor is potent in the enhancement of exogenous gene expression in mammalian cells.

14-3-3 Mediates Transcriptional Regulation by Modulating Nucleocytoplasmic Shuttling of Tobacco DNA-binding Protein Phosphatase-1

Tobacco DBP1 is the founding member of a novel class of plant transcription factors featuring sequence-specific DNA binding and protein phosphatase activity. To understand the mechanisms underlying the function of this family of transcriptional regulators, we have identified the tobacco 14-3-3 isoform G as the first protein interacting with a DBP factor. 14-3-3 recognition involves the N-terminal region of DBP1, which also supports the DNA binding activity attributed to DBP1. The relevance of this interaction is reinforced by its conservation in Arabidopsis plants, where the closest relative of DBP1 in this species also interacts with a homologous 14-3-3 protein through its N-terminal region. Furthermore, we show that in planta 14-3-3 G is directly involved in regulating DBP1 function by promoting nuclear export and subsequent cytoplasmic retention of DBP1 under conditions that in turn alleviate DBP1-mediated repression of target gene expression.

Suppressed Disassembly of Autolyzing p94/CAPN3 by N2A Connectin/Titin in a Genetic Reporter System

p94/calpain 3 is a skeletal muscle-specific member of the Ca(2+)-regulated cytosolic cysteine protease family, the calpains. Defective p94 protease activity originating from gene mutations causes a muscular dystrophy called calpainopathy, indicating the indispensability of p94 for muscle survival. Because of the existence of the p94-specific regions IS1 and IS2, p94 undergoes very rapid and exhaustive autolysis. To elucidate the physiological relevance of this unique activity, the autolytic profiles of p94 and the effect of the p94 binding protein, connectin/titin, on this process were investigated. In vitro analysis of p94 autolysis showed that autolysis in IS1 proceeds without immediate disassembly into fragments and that the newly identified cryptic autolytic site in IS2 is critical for disassembling autolyzed fragments. As a genetic system to assay p94 autolysis semiquantitatively, p94 was expressed in yeast as a hybrid protein between the DNA binding and activation domains of the yeast transcriptional activator Gal4. Transcriptional activation by the Gal4-p94:WT hybrid protein is precluded by p94 autolysis. Complete or partial loss of autolytic activity by C129S active site mutation, limb girdle muscular dystrophy type 2A pathogenic missense mutations, or PCR-based random mutagenesis could be detected by semiquantitative restoration of Gal4-dependent beta-galactosidase gene expression. Using this system, the N2A connectin fragment that binds to p94 was shown to suppress p94 autolytic disassembly. The proximity of the IS2 autolytic and connectin-binding sites in p94 suggested that N2A connectin suppresses IS2 autolysis. These data indicate the importance of p94-connectin interaction in the control of p94 functions by regulating autolytic decay of p94.

Tumor Necrosis Factor α-induced Desumoylation and Cytoplasmic Translocation of Homeodomain-interacting Protein Kinase 1 Are Critical for Apoptosis Signal-regulating Kinase 1-JNK/p38 Activation

The apoptosis signal-regulating kinase 1 (ASK1)-JNK/p38 signaling pathway is pivotal component in cell apoptosis and can be activated by a variety of death stimuli including tumor necrosis factor (TNF) alpha and oxidative stress (reactive oxygen species). However, the mechanism for ASK1 activation is not fully understood. We have recently identified ASK1-interacting protein (AIP1) as novel signal transducer in TNFalpha-induced ASK1 activation by facilitating dissociation of ASK1 from its inhibitor 14-3-3. In the present study, we employed yeast two-hybrid system using the N-terminal domain of AIP1 as bait and identified homeodomain-interacting protein kinase 1 (HIPK1) as an AIP1-associated protein. Interestingly, we showed that TNFalpha induced HIPK1 desumoylation concomitant with a translocation from nucleus to cytoplasm at 15 min followed by a return to nucleus by 60 min. The kinetics of HIPK1 translocation correlates with those of stress-induced ASK1-JNK/P38 activation. A specific JNK inhibitor blocked the reverse but not the initial translocation of HIPK1, suggesting that the initial translocation is an upstream event of ASK1-JNK/p38 signaling and JNK activation regulates the reverse translocation as a feedback mechanism. Consistently, expression of HIPK1 increased, whereas expression of a kinase-inactive form (HIPK1-D315N) or small interference RNA of HIPK1 decreased stress-induced ASK1-JNK/P38 activation without effects on IKK-NF-kappaB signaling. Moreover, a sumoylation-defective mutant of HIPK1 (KR5) localizes to the cytoplasm and is constitutively active in ASK1-JNK/P38 activation. Furthermore, HIPK1-KR5 induces dissociation of ASK1 from its inhibitors 14-3-3 and thioredoxin and synergizes with AIP1 to induce ASK1 activation. Our study suggests that TNFalpha-induced desumoylation and cytoplasmic translocation of HIPK1 are critical in TNFalpha-induced ASK1-JNK/p38 activation.

An Unexpected Role for Ubiquitylation of a Transcriptional Activator

The yeast transcriptional activator Gal4 has served as a paradigm for understanding how eukaryotic cells mount rapid transcriptional responses to environmental changes. In this issue of Cell, Muratani et al. (2005) provide evidence that Gal4 ubiquitylation and destruction are required for activation by Gal4. Surprisingly, this modification is required at a postinitiation step in transcription for the production of mRNAs that are correctly processed and fully functional for translation.

A Matter of Taste

Mating in Drosophila involves a stereotyped series of courtship behaviors in which the male fly successively orients toward the female, taps her with his foreleg, "sings," licks her, and finally attempts to copulate; courtship efficiency determines mating success. Courtship behavior is triggered by specific sensory cues, but the molecular mechanisms by which sensory stimuli are translated into behavioral responses remain unclear (see Giarratani and Vosshall). Bray and Amrein have now identified a male-specific receptor expressed in chemosensory foreleg neurons that appears to be required for efficient courtship. The authors used flies expressing the yeast transcriptional activator GAL4 under the control of the promoters of various members of a family of heterotrimeric GTP-binding protein (G protein)-coupled receptors ( Gr genes) that are expressed in subsets of gustatory receptor neurons to drive expression of reporters. One gene, Gr68a , was selectively expressed in chemosensory neurons of male-specific foreleg taste bristles. Inactivation of synaptic transmission from these neurons by selective expression of the tetanus toxin light-chain protein led to reduced courtship intensity, decreased mating efficiency, and lack of success in mating when competing with wild-type males. Knockdown of the GR68a receptor through RNA interference had similar effects. By quantifying performance of the steps involved in Drosophila courtship, the authors determined that the behavioral deficit occurred after orienting but before singing. Thus, G68a appears to be required for the recognition of the nonvolatile pheromones found on the abdomen of female Drosophila , and input from these pheromones during tapping appears to be required for successful completion of courtship behavior. S. Bray, H. Amrein, A putative Drosophila pheromone receptor expressed in male-specific taste neurons is required for efficient courtship. Neuron 39 , 1019-1029 (2003). [Online Journal] L. Giarratani, L. B. Vosshall, Toward a molecular description of pheromone perception. Neuron 39 , 881-883 (2003). [Online Journal]

Making a better RNAi vector for Drosophila: use of intron spacers

Double-stranded RNA induces sequence-specific inhibition of gene expression at a posttranscriptional level in eukaryotes (RNAi). This natural phenomenon has been developed into a tool for studying gene function in several model organisms, including Drosophila melanogaster. Transgenes bearing inverted repeats are able to exert an RNAi effect in Drosophila, but cloning difficulties and inconsistent silencing complicate the method. We have constructed a transgene containing inverted repeats separated by a functional intron such that mRNA produced by the transgene is predicted to form loopless hairpin RNA following splicing. A single copy of the transgene effectively and uniformly silences expression of a target gene ( white) in transgenic flies. We have developed a vector that is designed to produce intron-spliced hairpin RNA corresponding to any Drosophila gene. The vector is under control of the upstream activating sequence (UAS) of the yeast transcriptional activator GAL4. The UAS/GAL4 system allows hairpin RNA to conditionally silence gene expression in Drosophila in a tissue-specific manner. Moreover, the presence of the intron spacer greatly enhances the stability of inverted-repeat sequences in bacteria, facilitating the cloning procedure.

Efficient and heritable functional knock-out of an adult phenotype in Drosophila using a GAL4-driven hairpin RNA incorporating a heterologous spacer.

We have developed a modified RNA interference (RNAi) method for generating gene knock-outs in Drosophila melanogaster. We used the sequence of the yellow (y) locus to construct an inverted repeat that will form a double-stranded hairpin structure (y-IR) that is under the control of the upstream activating sequence (UAS) of the yeast transcriptional activator GAL4. Hairpins are extremely difficult to manipulate in Escherichia coli, so our method makes use of a heterologous 330 bp spacer encoding sequences from green fluorescent protein to facilitate the cloning steps. When the UAS-y-IR hairpin is expressed under the control of different promoter-GAL4 fusions, a high frequency of y pigment phenocopies is obtained in adults. Consequently this method for producing gene knock-outs has several advantages over previous methods in that it is applicable to any gene within the fly genome, greatly facilitates cloning of the hairpin, can be used if required with GAL4 drivers to avoid lethality or to induce RNAi in a specific developmental stage and/or tissue, is useful for generating knock-outs of adult phenotypes as reported here and, finally, the system can be manipulated to investigate the trans-acting factors that are involved in the RNAi mechanism.

Identification of transactivation, repression, and protein-protein interaction domains using GAL4-fusion proteins.

The Epstein-Barr viral nuclear antigen 2 (EBNA2) latency protein is a transcriptional activator that plays a critical role in regulation of Epstein-Barr virus (EBV) latency gene expression and in EBV-induced B-cell immortalization. GAL4-fusion constructions have been instrumental in elucidating the mechanism of action of EBNA2 (, , , ). Regulatory proteins are modular in structure with separate domains for functions such as promoter targeting, transcriptional activation, transcriptional repression, and protein-protein interactions. Functional chimeras can be generated by fusing domains from different proteins. In the case of transcriptional activation, the transactivator protein must be targeted to the responsive promoter and contain an activation domain that either directly or indirectly interacts with the core cellular transcriptional machinery to increase transcriptional efficiency. Early work on transcriptional transactivators demonstrated the modular nature of these proteins by showing that the DNA binding domain of the yeast transcriptional activator GAL4, GAL4 (aa1-147) could be fused to a variety of yeast, cellular, bacterial, and viral sequences to reconstitute functional transcriptional activator proteins (,). Subsequently, GAL4-fusions became the standard experimental approach for identifying activation domains.