scholarly journals Pitaya HpWRKY3 Is Associated with Fruit Sugar Accumulation by Transcriptionally Modulating Sucrose Metabolic Genes HpINV2 and HpSuSy1

2019 ◽  
Vol 20 (8) ◽  
pp. 1890 ◽  
Author(s):  
Wei Wei ◽  
Mei-nv Cheng ◽  
Liang-jie Ba ◽  
Run-xi Zeng ◽  
Dong-lan Luo ◽  
...  
Keyword(s):  
Consumer Preferences ◽  
Wrky Transcription Factor ◽  
Sugar Accumulation ◽  
Fruit Maturation ◽  
Mobility Shift ◽  
Metabolic Genes ◽  
Similar Expression Pattern ◽  
Gel Mobility Shift ◽  
Upstream Regulators ◽  
Shed Light

Sugar level is an important determinant of fruit taste and consumer preferences. However, upstream regulators that control sugar accumulation during fruit maturation are poorly understood. In the present work, we found that glucose is the main sugar in mature pitaya (Hylocereus) fruit, followed by fructose and sucrose. Expression levels of two sucrose-hydrolyzing enzyme genes HpINV2 and HpSuSy1 obviously increased during fruit maturation, which were correlated well with the elevated accumulation of glucose and fructose. A WRKY transcription factor HpWRKY3 was further identified as the putative binding protein of the HpINV2 and HpSuSy1 promoters by yeast one-hybrid and gel mobility shift assays. HpWRKY3 was localized exclusively in the nucleus and possessed trans-activation ability. HpWRKY3 exhibited the similar expression pattern with HpINV2 and HpSuSy1. Finally, transient expression assays in tobacco leaves showed that HpWRKY3 activated the expressions of HpINV2 and HpSuSy1. Taken together, we propose that HpWRKY3 is associated with pitaya fruit sugar accumulation by activating the transcriptions of sucrose metabolic genes. Our findings thus shed light on the transcriptional mechanism that regulates the sugar accumulation during pitaya fruit quality formation.

Snapshot blotting: The transfer of nucleic acids and nucleoprotein complexes from electrophoresis gels to EM grids

1992 ◽  
Vol 50 (1) ◽  
pp. 762-763
Author(s):  
Stephen D. Jett
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Nucleic Acid Binding ◽  
Mobility Shift ◽  
Carbon Coated ◽  
Nucleoprotein Complexes ◽  
Mobility Shift Assay ◽  
Protein Nucleic Acid ◽  
Gel Mobility Shift ◽  
Nucleic Acid Interactions

The electrophoresis gel mobility shift assay is a popular method for the study of protein-nucleic acid interactions. The binding of proteins to DNA is characterized by a reduction in the electrophoretic mobility of the nucleic acid. Binding affinity, stoichiometry, and kinetics can be obtained from such assays; however, it is often desirable to image the various species in the gel bands using TEM. Present methods for isolation of nucleoproteins from gel bands are inefficient and often destroy the native structure of the complexes. We have developed a technique, called “snapshot blotting,” by which nucleic acids and nucleoprotein complexes in electrophoresis gels can be electrophoretically transferred directly onto carbon-coated grids for TEM imaging.

scholarly journals The transcription factor OpWRKY2 positively regulates the biosynthesis of the anticancer drug camptothecin in Ophiorrhiza pumila

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Xiaolong Hao ◽  
Chenhong Xie ◽  
Qingyan Ruan ◽  
Xichen Zhang ◽  
Chao Wu ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Anticancer Activity ◽  
Time Course ◽  
Indole Alkaloid ◽  
Fold Increase ◽  
Wrky Transcription Factor ◽  
Mobility Shift ◽  
Pathway Gene ◽  
Low Concentrations ◽  
Encoding Gene

AbstractThe limited bioavailability of plant-derived natural products with anticancer activity poses major challenges to the pharmaceutical industry. An example of this is camptothecin, a monoterpene indole alkaloid with potent anticancer activity that is extracted at very low concentrations from woody plants. Recently, camptothecin biosynthesis has been shown to become biotechnologically amenable in hairy-root systems of the natural producer Ophiorrhiza pumila. Here, time-course expression and metabolite analyses were performed to identify novel transcriptional regulators of camptothecin biosynthesis in O. pumila. It is shown here that camptothecin production increased over cultivation time and that the expression pattern of the WRKY transcription factor encoding gene OpWRKY2 is closely correlated with camptothecin accumulation. Overexpression of OpWRKY2 led to a more than three-fold increase in camptothecin levels. Accordingly, silencing of OpWRKY2 correlated with decreased camptothecin levels in the plant. Further detailed molecular characterization by electrophoretic mobility shift, yeast one-hybrid and dual-luciferase assays showed that OpWRKY2 directly binds and activates the central camptothecin pathway gene OpTDC. Taken together, the results of this study demonstrate that OpWRKY2 acts as a direct positive regulator of camptothecin biosynthesis. As such, a feasible strategy for the over-accumulation of camptothecin in a biotechnologically amenable system is presented.

scholarly journals FarRRegulates the farAB-Encoded Efflux Pump of Neisseriagonorrhoeae via an MtrR RegulatoryMechanism

2003 ◽  
Vol 185 (24) ◽  
pp. 7145-7152 ◽  
Author(s):  
E.-H. Lee ◽  
C. Rouquette-Loughlin ◽  
J. P. Folster ◽  
W. M. Shafer
Keyword(s):  
Regulatory Mechanism ◽  
Efflux Pump ◽  
Regulatory Protein ◽  
Transcriptional Repressors ◽  
Sequence Database ◽  
Mobility Shift ◽  
Pump System ◽  
Regulatory Cascade ◽  
Genome Sequence Database ◽  
Gel Mobility Shift

ABSTRACT The farAB operon of Neisseria gonorrhoeae encodes an efflux pump which mediates gonococcal resistance to antibacterial fatty acids. It was previously observed that expression of the farAB operon was positively regulated by MtrR, which is a repressor of the mtrCDE-encoded efflux pump system (E.-H. Lee and W. M. Shafer, Mol. Microbiol. 33:839-845, 1999). This regulation was believed to be indirect since MtrR did not bind to the farAB promoter. In this study, computer analysis of the gonococcal genome sequence database, lacZ reporter fusions, and gel mobility shift assays were used to elucidate the regulatory mechanism by which expression of the farAB operon is modulated by MtrR in gonococci. We identified a regulatory protein belonging to the MarR family of transcriptional repressors and found that it negatively controls expression of farAB by directly binding to the farAB promoter. We designated this regulator FarR to signify its role in regulating the farAB operon. We found that MtrR binds to the farR promoter, thereby repressing farR expression. Hence, MtrR regulates farAB in a positive fashion by modulating farR expression. This MtrR regulatory cascade seems to play an important role in adjusting levels of the FarAB and MtrCDE efflux pumps to prevent their excess expression in gonococci.

Influence of homologous phasins (PhaP) on PHA accumulation and regulation of their expression by the transcriptional repressor PhaR in Ralstonia eutropha H16

Microbiology ◽  
2005 ◽  
Vol 151 (3) ◽  
pp. 825-833 ◽  
Author(s):  
Markus Pötter ◽  
Helena Müller ◽  
Alexander Steinbüchel
Keyword(s):  
Ralstonia Eutropha ◽  
Current Model ◽  
Transcriptional Repressor ◽  
Start Codon ◽  
Wild Type ◽  
Mobility Shift ◽  
Intergenic Regions ◽  
Gel Mobility Shift ◽  
Dnasei Footprinting ◽  
Similar Growth

Phasins play an important role in the formation of poly(3-hydroxybutyrate) [poly(3HB)] granules and affect their size. Recently, three homologues of the phasin protein PhaP1 were identified in Ralstonia eutropha strain H16. The functions of PhaP2, PhaP3 and PhaP4 were examined by analysis of R. eutropha H16 deletion strains (ΔphaP1, ΔphaP2, ΔphaP3, ΔphaP4, ΔphaP12, ΔphaP123 and ΔphaP1234). When cells were grown under conditions permissive for poly(3HB) accumulation, the wild-type strain and all single-phasin negative mutants (ΔphaP2, ΔphaP3 and ΔphaP4), with the exception of ΔphaP1, showed similar growth and poly(3HB) accumulation behaviour, and also the size and number of the granules were identical. The single ΔphaP1 mutant and the ΔphaP12, ΔphaP123 and ΔphaP1234 mutants showed an almost identical growth behaviour; however, they accumulated poly(3HB) at a significantly lower level than wild-type and the single ΔphaP2, ΔphaP3 or ΔphaP4 mutants. Gel-mobility-shift assays and DNaseI footprinting experiments demonstrated the capability of the transcriptional repressor PhaR to bind to a DNA region +36 to +46 bp downstream of the phaP3 start codon. The protected sequence exhibited high similarity to the binding sites of PhaR upstream of phaP1, which were identified recently. In contrast, PhaR did not bind to the upstream or intergenic regions of phaP2 and phaP4, thus indicating that the expression of these two phasins is regulated in a different way. Our current model for the regulation of phasins in R. eutropha strain H16 was extended and confirmed.

The clpB gene of Bifidobacterium breve UCC 2003: transcriptional analysis and first insights into stress induction

Microbiology ◽  
2005 ◽  
Vol 151 (9) ◽  
pp. 2861-2872 ◽  
Author(s):  
Marco Ventura ◽  
John G. Kenny ◽  
Ziding Zhang ◽  
Gerald F. Fitzgerald ◽  
Douwe van Sinderen
Keyword(s):  
3D Structure ◽  
Binding Mode ◽  
Dna Interaction ◽  
Transcriptional Analysis ◽  
Amino Acid Residues ◽  
Mobility Shift ◽  
Bifidobacterium Breve ◽  
Gel Mobility Shift ◽  
Slot Blot Analysis

The so-called clp genes, which encode components of the Clp proteolytic complex, are widespread among bacteria. The Bifidobacterium breve UCC 2003 genome contains a clpB gene with significant homology to predicted clpB genes from other members of the Actinobacteridae group. The heat- and osmotic-inducibility of the B. breve UCC 2003 clpB homologue was verified by slot-blot analysis, while Northern blot and primer extension analyses showed that the clpB gene is transcribed as a monocistronic unit with a single promoter. The role of a hspR homologue, known to control the regulation of clpB and dnaK gene expression in other high G+C content bacteria was investigated by gel mobility shift assays. Moreover the predicted 3D structure of HspR provides further insight into the binding mode of this protein to the clpB promoter region, and highlights the key amino acid residues believed to be involved in the protein–DNA interaction.

scholarly journals Interactions between Integrase and Excisionase in the Phage Lambda Excisive Nucleoprotein Complex

2002 ◽  
Vol 184 (18) ◽  
pp. 5200-5203 ◽  
Author(s):  
Eun Hee Cho ◽  
Richard I. Gumport ◽  
Jeffrey F. Gardner
Keyword(s):  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Mobility Shift ◽  
Host Chromosome ◽  
Amino Terminal ◽  
Nucleoprotein Complex ◽  
Carboxyl Terminal ◽  
Α Helix ◽  
Gel Mobility Shift ◽  
Amino Terminal Domain

ABSTRACT Bacteriophage lambda site-specific recombination comprises two overall reactions, integration into and excision from the host chromosome. Lambda integrase (Int) carries out both reactions. During excision, excisionase (Xis) helps Int to bind DNA and introduces a bend in the DNA that facilitates formation of the proper excisive nucleoprotein complex. The carboxyl-terminal α-helix of Xis is thought to interact with Int through direct protein-protein interactions. In this study, we used gel mobility shift assays to show that the amino-terminal domain of Int maintained cooperative interactions with Xis. This finding indicates that the amino-terminal arm-type DNA binding domain of Int interacts with Xis.

Positive and negative transcriptional elements of the human type IV collagenase gene

1990 ◽  
Vol 10 (12) ◽  
pp. 6524-6532
Author(s):  
S M Frisch ◽  
J H Morisaki
Keyword(s):  
Core Promoter ◽  
Type Iv Collagenase ◽  
Mobility Shift ◽  
Specific Expression ◽  
Enhancer Element ◽  
Type Iv ◽  
Dnase I Footprinting ◽  
Gel Mobility Shift ◽  
Transcriptional Elements ◽  
Mcf 7

Proteolysis by type IV collagenase (T4) has been implicated in the process of tumor metastasis. The T4 gene is expressed in fibroblasts, but not in normal epithelial cells, and its expression is specifically repressed by the E1A oncogene of adenovirus. We present an investigation of the transcriptional elements responsible for basal, E1A-repressible, and tissue-specific expression. 5'-Deletion analysis, DNase I footprinting, and gel mobility shift assays revealed a strong, E1A-repressible enhancer element, r2, located about 1,650 bp upstream of the start site. This enhancer bound a protein with binding specificity very similar to that of the transcription factor AP-2. A potent silencer sequence was found 2 to 5 bp downstream of this enhancer. The silencer repressed transcription from either r2 or AP-1 enhancer elements and in the context of either type IV collagenase or thymidine kinase (tk) gene core promoters; enhancerless transcription from the latter core promoter was also repressed. Comprising the silencer were two contiguous, autonomously functioning silencer elements. Negative regulation of T4 transcription by at least two factors was demonstrated. mcf-7 proteins specifically binding both elements were detected by gel mobility shift assays; a protein of approximately 185 kDa that bound to one of these elements was detected by DNA-protein cross-linking. The silencer repressed transcription, in an r2 enhancer-tk promoter context, much more efficiently in T4-nonproducing cells (mcf-7 or HeLa) than in T4-producing cells (HT1080), suggesting that cell type-specific silencing may contribute to the regulation of this gene.

The Aspergillus nidulans abaA gene encodes a transcriptional activator that acts as a genetic switch to control development

1994 ◽  
Vol 14 (4) ◽  
pp. 2503-2515
Author(s):  
A Andrianopoulos ◽  
W E Timberlake
Keyword(s):  
Aspergillus Nidulans ◽  
Transcriptional Activation ◽  
Expression System ◽  
Regulatory Gene ◽  
Binding Motif ◽  
Mobility Shift ◽  
Genetic Switch ◽  
Regulatory Regions ◽  
Cis Acting ◽  
Gel Mobility Shift

The Aspergillus nidulans abaA gene encodes a protein containing an ATTS DNA-binding motif and is required for the terminal stages of conidiophore development. Results from gel mobility shift and protection, missing-contact, and interference footprint assays showed that AbaA binds to the sequence 5'-CATTCY-3', where Y is a pyrimidine, making both major- and minor-groove contacts. Multiple AbaA binding sites are present in the cis-acting regulatory regions of several developmentally controlled structural genes as well as those of the upstream regulatory gene brlA, the downstream regulatory gene wetA, and abaA itself. These cis-acting regulatory regions confer AbaA-dependent transcriptional activation in a heterologous Saccharomyces cerevisiae gene expression system. From these observations, we propose that the AbaA transcription factor establishes a novel set of feedback regulatory loops responsible for determination of conidiophore development.

The orphan nuclear receptor NGFI-B regulates expression of the gene encoding steroid 21-hydroxylase

1993 ◽  
Vol 13 (2) ◽  
pp. 861-868
Author(s):  
T E Wilson ◽  
A R Mouw ◽  
C A Weaver ◽  
J Milbrandt ◽  
K L Parker
Keyword(s):  
Nuclear Receptor ◽  
Transcriptional Activation ◽  
Core Promoter ◽  
Orphan Nuclear Receptor ◽  
Recognition Sequence ◽  
Mobility Shift ◽  
Adrenocortical Cells ◽  
Gene Encoding ◽  
Adult Rat ◽  
Gel Mobility Shift

As part of its trophic action to maintain the steroidogenic capacity of adrenocortical cells, corticotropin (ACTH) increases the transcription of the cytochrome P-450 steroid hydroxylase genes, including the gene encoding steroid 21-hydroxylase (21-OHase). We previously identified several promoter elements that regulate 21-OHase gene expression in mouse Y1 adrenocortical tumor cells. One of these elements, located at nucleotide -65, closely resembles the recognition sequence of the orphan nuclear receptor NGFI-B, suggesting that NGFI-B regulates this essential steroidogenic enzyme. To explore this possibility, we first used in situ hybridization to demonstrate high levels of NGFI-B transcripts in the adrenal cortex of the adult rat. In cultured mouse Y1 adrenocortical cells, treatment with ACTH, the major regulator of 21-OHase transcription, rapidly increased NGFI-B expression. Gel mobility shift and DNase I footprinting experiments showed that recombinantly expressed NGFI-B interacts specifically with the 21-OHase -65 element and identified one complex formed by Y1 extracts and the 21-OHase -65 element that contains NGFI-B. Expression of NGFI-B significantly augmented the activity of the intact 21-OHase promoter, while mutations of the -65 element that abolish NGFI-B binding markedly diminished NGFI-B-mediated transcriptional activation. Specific mutations of NGFI-B shown previously to impair either DNA binding or transcriptional activation diminished the effect of NGFI-B coexpression on 21-OHase expression. Finally, an oligonucleotide containing the NGFI-B response element conferred ACTH response to a core promoter from the prolactin gene, showing that this element is sufficient for ACTH induction. Collectively, these results identify a cellular promoter element that is regulated by NGFI-B and implicate NGFI-B in the transcriptional induction of 21-OHase by ACTH.

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