Showing papers on "Upstream activating sequence published in 2008"
TL;DR: It is proposed that PROMPT transcription is a common characteristic of RNA polymerase II (RNAPII) transcribed genes with a possible regulatory potential and is positively correlated with gene activity.
Abstract: Studies have shown that the bulk of eukaryotic genomes is transcribed. Transcriptome maps are frequently updated, but low-abundant transcripts have probably gone unnoticed. To eliminate RNA degradation, we depleted the exonucleolytic RNA exosome from human cells and then subjected the RNA to tiling microarray analysis. This revealed a class of short, polyadenylated and highly unstable RNAs. These promoter upstream transcripts (PROMPTs) are produced approximately 0.5 to 2.5 kilobases upstream of active transcription start sites. PROMPT transcription occurs in both sense and antisense directions with respect to the downstream gene. In addition, it requires the presence of the gene promoter and is positively correlated with gene activity. We propose that PROMPT transcription is a common characteristic of RNA polymerase II (RNAPII) transcribed genes with a possible regulatory potential.
725 citations
TL;DR: The Gal4 gene trap and enhancer trap approaches together with various UAS lines should be important tools for investigating roles of genes and cells in vertebrate systems for a long time.
Abstract: Targeted gene expression by the Gal4-UAS system is a powerful methodology for analyzing function of genes and cells in vivo and has been extensively used in genetic studies in Drosophila. On the other hand, the Gal4-UAS system had not been applied effectively to vertebrate systems for a long time mainly due to the lack of an efficient transgenesis method. Recently, a highly efficient transgenesis method using the medaka fish Tol2 transposable element was developed in zebrafish. Taking advantage of the Tol2 transposon system, we and other groups developed the Gal4 gene trap and enhancer trap methods and established various transgenic fish expressing Gal4 in specific cells. By crossing such Gal4 lines with transgenic fish lines harboring various reporter genes and effector genes downstream of UAS (upstream activating sequence), specific cells can be visualized and manipulated in vivo by targeted gene expression. Thus, the Gal4 gene trap and enhancer trap approaches together with various UAS lines should be important tools for investigating roles of genes and cells in vertebrates.
152 citations
TL;DR: The GAL4 system is described, highlighting the properties that make it a powerful tool for the analysis of gene function in Drosophila and higher organisms.
Abstract: Over the past decade the adoption and refinement of the GAL4 system by the Drosophila field has resulted in a wide array of tools with which the researcher can drive transgene expression in a precise spatiotemporal pattern. The GAL4 system relies on two components: (1) GAL4, a transcriptional activator from yeast, which is expressed in a tissue-specific manner and (2) a transgene under the control of the upstream activation sequence that is bound by GAL4 (UASG). The two components are brought together in a simple genetic cross. In the progeny of the cross, the transgene is only transcribed in those cells or tissues expressing the GAL4 protein. Recent modifications of the GAL4 system have improved the control of both the initiation and the spatial restriction of transgene expression. Here we describe the GAL4 system highlighting the properties that make it a powerful tool for the analysis of gene function in Drosophila and higher organisms.
151 citations
TL;DR: To improve the utility of the GeneSwitch GAL4 system of Drosophila, a large-scale enhancer-trap screen for insertions that yielded nervous system expression was performed and revealed novel patterns of sensory neurons, interneurons, and glia.
Abstract: There is a critical need for genetic methods for the inducible expression of transgenes in specific cells during development. A promising approach for this is the GeneSwitch GAL4 system of Drosophila. With GeneSwitch GAL4 the expression of upstream activating sequence (UAS) effector lines is controlled by a chimeric GAL4 protein that becomes active in the presence of the steroid RU486 (mifepristone). To improve the utility of this expression system, we performed a large-scale enhancer-trap screen for insertions that yielded nervous system expression. A total of 204 GeneSwitch GAL4 lines with various larval expression patterns in neurons, glia, and/or muscle fibers were identified for chromosomes I–III. All of the retained lines show increased activity when induced with RU486. Many of the lines reveal novel patterns of sensory neurons, interneurons, and glia. There were some tissue-specific differences in background expression, with muscles and glia being more likely to show activity in the absence of the inducing agent. However, >90% of the neuron-specific driver lines showed little or no background activity, making them particularly useful for inducible expression studies.
136 citations
TL;DR: It is proposed that miR169 and uORF1p play essential, sequential, and nonredundant roles in regulating MtHAP2-1 expression and contributes to a better understanding of the action of upstream ORFs (uORFs) in the regulation of gene expression.
Abstract: MtHAP2-1 is a CCAAT-binding transcription factor from the model legume Medicago truncatula. We previously showed that MtHAP2-1 expression is regulated both spatially and temporally by microRNA169. Here we present a novel regulatory mechanism controlling MtHAP2-1 expression. Alternative splicing of an intron in the MtHAP2-1 5'leader sequence (LS) becomes predominant during the development of root nodules, leading to the production of a small peptide, uORF1p. Our results indicate that binding of uORF1p to MtHAP2-1 5'LS mRNA leads to reduced accumulation of the MtHAP2-1 transcript and may contribute to spatial restriction of MtHAP2-1 expression within the nodule. We propose that miR169 and uORF1p play essential, sequential, and nonredundant roles in regulating MtHAP2-1 expression. Importantly, in contrast to previously described cis-acting uORFs, uORF1p is able to act in trans to down-regulate gene expression. Our work thus contributes to a better understanding of the action of upstream ORFs (uORFs) in the regulation of gene expression.
114 citations
TL;DR: It is shown that Sus1 is required during transcription elongation and is associated with the elongating form of RNA Polymerase II (RNAP II) phosphorylated on Ser5 and Ser2 of the C-terminal domain (CTD), and that Sgf73 is necessary for association of Sus1 with both SAGA and TREX2, and that its absence dramatically reduces Sus1 occupancy of UAS and ORF sequences.
Abstract: Gene transcription, RNA biogenesis, and mRNA transport constitute a complicated process essential for all eukaryotic cells. The transcription/export factor Sus1 plays a key role in coupling transcription activation with mRNA export, and it resides in both the SAGA and TREX2 complexes. Moreover, Sus1 is responsible for GAL1 gene gating at the nuclear periphery, which is important for its transcriptional status. Here, we show that Sus1 is required during transcription elongation and is associated with the elongating form of RNA Polymerase II (RNAP II) phosphorylated on Ser5 and Ser2 of the C-terminal domain (CTD). In addition, Sus1 copurifies with the essential mRNA export factors Yra1 and Mex67, which bind to the mRNA cotranscriptionally. Consistently, ChIP analysis reveals that Sus1 is present at coding regions dependent on transcription in a manner stimulated by Kin28-dependent CTD phosphorylation. Strikingly, eliminating the TREX2 component Sac3 or the SAGA subunit Ubp8 partially impairs Sus1 targeting to coding sequences and upstream activating sequences (UAS). We found, unexpectedly, that Sgf73 is necessary for association of Sus1 with both SAGA and TREX2, and that its absence dramatically reduces Sus1 occupancy of UAS and ORF sequences. Our results reveal that Sus1 plays a key role in coordinating gene transcription and mRNA export by working at the interface between the SAGA and TREX2 complexes during transcription elongation.
106 citations
TL;DR: It is shown that in Saccharomyces cerevisiae tRNA genes (tDNAs) and even partially assembled Pol III complexes containing only the transcription factor TFIIIC can exhibit chromatin boundary functions both as heterochromatin barriers and as insulators to gene activation.
Abstract: Chromosomal sites of RNA polymerase III (Pol III) transcription have been demonstrated to have “extratranscriptional” functions, as the assembled Pol III complex can act as chromatin boundaries or pause sites for replication forks, can alter nucleosome positioning or affect transcription of neighboring genes, and can play a role in sister chromatid cohesion. Several studies have demonstrated that assembled Pol III complexes block the propagation of heterochromatin-mediated gene repression. Here we show that in Saccharomyces cerevisiae tRNA genes (tDNAs) and even partially assembled Pol III complexes containing only the transcription factor TFIIIC can exhibit chromatin boundary functions both as heterochromatin barriers and as insulators to gene activation. Both the TRT2 tDNA and the ETC4 site which binds only the TFIIIC complex prevented an upstream activation sequence from activating the GAL promoters in our assay system, effectively acting as chromatin insulators. Additionally, when placed downstream from the heterochromatic HMR locus, ETC4 blocked the ectopic spread of Sir protein-mediated silencing, thus functioning as a barrier to repression. Finally, we show that TRT2 and the ETC6 site upstream of TFC6 in their natural contexts display potential insulator-like functions, and ETC6 may represent a novel case of a Pol III factor directly regulating a Pol II promoter. The results are discussed in the context of how the TFIIIC transcription factor complex may function to demarcate chromosomal domains in yeast and possibly in other eukaryotes.
90 citations
TL;DR: The mapped polyadenylation sites of the upstream, unstable short transcripts form a heterogeneous family of RNAs of ≈200 nucleotides extend the emerging concept that distinct modes of termination by RNA polymerase II exist and that the distance of the terminator from the promoter, as well as its sequence, is important for the pathway chosen.
Abstract: The IMD2 gene in Saccharomyces cerevisiae is regulated by intracellular guanine nucleotides. Regulation is exerted through the choice of alternative transcription start sites that results in synthesis of either an unstable short transcript terminating upstream of the start codon or a full-length productive IMD2 mRNA. Start site selection is dictated by the intracellular guanine nucleotide levels. Here we have mapped the polyadenylation sites of the upstream, unstable short transcripts that form a heterogeneous family of RNAs of ≈200 nucleotides. The switch from the upstream to downstream start sites required the Rpb9 subunit of RNA polymerase II. The enzyme's ability to locate the downstream initiation site decreased exponentially as the start was moved downstream from the TATA box. This suggests that RNA polymerase II's pincer grip is important as it slides on DNA in search of a start site. Exosome degradation of the upstream transcripts was highly dependent upon the distance between the terminator and promoter. Similarly, termination was dependent upon the Sen1 helicase when close to the promoter. These findings extend the emerging concept that distinct modes of termination by RNA polymerase II exist and that the distance of the terminator from the promoter, as well as its sequence, is important for the pathway chosen.
83 citations
TL;DR: The study demonstrated that the activation of MMP-9 promoter is dependent upon interactions of Fra-1/c-Jun with Stat3, and a juxtaposed Stat3/AP-1 element plays a crucial role in the manner of enhancersome in theactivation of M MP-9 gene.
79 citations
TL;DR: These findings suggest that the inhibitory effect on T7 transcription results from Z-DNA formation in the (CG)14 sequence rather than from an effect of the sequence composition or from hairpin formation in either the DNA or the RNA product.
Abstract: DNA sequences capable of forming unusual secondary structures can be a source of genomic instability. In some cases that instability might be affected by transcription, as recently shown for the Z-DNA forming sequence (CG)(14), which causes genomic instability both in mammalian cells and in bacteria, and this effect increases with its transcription. We have investigated the effect of this (CG)(14) sequence on transcription with T7 RNA polymerase in vitro. We detected partial transcription blockage within the sequence; the blockage increased with negative supercoiling of the template DNA. This effect was not observed in a control self-complementary sequence of identical length and base composition as the (CG)(14) sequence, when the purine-pyrimidine alternation required for Z-DNA formation was disrupted. These findings suggest that the inhibitory effect on T7 transcription results from Z-DNA formation in the (CG)(14) sequence rather than from an effect of the sequence composition or from hairpin formation in either the DNA or the RNA product.
55 citations
TL;DR: Novel regulatory sequences for human (h) SSTR2 transcription are demonstrated and the existence of a novel upstream promoter for the hSSTR2 gene that is regulated by epigenetic modifications is revealed, suggesting for complex control of thehSSTR 2 transcription.
Abstract: Somatostatin is a neuropeptide that inhibits exocrine and endocrine secretions of several hormones and negatively regulates cell proliferation. These events are mediated through somatostatin engagement on one of five G protein-coupled receptors named SSTR1 to STTR5. Somatostatin binding to SSTR2 mediates predominantly antisecretory and antiproliferative effects; two important biological activities in the gastroenteropancreatic endocrine and exocrine system. Herein we demonstrate novel regulatory sequences for human (h) SSTR2 transcription. By genomic DNA sequence analysis, we reveal two CpG islands located 3.8 kb upstream from the transcription start site. We identify a novel transcription start site and a promoter region within one of these CpG islands. We demonstrate that two epigenetic modifications, DNA methylation and histone acetylation, regulate the activation of hSSTR2 upstream promoter. Furthermore, we show that the transcription from this upstream promoter region directly correlates to hSSTR2 mRNA expression in various human cell lines. A combined treatment of a demethylating agent, 5-aza-2-deoxycytidine and a histone deacetylase inhibitor, trichostatin A, leads to increased expression of hSSTR2 mRNA in cell lines in which the CpG island is methylated. The epigenetic regulation of this promoter region results in differential expression of hSSTR2 mRNA in human cell lines. This study reveals the existence of a novel upstream promoter for the hSSTR2 gene that is regulated by epigenetic modifications, suggesting for complex control of the hSSTR2 transcription.
TL;DR: It is concluded that CK2 triggers the IRF-4 and p65 pathways independently to serve as a master regulator of IL-1β transcription, and that activation of both NF-κB and IRf-4 depends on CK2 kinase activity.
Abstract: IL-1beta is a key proinflammatory cytokine with roles in multiple diseases. Monocytes package the IL-1beta promoter into a "poised architecture" characterized by a histone-free transcription start site and constitutive transcription factor associations. Upon LPS stimulation, multiple proteins inducibly associate with the IL-1beta gene. To understand how the complex combination of constitutive and inducible transcription factors activate the IL-1beta gene from a poised structure, we measured temporal changes in NF-kappaB and IFN regulatory factor (IRF) association with IL-1beta regulatory elements. Association of the p65 subunit of NF-kappaB peaks 30-60 min post-monocyte stimulation, and it shortly precedes IRF-4 recruitment to the IL-1beta enhancer and maximal mRNA production. In contrast, IRF-8/enhancer association decreases poststimulation. To test the importance of delayed IRF-4/enhancer association, we introduced a mutated PU.1 protein shown to prevent PU.1-mediated IRF-4 recruitment to the enhancer sequence. Mutated PU.1 initially increased IL-1beta mRNA followed by decreased mRNA levels 2-3 h poststimulation. Taken together, these data support a dynamic model of IL-1beta transcriptional activation in which a combination of IRF-8 and p65 drives the initial phase of IL-1beta transcription, while PU.1-mediated IRF-4 recruitment to the enhancer is important for the second phase. We further demonstrate that activation of both NF-kappaB and IRF-4 depends on CK2 kinase activity. Because IRF-4/enhancer association requires CK2 but not p65 activation, we conclude that CK2 triggers the IRF-4 and p65 pathways independently to serve as a master regulator of IL-1beta transcription.
TL;DR: It is demonstrated for the first time that a cytokine gene can be regulated by cis-acting regions much further upstream than previously examined, and the control of IL6 transcription is likely to beregulated by a complex mechanism of modular cis-regulatory elements.
TL;DR: The results demonstrate the important role Sp1 and Sp3 plays in regulating the expression of human EC-SOD in the lung and show strong activation of luciferase gene expression.
Abstract: The molecular mechanisms that govern the transcription of human extracellular superoxide dismutase (EC-SOD), the major extracellular antioxidant enzyme, are largely unknown. To elucidate the mechanisms involved in human EC-SOD gene regulation and expression, we localized multiple transcription start sites to a finite region located 3.9 kb upstream of the ATG initiation codon. Within this segment, we subcloned a 2.7-kb fragment upstream of a luciferase reporter gene; the resulting construct exhibited strong in vivo promoter activity in two lung-derived cell lines. Deletion analysis of the EC-SOD 5′-flanking sequences identified a minimal 0.3-kb region that had strong basal promoter activity. Computer sequence analysis revealed a putative Sp1-like binding site within the EC-SOD proximal promoter region that lacked a TATA-box and showed a high frequency of GC nucleotides. Binding of Sp1 and Sp3 transcription factors to the EC-SOD promoter was confirmed by DNase I footprint analysis, electophoretic mobility shift assay, and competition and supershift assays. Cotransfection of the EC-SOD promoter–luciferase reporter constructs with plasmids encoding Sp1 and Sp3 into Sp-deficient insect SL2 cells showed strong activation of luciferase gene expression. The occupancy of the EC-SOD promoter by Sp1/Sp3 and RNA polymerase II in vivo was determined by chromatin immunoprecipitation assay and correlated well with levels of EC-SOD expression in lung epithelial cells (A549) and pulmonary fibroblasts (MRC5). Collectively, our results demonstrate the important role Sp1 and Sp3 plays in regulating the expression of human EC-SOD in the lung.
TL;DR: This work has mapped and begun studies of the transcriptional control regions of the cephalochordate amphioxus MEF2 gene, suggesting that this mode of autoregulatory control is conserved among higher metazoan MEf2 genes.
TL;DR: Results indicate that lipase expression in Pseudomonas alcaligenes is under the control of the LipQR two-component system, strongly suggesting that LipR acts as a positive regulator of lipaseexpression.
Abstract: Preliminary observations in a large-scale fermentation process suggested that the lipase expression of Pseudomonas alcaligenes can be switched on by the addition of certain medium components, such as soybean oil. In an attempt to elucidate the mechanism of induction of lipase expression, we have set up a search method for genes controlling lipase expression by use of a cosmid library containing fragments of P. alcaligenes genomic DNA. A screen for lipase hyperproduction resulted in the selection of multiple transformants, of which the best-producing strains comprised cosmids that shared an overlapping genomic fragment. Within this fragment, two previously unidentified genes were found and named lipQ and lipR. Their encoded proteins belong to the NtrBC family of regulators that regulate gene expression via binding to a specific upstream activator sequence (UAS). Such an NtrC-like UAS was identified in a previous study in the P. alcaligenes lipase promoter, strongly suggesting that LipR acts as a positive regulator of lipase expression. The regulating role could be confirmed by down-regulated lipase expression in a strain with an inactivated lipR gene and a threefold increase in lipase yield in a large-scale fermentation when expressing the lipQR operon from the multicopy plasmid pLAFR3. Finally, cell extracts of a LipR-overexpressing strain caused a retardation of the lipase promoter fragment in a band shift assay. Our results indicate that lipase expression in Pseudomonas alcaligenes is under the control of the LipQR two-component system.
TL;DR: The data establish an animal model that mimics some of the features of human mtDNA depletion syndromes, and brains of larvae over‐expressing pol γ‐α showed evidence of increased cell death that correlates with the observed phenotypes.
Abstract: DNA polymerase γ (pol γ) is the sole DNA polymerase devoted to mitochondrial DNA (mtDNA) replication. We have characterized the molecular and physiological effects of over-expression of the catalytic subunit of pol γ, pol γ-α, in the nervous system of Drosophila melanogaster using the upstream activation sequence (UAS)/yeast transcriptional activator by binding to UAS (GAL4) system. Tissue-specific over-expression of pol γ-α was confirmed by immunoblot analysis, whereas the very low levels of endogenous protein are undetectable in UAS or GAL4 control lines. The transgenic flies over-expressing pol γ-α in the nervous system showed a moderate increase in pupal lethality, and a significant decrease in the median life span of adult flies. Moreover, these flies displayed a decrease in the rate of synthesis of mtDNA, which is accompanied by a significant mtDNA depletion, and a corresponding decrease in the levels of mitochondrial transcription factor A (mtTFA). Biochemical analysis showed an oxidative phosphorylation (OXPHOS) defect in transgenic flies, which were more susceptible to oxidative stress. Although we did not detect apoptosis in the nervous system of adult transgenic flies, brains of larvae over-expressing pol γ-α showed evidence of increased cell death that correlates with the observed phenotypes. Our data establish an animal model that mimics some of the features of human mtDNA depletion syndromes.
TL;DR: A novel physiologically relevant transcriptional mechanism for the reciprocal regulation of the SREBP‐1a expression is revealed and a functional role for the 3 GC‐boxes containing overlapping sites for the Sp1 and EGR‐1 transcription factors is discovered.
TL;DR: Expression of the eglA gene, encoding for a major endoglucanse EG A in Aspergillus nidulans, is induced by cellulose and cellobiose, but not by xylose, suggesting that induction is independent of XlnR, a transcriptional activator of xylanolytic and cellulolytic genes in As pergillus.
Abstract: Expression of the eglA gene, encoding for a major endoglucanse EG A in Aspergillus nidulans, is induced by cellulose and cellobiose, but not by xylose. This suggests that induction is independent of XlnR, a transcriptional activator of xylanolytic and cellulolytic genes in Aspergillus.Mutational analysis of the eglA promoter was performed to identify the novel cis-element responsible for XlnR-independent induction. The region spanning −153 to −138 (CCGTACCTTTTTAGGA), designated CeRE(Cellulose Responsive Element), was found to be the upstream activating element essential to inductive expression of the eglA gene.
TL;DR: It is found that at least part of the genomic response to GAL3 appears to be caused by effects of GAL4 on stress and immune response pathways, including genes that encode components of important signaling pathways.
Abstract: The yeast transcription factor GAL4 is widely used in Drosophila genetics to misexpress genes that are under control of the yeast upstream activator sequence (UAS). Here we show that high levels of GAL4 change the expression of many Drosophila genes in a UAS-independent manner, including genes that encode components of important signaling pathways. We find that at least part of the genomic response to GAL4 appears to be caused by effects of GAL4 on stress and immune response pathways. Finally, using the transcription factor Senseless as an example, we demonstrate how an interaction between GAL4 and a GAL4-driven protein can impede the use of the GAL4/UAS system in experiments aimed at determining the transcriptional response to a misexpressed gene.
TL;DR: This study characterize for the first time the basal activity of the human PRDM1 promoter, through which several factors, including SP1, SP3 and early growth response 1, modulate its expression through a conserved GC‐box.
Abstract: The positive regulatory domain containing 1, encoded by the PRDM1 gene, is a transcriptional repressor considered as a master regulator that is required and sufficient for plasma cell differentiation. In the present study we have performed sequence analysis of the upstream region of the human PRDM1 gene to detect the minimal promoter region necessary for PRDM1 gene transcription. This region comprises the region upstream of the initiation site, as well as the first exon. Collectively, deletion and mutation analysis in conjunction with luciferase reporter assays, EMSA and supershift assays identified a phylogenetically conserved GC-box as an essential element for PRDM1 expression. This GC-box element matches to a binding site for multiple transcription factors such as SP1 and SP3 isoforms as well as early growth response 1. Chromatin immunoprecipitation assays confirmed the in vivo binding capability of these factors to the human PRDM1 promoter. These studies together characterize for the first time the basal activity of the human PRDM1 promoter, through which several factors, including SP1, SP3 and early growth response 1, modulate its expression through a conserved GC-box.
TL;DR: Results demonstrate that GATA4 and Nkx2.5 could be one of the upstream regulators of Id2, and suggest that Id proteins may play a regulatory role in cardiogenesis, and identify two motifs located at −497/−502 and −264/−270 were functionally important for Id2 promoter activation by GATA 4 and NKx 2.5.
Abstract: Inhibitor of differentiation/DNA binding (Id) proteins function as a regulator of helix-loop-helix proteins participating in cell lineage commitment and differentiation. Here, we observed a marked induction of Id2 during cardiomyocyte differentiation from P19CL6 murine embryonic teratocarcinoma stem cells, prompting us to investigate the upstream regulatory mechanism of Id2 induction. Computer analysis of Id2 promoter and subsequent electrophoretic mobility shift assay revealed several binding sites for GATA4 and Nkx2.5 within the Id2 promoter. By further deletion and mutation analysis of the respective binding site, we identified that two motifs located at -497/-502 and -264/-270 were functionally important for Id2 promoter activation by GATA4 and Nkx2.5, respectively. Overexpression of GATA4 and/or Nkx2.5 induced not only Id2 promoter activity but also Id2 protein expression. Additionally, Id proteins significantly inhibit the GATA4 and Nkx2.5-dependent transcription, suggesting Id proteins may play a regulatory role in cardiogenesis. Collectively, our results demonstrate that GATA4 and Nkx2.5 could be one of the upstream regulators of Id2.
TL;DR: In this paper, the authors identify factors regulating PRm1 within the megakaryocytic human erythroleukemia 92.1.7 cell line, and demonstrate that Sp1, Egr1, and NF-E2 regulate core Prm1 activity, whereas GATA-1 and Ets-1 act as critical upstream activators.
TL;DR: JDP2 physically interacts (MI:0218) with ATF2 (uniprotkb:P15336) and c-JUN ( uniprot kb:P05412) by anti tag coimmunoprecipitation.
TL;DR: It is suggested that the ultraconservation may reflect the importance of finely tuned regulation of alternate transcription of FBN1 and that the sequences involved have been under negative selective pressure for at least the last 180 million years of mammalian evolution.
TL;DR: In this paper, a quantitative real-time RT-PCR method was developed for determining the differential expression of C. rugosa lipase family genes in response to various environmental and nutritional factors.
Abstract: Five lipase genes have been identified and sequenced from Candida rugosa. However, as the sequences of LIP multigene family are extremely closely related, it is difficult to characterize the expression spectrum of LIP genes. In the present work we have cloned, sequenced, and analyzed the promoters of these five LIP isoform genes, and several putative transcriptional elements including oleate response element (ORE) and upstream activation sequence 1 (UAS1) were identified. A quantitative real-time RT-PCR method was developed for determining the differential expression of C. rugosa lipase family genes in response to various environmental and nutritional factors. While all five LIP genes display significant changes in mRNA expression under oleic acid and/or olive oil culture conditions, LIP2 showed the strongest induction (456-fold) in response to oleic acid. LIP transcription and promoter regulation were studied by assaying the beta-galactosidase activities of promoter-lacZ fusions in Saccharomyces cerevisiae. Three of the LIP genes, LIP3, LIP4, and LIP5, showed significant induction by oleic acid, and their ORE and UAS1 elements are essential for induction by oleic acid. Together, this suggests that the multiple lipase expression profiles may be due to differential transcriptional regulation of the LIP genes in response to environment or nutritional factors.
TL;DR: It is shown that, when Pip2 is missing, Oaf 1 may form homodimers which apparently substitute for the Oaf1-Pip2 heterodimer, and some of the oleate-inducible genes undergo activation even in the absence of Pip2.
TL;DR: It is demonstrated that the expression of the aub gene and protein increase in aubsting mutants is demonstrated and an increased amount of AUBERGINE interferes with the regular functioning of the somatic RNAi pathway.
Abstract: 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 aubsting mutants. We used a genetic method to test whether aubsting 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 yellow1 (y1) 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.
TL;DR: This work measured silencing in different tissues and stages of development and found variable silencing of GFP expression driven by the same driver, suggesting that trans-inactivation may not be possible until later in development.
Abstract: The brownDominant (bwD) allele of Drosophila contains a heterochromatic block that causes the locus to interact with centric heterochromatin. This association silences bw+ in heterozygotes (trans-inactivation) and is dependent on nuclear organizational changes later in development, suggesting that trans-inactivation may not be possible until later in development. To study this, a P element containing an upstream activating sequence (UAS)–GFP reporter was inserted 5 kb from the bwD insertion site. Seven different GAL4 driver lines were used and GFP fluorescence was compared in the presence or the absence of bwD. We measured silencing in different tissues and stages of development and found variable silencing of GFP expression driven by the same driver. When UAS–GFP was not expressed until differentiation in the eye imaginal disc it was more easily trans-inactivated than when it was expressed earlier in undifferentiated cells. In contrast to some studies by other workers on silencing in cis, we did not find consistent correlation of silencing with level of expression or evidence of relaxation of silencing with terminal differentiation. We suggest that such contrasting results may be attributed to a potentially different role played by nuclear organization in cis and trans position-effect variegation.
TL;DR: This protocol describes the generation of driver and reporter lines for use with the GAL4 system in Drosophila and an alternative strategy for integration using the phiC31 system is provided.
Abstract: INTRODUCTIONThe GAL4 system is a method for ectopic gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns. This protocol describes the generation of driver and reporter lines for use with the GAL4 system in Drosophila. A promoter-GAL4 fusion is constructed using a P-element transformable vector, and a GAL4-responsive target gene is created via generation of an upstream activation sequence (UAS)-reporter construct. An alternative strategy for integration using the phiC31 system is also provided. Transformant lines are generated using standard procedures for microinjection.