scholarly journals Optimised Heterologous Expression and Functional Analysis of the Yersinia pestis F1-Capsular Antigen Regulator Caf1R

2021 ◽  
Vol 22 (18) ◽  
pp. 9805
Author(s):  
Dharmender K. Gahlot ◽  
Gyles Ifill ◽  
Sheila MacIntyre
Keyword(s):  
Yersinia Pestis ◽  
Vaccine Development ◽  
Recombinant Expression ◽  
Bacterial Pathogen ◽  
Clinical Value ◽  
Mobility Shift ◽  
E Coli ◽  
Capsular Antigen ◽  
Mobility Shift Assay

The bacterial pathogen, Yersinia pestis, has caused three historic pandemics and continues to cause small outbreaks worldwide. During infection, Y. pestis assembles a capsule-like protective coat of thin fibres of Caf1 subunits. This F1 capsular antigen has attracted much attention due to its clinical value in plague diagnostics and anti-plague vaccine development. Expression of F1 is tightly regulated by a transcriptional activator, Caf1R, of the AraC/XylS family, proteins notoriously prone to aggregation. Here, we have optimised the recombinant expression of soluble Caf1R. Expression from the native and synthetic codon-optimised caf1R cloned in three different expression plasmids was examined in a library of E. coli host strains. The functionality of His-tagged Caf1R was demonstrated in vivo, but insolubility was a problem with overproduction. High levels of soluble MBP-Caf1R were produced from codon optimised caf1R. Transcriptional-lacZ reporter fusions defined the PM promoter and Caf1R binding site responsible for transcription of the cafMA1 operon. Use of the identified Caf1R binding caf DNA sequence in an electrophoretic mobility shift assay (EMSA) confirmed correct folding and functionality of the Caf1R DNA-binding domain in recombinant MBP-Caf1R. Availability of functional recombinant Caf1R will be a valuable tool to elucidate control of expression of F1 and Caf1R-regulated pathophysiology of Y. pestis.

scholarly journals Identification of Chlamydia trachomatis Genomic Sequences Recognized by Chlamydial Divalent Cation-Dependent Regulator A (DcrA)

2005 ◽  
Vol 187 (2) ◽  
pp. 443-448 ◽  
Author(s):  
Annette Rau ◽  
Susan Wyllie ◽  
Judy Whittimore ◽  
Jane E. Raulston
Keyword(s):  
Chlamydia Trachomatis ◽  
Divalent Cation ◽  
Open Reading Frames ◽  
Genomic Sequences ◽  
Mobility Shift ◽  
Reading Frame ◽  
E Coli ◽  
Resultant Data ◽  
Mobility Shift Assay ◽  
Titration Assay

ABSTRACT The Chlamydia trachomatis divalent cation-dependent regulator (DcrA), encoded by open reading frame CT296, is a distant relative of the ferric uptake regulator (Fur) family of iron-responsive regulators. Chlamydial DcrA specifically binds to a consensus Escherichia coli Fur box and is able to complement an E. coli Fur mutant. In this report, the E. coli Fur titration assay (FURTA) was used to locate chlamydial genomic sequences that are recognized by E. coli Fur. The predictive regulatory regions of 28 C. trachomatis open reading frames contained sequences functionally recognized by E. coli Fur; targets include components of the type III secretion pathway, elements involved in envelope and cell wall biogenesis, predicted transport proteins, oxidative defense enzymes, and components of metabolic pathways. Selected FURTA-positive sequences were subsequently examined for recognition by C. trachomatis DcrA using an electrophoretic mobility shift assay. The resultant data show that C. trachomatis DcrA binds to native chlamydial genomic sequences and, overall, substantiate a functional relationship between chlamydial DcrA and the Fur family of regulators.

scholarly journals Mutational and in vitro protein-binding studies on centromere DNA from Saccharomyces cerevisiae.

1987 ◽  
Vol 7 (12) ◽  
pp. 4522-4534 ◽  
Author(s):  
R Ng ◽  
J Carbon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Binding ◽  
In Vitro Assays ◽  
Binding Studies ◽  
Mobility Shift ◽  
Sequence Element ◽  
Sequence Elements ◽  
Mobility Shift Assay

Centromeres on chromosomes in the yeast Saccharomyces cerevisiae contain approximately 140 base pairs (bp) of DNA. The functional centromere (CEN) region contains three important sequence elements (I, PuTCACPuTG; II, 78 to 86 bp of high-AT DNA; and III, a conserved 25-bp sequence with internal bilateral symmetry). Various point mutations or deletions in the element III region have a profound effect on CEN function in vivo, indicating that this DNA region is a key protein-binding site. This has been confirmed by the use of two in vitro assays to detect binding of yeast proteins to DNA fragments containing wild-type or mutationally altered CEN3 sequences. An exonuclease III protection assay was used to demonstrate specific binding of proteins to the element III region of CEN3. In addition, a gel DNA fragment mobility shift assay was used to characterize the binding reaction parameters. Sequence element III mutations that inactivate CEN function in vivo also prevent binding of proteins in the in vitro assays. The mobility shift assay indicates that double-stranded DNAs containing sequence element III efficiently bind proteins in the absence of sequence elements I and II, although the latter sequences are essential for optimal CEN function in vivo.

Regulatory factors controlling transcription of Saccharomyces cerevisiae IXR1 by oxygen levels: a model of transcriptional adaptation from aerobiosis to hypoxia implicating ROX1 and IXR1 cross-regulation

2009 ◽  
Vol 425 (1) ◽  
pp. 235-243 ◽  
Author(s):  
Raquel  Castro-Prego ◽  
Mónica Lamas-Maceiras ◽  
Pilar Soengas ◽  
Isabel Carneiro ◽  
Isabel González-Siso ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Oxygen Availability ◽  
Mrna Levels ◽  
Mobility Shift ◽  
Hypoxic Conditions ◽  
Cross Links ◽  
Mobility Shift Assay ◽  
Transcriptional Adaptation

Ixr1p from Saccharomyces cerevisiae has been previously studied because it binds to DNA containing intrastrand cross-links formed by the anticancer drug cisplatin. Ixr1p is also a transcriptional regulator of anaerobic/hypoxic genes, such as SRP1/TIR1, which encodes a stress-response cell wall manoprotein, and COX5B, which encodes the Vb subunit of the mitochondrial complex cytochrome c oxidase. However, factors controlling IXR1 expression remained unexplored. In the present study we show that IXR1 mRNA levels are controlled by oxygen availability and increase during hypoxia. In aerobiosis, low levels of IXR1 expression are maintained by Rox1p repression through the general co-repressor complex Tup1–Ssn6. Ixr1p itself is necessary for full IXR1 expression under hypoxic conditions. Deletion analyses have identified the region in the IXR1 promoter responsible for this positive auto-control (nucleotides −557 to −376). EMSA (electrophoretic mobility-shift assay) and ChIP (chromatin immunoprecipitation) assays show that Ixr1p binds to the IXR1 promoter both in vitro and in vivo. Ixr1p is also required for hypoxic repression of ROX1 and binds to its promoter. UPC2 deletion has opposite effects on IXR1 and ROX1 transcription during hypoxia. Ixr1p is also necessary for resistance to oxidative stress generated by H2O2. IXR1 expression is moderately activated by H2O2 and this induction is Yap1p-dependent. A model of IXR1 regulation as a relay for sensing different signals related to change in oxygen availability is proposed. In this model, transcriptional adaptation from aerobiosis to hypoxia depends on ROX1 and IXR1 cross-regulation.

scholarly journals Transcriptional and Functional Analysis of the Neisseria gonorrhoeae Fur Regulon

2009 ◽  
Vol 192 (1) ◽  
pp. 77-85 ◽  
Author(s):  
Lydgia A. Jackson ◽  
Thomas F. Ducey ◽  
Michael W. Day ◽  
Jeremy B. Zaitshik ◽  
Joshua Orvis ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Transcriptional Control ◽  
Cell Communication ◽  
Essential Element ◽  
Open Reading Frames ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Sequence Logos ◽  
Mobility Shift Assay

ABSTRACT To ensure survival in the host, bacteria have evolved strategies to acquire the essential element iron. In Neisseria gonorrhoeae, the ferric uptake regulator Fur regulates metabolism through transcriptional control of iron-responsive genes by binding conserved Fur box (FB) sequences in promoters during iron-replete growth. Our previous studies showed that Fur also controls the transcription of secondary regulators that may, in turn, control pathways important to pathogenesis, indicating an indirect role for Fur in controlling these downstream genes. To better define the iron-regulated cascade of transcriptional control, we combined three global strategies—temporal transcriptome analysis, genomewide in silico FB prediction, and Fur titration assays (FURTA)—to detect genomic regions able to bind Fur in vivo. The majority of the 300 iron-repressed genes were predicted to be of unknown function, followed by genes involved in iron metabolism, cell communication, and intermediary metabolism. The 107 iron-induced genes encoded hypothetical proteins or energy metabolism functions. We found 28 predicted FBs in FURTA-positive clones in the promoters and within the open reading frames of iron-repressed genes. We found lower levels of conservation at critical thymidine residues involved in Fur binding in the FB sequence logos of FURTA-positive clones with intragenic FBs than in the sequence logos generated from FURTA-positive promoter regions. In electrophoretic mobility shift assay studies, intragenic FBs bound Fur with a lower affinity than intergenic FBs. Our findings further indicate that transcription under iron stress is indirectly controlled by Fur through 12 potential secondary regulators.

scholarly journals Use of In Vivo-Induced Antigen Technology for Identification of Escherichia coli O157:H7 Proteins Expressed during Human Infection

2005 ◽  
Vol 73 (5) ◽  
pp. 2665-2679 ◽  
Author(s):  
Manohar John ◽  
Indira T. Kudva ◽  
Robert W. Griffin ◽  
Allen W. Dodson ◽  
Bethany McManus ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Vaccine Development ◽  
Human Infection ◽  
Escherichia Coli O157 ◽  
Standard Laboratory ◽  
E Coli ◽  
Phase Culture ◽  
Stool Specimens

ABSTRACT Using in vivo-induced antigen technology (IVIAT), a modified immunoscreening technique that circumvents the need for animal models, we directly identified immunogenic Escherichia coli O157:H7 (O157) proteins expressed either specifically during human infection but not during growth under standard laboratory conditions or at significantly higher levels in vivo than in vitro. IVIAT identified 223 O157 proteins expressed during human infection, several of which were unique to this study. These in vivo-induced (ivi) proteins, encoded by ivi genes, mapped to the backbone, O islands (OIs), and pO157. Lack of in vitro expression of O157-specific ivi proteins was confirmed by proteomic analysis of a mid-exponential-phase culture of E. coli O157 grown in LB broth. Because ivi proteins are expressed in response to specific cues during infection and might help pathogens adapt to and counter hostile in vivo environments, those identified in this study are potential targets for drug and vaccine development. Also, such proteins may be exploited as markers of O157 infection in stool specimens.

Cyclic AMP receptor protein is a repressor of adenylyl cyclase gene cyaA in Yersinia pestis

2013 ◽  
Vol 59 (5) ◽  
pp. 304-310 ◽  
Author(s):  
Shi Qu ◽  
Yiquan Zhang ◽  
Lei Liu ◽  
Li Wang ◽  
Yanping Han ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Adenylyl Cyclase ◽  
Yersinia Pestis ◽  
Core Promoter ◽  
Growth Conditions ◽  
Receptor Protein ◽  
Camp Production ◽  
Mobility Shift ◽  
Cyclic Amp Receptor Protein ◽  
Mobility Shift Assay

Yersinia pestis is one of the most dangerous pathogens. The cyclic AMP receptor protein (CRP) is required for the full virulence of Y. pestis, and it acts as a transcriptional regulator to control a large regulon, which includes several virulence-associated genes. The regulatory action of CRP is triggered only by binding to the small molecule cofactor cyclic AMP (cAMP). cAMP is synthesized from adenosine triphosphate by the adenylyl cyclase encoded by cyaA. In the present work, the regulation of crp and cyaA by CRP was investigated by primer extension, LacZ fusion, electrophoretic mobility shift assay, and DNase I footprinting. No transcriptional regulatory association between CRP and its own gene could be detected under the growth conditions tested. In contrast, CRP bound to a DNA site overlapping the core promoter −10 region of cyaA to repress the cyaA transcription. The determination of cellular cAMP levels further verified that CRP negatively controlled cAMP production. Repression of cAMP production by CRP through acting on the cAMP synthesase gene cyaA would represent a mechanism of negative automodulation of cellular CRP function.

scholarly journals Transcript analysis reveals an extended regulon and the importance of protein–protein co-operativity for the Escherichia coli methionine repressor

2006 ◽  
Vol 396 (2) ◽  
pp. 227-234 ◽  
Author(s):  
Ferenc Marincs ◽  
Iain W. Manfield ◽  
Jonathan A. Stead ◽  
Kenneth J. Mcdowall ◽  
Peter G. Stockley
Keyword(s):  
Escherichia Coli ◽  
Gene Replacement ◽  
Dna Arrays ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Metabolic Role ◽  
Mobility Shift Assay

We have used DNA arrays to investigate the effects of knocking out the methionine repressor gene, metJ, on the Escherichia coli transcriptome. We assayed the effects in the knockout strain of supplying wild-type or mutant MetJ repressors from an expression plasmid, thus establishing a rapid assay for in vivo effects of mutations characterized previously in vitro. Repression is largely restricted to known genes involved in the biosynthesis and uptake of methionine. However, we identified a number of additional genes that are significantly up-regulated in the absence of repressor. Sequence analysis of the 5′ promoter regions of these genes identified plausible matches to met-box sequences for three of these, and subsequent electrophoretic mobility-shift assay analysis showed that for two such loci their repressor affinity is higher than or comparable with the known metB operator, suggesting that they are directly regulated. This can be rationalized for one of the loci, folE, by the metabolic role of its encoded enzyme; however, the links to the other regulated loci are unclear, suggesting both an extension to the known met regulon and additional complexity to the role of the repressor. The plasmid gene replacement system has been used to examine the importance of protein–protein co-operativity in operator saturation using the structurally characterized mutant repressor, Q44K. In vivo, there are detectable reductions in the levels of regulation observed, demonstrating the importance of balancing protein–protein and protein–DNA affinity.

scholarly journals Ginsenoside Rg3 Sensitizes Colorectal Cancer to Radiotherapy through Downregulation of Proliferative and Angiogenic Biomarkers

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Taiguo Liu ◽  
Lina Duo ◽  
Ping Duan
Keyword(s):  
Quality Of Life ◽  
Colorectal Cancer ◽  
Gene Products ◽  
Ginsenoside Rg3 ◽  
Mobility Shift ◽  
Antitumor Effects ◽  
Fractionated Radiotherapy ◽  
Mobility Shift Assay

Background. Radiation therapy is an important mode of colorectal cancer treatment. However, most people die of local recurrence after tumors become resistant to radiotherapy, and little progress has been made in treating radiotherapy-resistant colorectal cancer. Hence, novel agents that are nontoxic and can sensitize colorectal cancer to radiotherapy are urgently needed. Ginsenoside Rg3, a saponin extracted from ginseng, shows cytotoxicity against a variety of cancer cells through suppression of pathways linked to oncogenesis, including cell survival, proliferation, invasion, and angiogenesis. In this article, we investigated whether Rg3 can sensitize colorectal cancer to radiation in vivo. Methods and Materials. We established CT-26 xenografts in BALB/c mice and treated them with vehicle, Rg3, radiation, and combined Rg3 + radiation. Mouse quality of life, survival, tumor volumes, and inhibitive rates were estimated. NF-κB activation was ascertained using electrophoretic mobility shift assay and immunohistochemistry. We also tested for markers of proliferation, angiogenesis, and invasion using immunohistochemistry and Western blot analysis. Results. Rg3 significantly enhanced the efficacy of fractionated radiotherapy by improving the quality of life of mice. Moreover, tumors from mice xenografted with CT-26 cells and treated with combined Rg3 + radiotherapy showed significantly lower tumor volumes (P<0.01 versus controls; P<0.05 versus radiation alone), NF-κB activation, and expression of NF-κB-regulated gene products (cyclin D1, survivin, cyclooxygenase-2 (COX-2), and vascular endothelial growth factor (VEGF)) compared with controls. The combination treatment was also effective in suppressing angiogenesis, as indicated by lower CD31+ microvessel density compared with controls (P<0.05). Conclusion. Our results suggest that Rg3 enhances the antitumor effects of radiotherapy for colorectal cancer by suppressing NF-κB and NF-κB-regulated gene products, leading to inhibition of tumors and prolongation of the lifespan of CT-26 xenograft BALB/c mice.

Mutational and in vitro protein-binding studies on centromere DNA from Saccharomyces cerevisiae

1987 ◽  
Vol 7 (12) ◽  
pp. 4522-4534
Author(s):  
R Ng ◽  
J Carbon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Binding ◽  
In Vitro Assays ◽  
Binding Studies ◽  
Mobility Shift ◽  
Sequence Element ◽  
Sequence Elements ◽  
Mobility Shift Assay

Centromeres on chromosomes in the yeast Saccharomyces cerevisiae contain approximately 140 base pairs (bp) of DNA. The functional centromere (CEN) region contains three important sequence elements (I, PuTCACPuTG; II, 78 to 86 bp of high-AT DNA; and III, a conserved 25-bp sequence with internal bilateral symmetry). Various point mutations or deletions in the element III region have a profound effect on CEN function in vivo, indicating that this DNA region is a key protein-binding site. This has been confirmed by the use of two in vitro assays to detect binding of yeast proteins to DNA fragments containing wild-type or mutationally altered CEN3 sequences. An exonuclease III protection assay was used to demonstrate specific binding of proteins to the element III region of CEN3. In addition, a gel DNA fragment mobility shift assay was used to characterize the binding reaction parameters. Sequence element III mutations that inactivate CEN function in vivo also prevent binding of proteins in the in vitro assays. The mobility shift assay indicates that double-stranded DNAs containing sequence element III efficiently bind proteins in the absence of sequence elements I and II, although the latter sequences are essential for optimal CEN function in vivo.

scholarly journals Binding of CCCTC-binding factor in vivo to the region located between Rep* and the C promoter of Epstein–Barr virus is unaffected by CpG methylation and does not correlate with Cp activity

2009 ◽  
Vol 90 (5) ◽  
pp. 1183-1189 ◽  
Author(s):  
Daniel Salamon ◽  
Ferenc Banati ◽  
Anita Koroknai ◽  
Mate Ravasz ◽  
Kalman Szenthe ◽  
...  
Keyword(s):  
Cell Lines ◽  
Epstein Barr Virus ◽  
Cpg Methylation ◽  
Ctcf Binding ◽  
Mobility Shift ◽  
Barr Virus ◽  
Binding Factor ◽  
Epstein Barr ◽  
Mobility Shift Assay

In this study, the binding of the insulator protein CCCTC-binding factor (CTCF) to the region located between Rep* and the C promoter (Cp) of Epstein–Barr virus (EBV) was analysed using chromatin immunoprecipitation and in vivo footprinting. CTCF binding was found to be independent of Cp usage in cell lines corresponding to the major EBV latency types. Bisulfite sequencing and an electrophoretic mobility-shift assay (using methylated and unmethylated probes) revealed that CTCF binding was insufficient to induce local CpG demethylation in certain cell lines and was unaffected by CpG methylation in the region between Rep* and Cp. In addition, CTCF binding to the latency promoter, Qp, did not correlate with Qp activity.

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