scholarly journals Transient expression in tobacco Bright Yellow 2 cells and pollen grains: A fast, efficient and reliable system for functional promoter analysis of plant genes

2010 ◽  
Vol 62 (1) ◽  
pp. 57-62 ◽  
Author(s):  
Ana Bratic ◽  
Dragana Majic ◽  
Jelena Samardzic ◽  
M.W. Kragl ◽  
Vesna Maksimovic
Keyword(s):  
Gene Expression ◽  
Transient Expression ◽  
Dna Sequences ◽  
Promoter Activity ◽  
Pollen Grains ◽  
Promoter Strength ◽  
Reliable System ◽  
Bright Yellow ◽  
Metallothionein Promoter ◽  
Plant Genes

Gene expression is mediated by DNA sequences directly upstream from the coding sequences, recruited transcription factors and RNA polymerase in a spatially-defined manner. Understanding promoter strength and regulation would enhance our understanding of gene expression. The goal of this study was to develop a fast, efficient and reliable method for testing basal promoter activity and identifying core sequences within its pollen specific elements. In this paper we examined the functionality of buckwheat metallothionein promoter by a histochemical GUS assay in two transient expression systems: BY2 cells and pollen grains. Strong promoter activity was observed in both systems.

scholarly journals Augmentation of gamma-globin gene promoter activity by carboxylic acids and components of the human beta-globin locus control region

Blood ◽  
1994 ◽  
Vol 84 (11) ◽  
pp. 3929-3935 ◽  
Author(s):  
S Safaya ◽  
A Ibrahim ◽  
RF Rieder
Keyword(s):  
Gene Expression ◽  
Organic Acids ◽  
Carboxylic Acids ◽  
Transient Expression ◽  
Promoter Activity ◽  
Globin Gene ◽  
Gene Promoter ◽  
K562 Cells ◽  
Hemoglobin Synthesis ◽  
Gamma Globin

Butyric acid increases fetal hemoglobin synthesis in adult animals and in erythroid cells in culture and induces the gamma-globin gene promoter in transient expression experiments in K562 cells (McDonagh KT, Nienhuis AW, Blood 78:255a, 1992 [abstr, suppl 1]). We compared the effect of butyrate and other short-chain carboxylic acids in transient expression studies with K562 cells using an expression plasmid bearing a luciferase reporter gene driven by the normal human A gamma-globin gene promoter. Butyrate (4 carbons) increased the activity of the human A gamma-globin gene promoter up to 123 times. Marked augmentation of the normal gamma-promoter activity was also noted with 5-carbon valeric acid (up to 394 times) and 3-carbon propionic acid (up to 129 times). The branched isobutyric acid as well as phenylacetate showed less ability to increase promoter activity. Addition of the tandemly repeated AP-1/NF-E2 (AP) enhancer sequences from hypersensitive site 2 (HS2) of the locus control region (LCR) increased gamma-promoter activity up to 24 times. Addition of a nearby 16-bp conserved motif (CM) in HS2 (Safaya S, Rieder RF, Blood 78:146a, 1992 [abstr, suppl 1]) to the AP-containing plasmid construct further increased gamma-promoter activity. In the presence of butyrate, the plasmid bearing both the AP and CM sequences showed gene expression up to 477 times greater than that of the basal gamma-promoter-driven luciferase plasmid in the absence of inducer. A plasmid bearing the herpes simplex thymidine kinase promoter was also tested and gene expression was markedly increased by the same organic acids. MEL cells responded to butyrate, valerate, and propionate with induction of hemoglobin synthesis. Responses to isobutyrate and 6-carbon caproate required higher concentrations of the compounds. Thus, other short-chain organic acids as well as butyrate increase gamma-promoter activity in the transient expression system, and this activity can be further augmented by incorporating LCR elements into the expression vector. Nonglobin promoters also respond to the same carboxylic acids.

scholarly journals Cyclosporin A-Sensitive Transcription Factor Egr-3 Regulates Fas Ligand Expression

1998 ◽  
Vol 18 (7) ◽  
pp. 3744-3751 ◽  
Author(s):  
Paul R. Mittelstadt ◽  
Jonathan D. Ashwell
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Dna Binding ◽  
Cyclosporin A ◽  
Binding Site ◽  
Transient Expression ◽  
Promoter Activity ◽  
Fas Ligand ◽  
Regulatory Element ◽  
Ligand Expression

ABSTRACT Activation-induced transcriptional upregulation of the ligand for Fas (FasL) and the resulting apoptosis of Fas-bearing cells constitute essential steps in a host of normal and pathological processes. Here we describe an activation-inducible cis-acting regulatory element in the fasL promoter that is required for gene expression. Oligonucleotide competition and antibody supershift analyses identified two activation-induced DNA-binding species: Egr-1 (NGFI-A, krox-24, zif268, TIS-8), a transcription factor that has been implicated in growth, differentiation, and apoptosis; and Egr-3 (PILOT), a transcription factor of no previously known function. Activation-induced expression of Egr-3, like that of FasL, was inhibited by cyclosporin A, whereas expression of Egr-1 was unaffected. Transient expression of Egr-3 alone increased fasL promoter activity in a cyclosporin A-insensitive manner, whereas expression of Egr-1 had little effect. Moreover, endogenous fasL mRNA was induced in nonlymphoid cells by forced expression of Egr-3 in the absence of any other stimulus. These studies identify a critical Egr family-binding site in the fasL promoter and demonstrate that activation-induced Egr-3, but not Egr-1, directly upregulatesfasL transcription in response to activating stimuli.

scholarly journals Functional analysis of the promoter of the mitochondrial phosphate carrier human gene: identification of activator and repressor elements and their transcription factors

2005 ◽  
Vol 391 (3) ◽  
pp. 613-621 ◽  
Author(s):  
Vito Iacobazzi ◽  
Vittoria Infantino ◽  
Paola Costanzo ◽  
Paola Izzo ◽  
Ferdinando Palmieri
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Hela Cell ◽  
Dna Sequences ◽  
Promoter Activity ◽  
Rna Binding ◽  
Binding Protein ◽  
Atp Synthesis ◽  
Nuclear Extracts ◽  
Phosphate Carrier

The phosphate carrier (PiC) catalyses the import of phosphate into mitochondria where it is needed for ATP synthesis. We have analysed the 5′-flanking region of the human PiC gene and found that it has a single transcriptional initiation site and lacks a TATA box. Through deletion analysis of the −1213/−25 nt region, we identified an activation domain (−223/−25) and an inhibition domain (−1017/−814). The most effective promoter activity in transfected HeLa cells corresponded to the region containing putative binding sites for Sp1 (−163/−142; where Sp1 stands for stimulating protein-1) and CREB (−138/−116; where CREB stands for cAMP-response-element-binding protein). These DNA sequences were active in gel-shift assays in the presence of HeLa cell nuclear extracts or recombinant Sp1 and CREB respectively. Forskolin increased PiC promoter activity via the CREB site. Both footprinting and transfection of deletion constructs of the inhibition region (−1017/−814) showed that PiC silencer activity extends over 25 nt (−943/−919), which specifically binds two proteins present in HeLa cell nuclear extracts. These transcription factors were purified by DNA affinity, analysed by MS and identified as p54nrb/NonO (nuclear RNA binding protein) and PSF (protein-associated splicing factor). The PiC silencer region cloned in front of the ferritin promoter conferred a strong inhibition to the heterologous promoter. These findings may provide insight into control of PiC gene expression in different cell types and under different growth conditions. To our knowledge, this is the first study to analyse the regulation of the PiC gene expression in any cell.

Sequence-Based Deep Learning Frameworks on Enhancer-Promoter Interactions Prediction

2020 ◽  
Vol 26 ◽  
Author(s):  
Xiaoping Min ◽  
Fengqing Lu ◽  
Chunyan Li
Keyword(s):  
Gene Expression ◽  
Deep Learning ◽  
Dna Sequences ◽  
Experimental Methods ◽  
Learning Methods ◽  
Comprehensive Review ◽  
Genomic Features ◽  
Disease Mechanisms ◽  
Evaluation Strategies ◽  
Learning Frameworks

: Enhancer-promoter interactions (EPIs) in the human genome are of great significance to transcriptional regulation which tightly controls gene expression. Identification of EPIs can help us better deciphering gene regulation and understanding disease mechanisms. However, experimental methods to identify EPIs are constrained by the fund, time and manpower while computational methods using DNA sequences and genomic features are viable alternatives. Deep learning methods have shown promising prospects in classification and efforts that have been utilized to identify EPIs. In this survey, we specifically focus on sequence-based deep learning methods and conduct a comprehensive review of the literatures of them. We first briefly introduce existing sequence-based frameworks on EPIs prediction and their technique details. After that, we elaborate on the dataset, pre-processing means and evaluation strategies. Finally, we discuss the challenges these methods are confronted with and suggest several future opportunities.

UBE2L6 is Involved in Cisplatin Resistance by Regulating the Transcription of ABCB6

2020 ◽  
Vol 20 (12) ◽  
pp. 1487-1496 ◽  
Author(s):  
Midori Murakami ◽  
Hiroto Izumi ◽  
Tomoko Kurita ◽  
Chiho Koi ◽  
Yasuo Morimoto ◽  
...  
Keyword(s):  
Gene Expression ◽  
Promoter Activity ◽  
Anticancer Agent ◽  
Cisplatin Resistance ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Molecular Target ◽  
Transcriptional Level ◽  
And Control ◽  
Resistant Cells

Background: Cisplatin is an important anticancer agent in cancer chemotherapy, but when resistant cells appear, treatment becomes difficult, and the prognosis is poor. Objective: In this study, we investigated the gene expression profile in cisplatin sensitive and resistant cells, and identified the genes involved in cisplatin resistance. Methods: Comparison of gene expression profiles revealed that UBE2L6 mRNA is highly expressed in resistant cells. To elucidate whether UBE2L6 is involved in the acquisition of cisplatin resistance, UBE2L6- overexpressing cells established from cisplatin-sensitive cells and UBE2L6-silenced cells developed from cisplatin- resistant cells were generated, and the sensitivity of cisplatin was examined. Results: The sensitivity of the UBE2L6-overexpressing cells did not change compared with the control cells, but the UBE2L6-silenced cells were sensitized to cisplatin. To elucidate the mechanism of UBE2L6 in cisplatin resistance, we compared the gene expression profiles of UBE2L6-silenced cells and control cells and found that the level of ABCB6 mRNA involved in cisplatin resistance was decreased. Moreover, ABCB6 promoter activity was partially suppressed in UBE2L6-silenced cells. Conclusion: These results suggest that cisplatin-resistant cells have upregulated UBE2L6 expression and contribute to cisplatin resistance by regulating ABCB6 expression at the transcriptional level. UBE2L6 might be a molecular target that overcomes cisplatin resistance.

scholarly journals Harnessing a Transient Gene Expression System in Nicotiana benthamiana to Explore Plant Agrochemical Transporters

Plants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 524
Author(s):  
Bingqi Wu ◽  
Zhiting Chen ◽  
Xiaohui Xu ◽  
Ronghua Chen ◽  
Siwei Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transient Expression ◽  
Nicotiana Benthamiana ◽  
Heterologous Gene Expression ◽  
Expression System ◽  
Functional Characterization ◽  
Transient Gene Expression ◽  
High Mobility ◽  
Gene Expression System

Functional characterization of plant agrichemical transporters provided an opportunity to discover molecules that have a high mobility in plants and have the potential to increase the amount of pesticides reaching damage sites. Agrobacterium-mediated transient expression in tobacco is simple and fast, and its protein expression efficiency is high; this system is generally used to mediate heterologous gene expression. In this article, transient expression of tobacco nicotine uptake permease (NtNUP1) and rice polyamine uptake transporter 1 (OsPUT1) in Nicotiana benthamiana was performed to investigate whether this system is useful as a platform for studying the interactions between plant transporters and pesticides. The results showed that NtNUP1 increases nicotine uptake in N. benthamiana foliar discs and protoplasts, indicating that this transient gene expression system is feasible for studying gene function. Moreover, yeast expression of OsPUT1 apparently increases methomyl uptake. Overall, this method of constructing a transient gene expression system is useful for improving the efficiency of analyzing the functions of plant heterologous transporter-encoding genes and revealed that this system can be further used to study the functions of transporters and pesticides, especially their interactions.

scholarly journals Gene Expression Space Shapes the Bioprocess Trade-Offs among Titer, Yield and Productivity

2021 ◽  
Vol 11 (13) ◽  
pp. 5859
Author(s):  
Fernando N. Santos-Navarro ◽  
Yadira Boada ◽  
Alejandro Vignoni ◽  
Jesús Picó
Keyword(s):  
Gene Expression ◽  
Gene Transcription ◽  
Performance Metrics ◽  
Cell Mass ◽  
Gene Copy ◽  
Substrate Uptake ◽  
Promoter Strength ◽  
Expression Levels ◽  
Trade Offs ◽  
Wide Range

Optimal gene expression is central for the development of both bacterial expression systems for heterologous protein production, and microbial cell factories for industrial metabolite production. Our goal is to fulfill industry-level overproduction demands optimally, as measured by the following key performance metrics: titer, productivity rate, and yield (TRY). Here we use a multiscale model incorporating the dynamics of (i) the cell population in the bioreactor, (ii) the substrate uptake and (iii) the interaction between the cell host and expression of the protein of interest. Our model predicts cell growth rate and cell mass distribution between enzymes of interest and host enzymes as a function of substrate uptake and the following main lab-accessible gene expression-related characteristics: promoter strength, gene copy number and ribosome binding site strength. We evaluated the differential roles of gene transcription and translation in shaping TRY trade-offs for a wide range of expression levels and the sensitivity of the TRY space to variations in substrate availability. Our results show that, at low expression levels, gene transcription mainly defined TRY, and gene translation had a limited effect; whereas, at high expression levels, TRY depended on the product of both, in agreement with experiments in the literature.

scholarly journals Polycombs and microRNA-223 regulate human granulopoiesis by transcriptional control of target gene expression

Blood ◽  
2012 ◽  
Vol 119 (17) ◽  
pp. 4034-4046 ◽  
Author(s):  
Giuseppe Zardo ◽  
Alberto Ciolfi ◽  
Laura Vian ◽  
Linda M. Starnes ◽  
Monia Billi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Sequences ◽  
Transcriptional Control ◽  
Transcriptional Level ◽  
Seed Region ◽  
Mrna Targets ◽  
Epigenetic Events ◽  
Evolutionarily Conserved ◽  
Lineage Decision ◽  
Chromatin Remodeling Complexes

Abstract Epigenetic modifications regulate developmental genes involved in stem cell identity and lineage choice. NFI-A is a posttranscriptional microRNA-223 (miR-223) target directing human hematopoietic progenitor lineage decision: NFI-A induction or silencing boosts erythropoiesis or granulopoiesis, respectively. Here we show that NFI-A promoter silencing, which allows granulopoiesis, is guaranteed by epigenetic events, including the resolution of opposing chromatin “bivalent domains,” hypermethylation, recruitment of polycomb (PcG)–RNAi complexes, and miR-223 promoter targeting activity. During granulopoiesis, miR-223 localizes inside the nucleus and targets the NFI-A promoter region containing PcGs binding sites and miR-223 complementary DNA sequences, evolutionarily conserved in mammalians. Remarkably, both the integrity of the PcGs-RNAi complex and DNA sequences matching the seed region of miR-223 are required to induce NFI-A transcriptional silencing. Moreover, ectopic miR-223 expression in human myeloid progenitors causes heterochromatic repression of NFI-A gene and channels granulopoiesis, whereas its stable knockdown produces the opposite effects. Our findings indicate that, besides the regulation of translation of mRNA targets, endogenous miRs can affect gene expression at the transcriptional level, functioning in a critical interface between chromatin remodeling complexes and the genome to direct fate lineage determination of hematopoietic progenitors.

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