Showing papers on "Upstream activating sequence published in 2020"

Expanding Toolbox for Genes Expression of Yarrowia lipolytica to Include Novel Inducible, Repressible, and Hybrid Promoters.

Abstract: Promoters are critical tools to precisely control gene expression for both synthetic biology and metabolic engineering Although Yarrowia lipolytica has demonstrated many industrially relevant advantages, promoter discovery efforts on this non-conventional yeast are limited due to the challenge in finding suitable inducible and repressible promoters Six copper-inducible promoters and five repressible promoters were isolated in this work Especially, Cu2+-repressible promoters showed relatively high activity under non-repressing conditions compared with a constitutive promoter, but the strength could be almost fully repressed by a supplement of a low content of Cu2+ The six Cu2+-inducible promoters were engineered to improve their dynamic regulation range with a tandem upstream activation sequence An engineered promoter was successfully used to construct a more productive pathway for production of a novel bioproduct, wax ester, than that used for both Cu2+-inducible promoter and constitutive promoter This study provides effective tools applicable to fine-tune the gene expression in this microbial host

Co-Expression of a Homologous Cytochrome P450 Reductase Is Required for In Vivo Validation of the Tetranychus urticae CYP392A16-Based Abamectin Resistance in Drosophila.

Abstract: Overexpression of the cytochrome P450 monooxygenase CYP392A16 has been previously associated with abamectin resistance using transcriptional analysis in the two-spotted spider mite Tetranychus urticae, an important pest species worldwide; however, this association has not been functionally validated in vivo despite the demonstrated ability of CYP392A16 to metabolize abamectin in vitro. We expressed CYP392A16 in vivo via a Gal4 transcription activator protein/Upstream Activating Sequence (GAL4/UAS) system in Drosophila melanogaster flies, driving expression with detoxification tissue-specific drivers. We demonstrated that CYP392A16 expression confers statistically significant abamectin resistance in toxicity bioassays in Drosophila only when its homologous redox partner, cytochrome P450 reductase (TuCPR), is co-expressed in transgenic flies. Our study shows that the Drosophila model can be further improved, to facilitate the functional analysis of insecticide resistance mechanisms acting alone or in combination.

A Role for Mediator Core in Limiting Coactivator Recruitment in Saccharomyces cerevisiae

Abstract: Mediator is an essential, multisubunit complex that functions as a transcriptional coactivator in yeast and other eukaryotic organisms. Mediator has four conserved modules, Head, Middle, Tail, and Kinase, and has been implicated in nearly all aspects of gene regulation. The Tail module has been shown to recruit the Mediator complex to the enhancer or upstream activating sequence (UAS) regions of genes via interactions with transcription factors, and the Kinase module facilitates the transition of Mediator from the UAS/enhancer to the preinitiation complex via protein phosphorylation. Here, we analyze expression of the Saccharomyces cerevisiae HO gene using a sin4 Mediator Tail mutation that separates the Tail module from the rest of the complex; the sin4 mutation permits independent recruitment of the Tail module to promoters without the rest of Mediator. Significant increases in recruitment of the SWI/SNF and SAGA coactivators to the HO promoter UAS were observed in a sin4 mutant, along with increased gene activation. These results are consistent with recent studies that have suggested that the Kinase module functions negatively to inhibit activation by the Tail. However, we found that Kinase module mutations did not mimic the effect of a sin4 mutation on HO expression. This suggests that at HO the core Mediator complex (Middle and Head modules) must play a role in limiting Tail binding to the promoter UAS and gene activation. We propose that the core Mediator complex helps modulate Mediator binding to the UAS regions of genes to limit coactivator recruitment and ensure proper regulation of gene transcription.

Identification and Characterization of a Novel Basic Helix-Loop-Helix Transcription Factor of Phospholipid Synthesis Regulation in Aspergillus niger.

Abstract: The synthesis of phospholipids relies on a sort of genes, whose promoter regions contain inositol-sensitive upstream activation sequence (UASINO) and are regulated by the basic helix-loop-helix (bHLH)-type ino2/ino4 transcription factor (TF) pair. Ten putative bHLH TFs have been found through whole genome sequencing of Aspergillus niger, but none of these TFs have been characterized. In this study, we identified and characterized the bHLH-type TF ino2(An02g04350) in A. niger. Electrophoretic mobility shift assay (EMSA) and yeast two-hybrid assay demonstrated that ino2 functions as a homodimer in UASINO genes (e.g., ino1 and cho1) and binds to opi1(An1g02370) in vitro. Real-time quantitative PCR of ino1 and quantification of total phospholipid indicated that the ino2 disruptant downregulated the transcription of ino1 and the amount of total cellular phosphatidylinositol. In addition, phenotype analyses showed that a loss of ino2 led to resistance to cell wall interference and DNA damage. Comparative transcriptome analyses showed that more than 1000 genes and GO terms associated with UASINO, endoplasmic reticulum-associated protein degradation, phosphatidylinositol synthesis, chitin synthesis, and fatty acid synthesis were differentially expressed in Δino2 compared to the wild type (WT). Taken together, these observations indicate that the bHLH TF ino2 functions as a homodimer that regulates the synthesis of phosphatidylinositol, fatty acid, and chitin and influences the homeostasis of the endoplasmic reticulum membrane.

Transcriptome analysis reveals the effects of transgenic expression of the Gal4 protein on normal gene expression in silkworm tissues

Abstract: The Gal4/upstream activating sequence(UAS) system, a well-known genetic tool, has been widely used to analyze gene function in many organisms, including the silkworm (Bombyx mori), a model lepidopteran insect. Several studies have suggested that Gal4 protein activation in tissues can negatively affect transgenic individuals; however, whether and to what extent the Gal4 protein affects normal endogenous gene expression have rarely been studied. Here, we analyzed the transcriptomes of transgenic silkworms expressing the Gal4 protein at high levels in both the wing disc (WD) and epidermis (EP) and investigated gene expression changes in both tissues. Overall, 24,593 genes were identified in the WD and EP libraries, and 2,025 and 2,488 were identified as significant differentially expressed genes(DEGs) in the WD and EP between the transgenic and control groups, respectively. These DEGs were further annotated by gene function classification and pathway assessment using public databases. In addition, 506 DEGs were shared (common) between both tissues. Of these, 97 genes were commonly upregulated, and 234 were commonly downregulated; many of them were annotated to be involved in metabolic processes such as “fat digestion and absorption”, “glycine, serine and threonine metabolism” and “glutathione metabolism” and in signal transduction pathways such as the “Rap1 signaling pathway”, “MAPK signaling pathway” and “Hippo signaling pathway”. Overall, this work enhances understanding of the effects of transgenic Gal4 protein expression on normal gene expression in silkworm tissues and suggests that researchers should pay attention to unexpected effects when using the Gal4/UAS system to study gene function.

Knockdown transgenic Drosophila and Parkinson's disease

Abstract: In 2000, by using the binary galactose 4 (GAL4)/upstream activating sequence (UAS) system, Feany and Bender were the first to report a transgenic Drosophila melanogaster fly model expressing familial Parkinson's disease (PD)-linked mutants (Alanine53Treonine and Alanine30Proline) of human alpha-synuclein. Amazingly, these mutant Drosophila (as was not the case with normal Drosophila) replicated the essential features of human PD including adult-onset loss of dopaminergic neurons, filamentous intraneuronal inclusions containing alpha-synuclein, and locomotor dysfunction. We provide highlights in the history of Drosophila PD research with special attention paid to the knockdown transgenic Drosophila GAL4/UAS RNA interference system to characterize and understand the response of mutant parkin, dj-1β, and Lrrk flies to oxidative stress phenomena induced by PQ2+ intoxication, thereby exploring potential therapeutic treatments.