scholarly journals Variability of the intracellular ionic environment of Escherichia coli. Differences between in vitro and in vivo effects of ion concentrations on protein-DNA interactions and gene expression.

1987 ◽  
Vol 262 (15) ◽  
pp. 7157-7164 ◽  
Author(s):  
B Richey ◽  
D S Cayley ◽  
M C Mossing ◽  
C Kolka ◽  
C F Anderson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Dna Interactions ◽  
Ion Concentrations ◽  
Ionic Environment ◽  
Protein Dna Interactions ◽  
In Vivo Effects

Alterations in Protein-DNA Interactions in the γ-Globin Gene Promoter in Response to Butyrate Therapy

Blood ◽  
1998 ◽  
Vol 92 (8) ◽  
pp. 2924-2933 ◽  
Author(s):  
Tohru Ikuta ◽  
Yuet Wai Kan ◽  
Paul S. Swerdlow ◽  
Douglas V. Faller ◽  
Susan P. Perrine
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Gene Promoter ◽  
Dna Interactions ◽  
Globin Mrna ◽  
Mobility Shift ◽  
Protein Dna Interactions ◽  
American Society ◽  
Globin Gene Expression

Abstract The mechanisms by which pharmacologic agents stimulate γ-globin gene expression in β-globin disorders has not been fully established at the molecular level. In studies described here, nucleated erythroblasts were isolated from patients with β-globin disorders before and with butyrate therapy, and globin biosynthesis, mRNA, and protein-DNA interactions were examined. Expression of γ-globin mRNA increased twofold to sixfold above baseline with butyrate therapy in 7 of 8 patients studied. A 15% to 50% increase in γ-globin protein synthetic levels above baseline γ globin ratios and a relative decrease in β-globin biosynthesis were observed in responsive patients. Extensive new in vivo footprints were detected in erythroblasts of responsive patients in four regions of the γ-globin gene promoter, designated butyrate-response elements gamma 1-4 (BRE-G1-4). Electrophoretic mobility shift assays using BRE-G1 sequences as a probe demonstrated that new binding of two erythroid-specific proteins and one ubiquitous protein, CP2, occurred with treatment in the responsive patients and did not occur in the nonresponder. The BRE-G1 sequence conferred butyrate inducibility in reporter gene assays. These in vivo protein-DNA interactions in human erythroblasts in which γ-globin gene expression is being altered strongly suggest that nuclear protein binding, including CP2, to the BRE-G1 region of the γ-globin gene promoter mediates butyrate activity on γ-globin gene expression. © 1998 by The American Society of Hematology.

scholarly journals In vitro and in vivo effects of the probiotic Escherichia coli strain M-17: immunomodulation and attenuation of murine colitis

2008 ◽  
Vol 100 (03) ◽  
pp. 530-541 ◽  
Author(s):  
Leo R. Fitzpatrick ◽  
Jeffrey Small ◽  
Robert A. Hoerr ◽  
Eileen F. Bostwick ◽  
Lynn Maines ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Coli Strain ◽  
Murine Colitis ◽  
In Vivo Effects

scholarly journals Mutagenesis of the Rns regulator of enterotoxigenic Escherichia coli reveals roles for a linker sequence and two helix–turn–helix motifs

Microbiology ◽  
2010 ◽  
Vol 156 (9) ◽  
pp. 2796-2806 ◽  
Author(s):  
Vivienne Mahon ◽  
Cyril J. Smyth ◽  
Stephen G. J. Smith
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Protein A ◽  
Diarrhoeal Disease ◽  
Enterotoxigenic Escherichia Coli ◽  
Insertion Mutagenesis

The pathogenesis of diarrhoeal disease due to human enterotoxigenic Escherichia coli absolutely requires the expression of fimbriae. The expression of CS1 fimbriae is positively regulated by the AraC-like protein Rns. AraC-like proteins are DNA-binding proteins that typically contain two helix–turn–helix (HTH) motifs. A program of pentapeptide insertion mutagenesis of the Rns protein was performed, and this revealed that both HTH motifs are required by Rns to positively regulate CS1 fimbrial gene expression. Intriguingly, a pentapeptide insertion after amino acid C102 reduced the ability of Rns to transactivate CS1 fimbrial expression. The structure of Rns in this vicinity (NACRS) was predicted to be disordered and thus might act as a flexible linker. This hypothesis was confirmed by deletion of this amino acid sequence from the Rns protein; a truncated protein that lacked this sequence was no longer functional. Strikingly, this sequence could be functionally substituted in vivo and in vitro by a flexible seven amino acid sequence from another E. coli AraC-like protein RhaS. Our data indicate that HTH motifs and a flexible sequence are required by Rns for maximal activation of fimbrial gene expression.

scholarly journals Scanning Mutagenesis of Mcm1: Residues Required for DNA Binding, DNA Bending, and Transcriptional Activation by a MADS-Box Protein

2000 ◽  
Vol 20 (1) ◽  
pp. 1-11 ◽  
Author(s):  
Thomas B. Acton ◽  
Janet Mead ◽  
Andrew M. Steiner ◽  
Andrew K. Vershon
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Serum Response Factor ◽  
Essential Gene ◽  
Dna Bending ◽  
Mads Box ◽  
Dna Interactions ◽  
Protein Dna Interactions

ABSTRACT MCM1 is an essential gene in the yeastSaccharomyces cerevisiae and is a member of the MADS-box family of transcriptional regulatory factors. To understand the nature of the protein-DNA interactions of this class of proteins, we have made a series of alanine substitutions in the DNA-binding domain of Mcm1 and examined the effects of these mutations in vivo and in vitro. Our results indicate which residues of Mcm1 are important for viability, transcriptional activation, and DNA binding and bending. Substitution of residues in Mcm1 which are highly conserved among the MADS-box proteins are lethal to the cell and abolish DNA binding in vitro. These positions have almost identical interactions with DNA in both the serum response factor-DNA and α2-Mcm1-DNA crystal structures, suggesting that these residues make up a conserved core of protein-DNA interactions responsible for docking MADS-box proteins to DNA. Substitution of residues which are not as well conserved among members of the MADS-box family play important roles in contributing to the specificity of DNA binding. These results suggest a general model of how MADS-box proteins recognize and bind DNA. We also provide evidence that the N-terminal extension of Mcm1 may have considerable conformational freedom, possibly to allow binding to different DNA sites. Finally, we have identified two mutants at positions which are critical for Mcm1-mediated DNA bending that have a slow-growth phenotype. This finding is consistent with our earlier results, indicating that DNA bending may have a role in Mcm1 function in the cell.

scholarly journals Investigation of exposure to lifestyle and environmental factors on cumulus-oocyte complex function

2021 ◽  
Author(s):  
◽  
Kelly Anne Campen
Keyword(s):  
Gene Expression ◽  
Oocyte Quality ◽  
Protein Levels ◽  
Time Interaction ◽  
Cumulus Oocyte Complex ◽  
In Vitro Exposure ◽  
In Vitro Effects ◽  
In Vivo Effects

<p>Pathways involved in bi-directional communication within the cumulus-oocyte complex (COC) include gap junction (GJ) communication, oocyte growth factor production, and glucose metabolism and are essential for oocyte health. Perturbation of these pathways may result in reduced oocyte quality due to altered COC function. Using rats as a model, in vitro effects of exposure to bisphenol A (BPA), caffeine, nicotine, ethanol, methylenedioxymeth- amphetamine (MDMA), or Δ⁹-tetrahydrocannabinol (THC) on COC function were investigated. Furthermore, MDMA was administered to rats to compare in vitro with in vivo effects.  The transfer of a fluorescent dye (calcein) from cumulus cells (CC) to the oocyte was used as a measure of GJ communication. Expression of CC-derived (Atr, Cx43, Cycs, Gfpt1, Pfkp) and oocyte-derived (Atr, Bmp15, Cx37, Gdf9) genes were measured using multiplex TaqMan quantitative PCR. Levels of CX43 and GDF9 proteins were quantified using Western blotting.  Optimisation of the GJ bioassay included the addition of phosphodiesterase inhibitors (rolipram and dipyridamole), and a 1 hour post-calcein incubation period to allow dye transfer. Quantification of gene expression in calcein-treated CC and oocytes was validated, enabling direct comparisons between GJ communication and gene expression.  To determine the in vitro effects, COC were incubated with test factors at high physiological concentrations over 25 hours. GJ communication decreased over time in control COC. This reduction was attenuated after exposure to BPA and nicotine, and partially by caffeine. Furthermore, exposure to ethanol maintained oocyte meiotic arrest, whereas MDMA and THC promoted meiotic resumption.  Oocyte-derived gene expression was mostly unaffected by in vitro exposure to the lifestyle and environmental factors, although a treatment x time interaction for Cx37 levels following nicotine exposure was observed. Of the CC-derived genes, Cx43 was the most sensitive where BPA, MDMA, and THC increased, and caffeine and ethanol decreased, expression. In CC, exposure to MDMA and THC increased Gfpt1 levels and exposure to MDMA resulted in a treatment x time interaction in Cycs and Pfkp expression.  In COC, caffeine increased CX43 protein levels after 1 hour. Nicotine initially reduced, but with time increased CX43 levels. Furthermore, CX43 levels decreased and increased after 25 hour exposures to ethanol and MDMA, respectively. GDF9 protein levels in COC exhibited wide within-treatment variation. Overall, BPA and caffeine reduced GDF9 levels after 1 hour whereas GDF9 levels were increased following exposure to BPA, caffeine, MDMA, and THC for 25 hours.  To determine in vivo effects, female rats were administered saline or 5 mg/kg/day MDMA for 3 days. COC from MDMA-treated rats had higher levels of CX43 protein but gene expression and meiotic reactivation were unaffected.  In conclusion, COC function was altered by in vitro exposure to BPA, caffeine, ethanol, nicotine, MDMA, and THC. Furthermore, in vivo exposure to MDMA elicits similar, albeit reduced, effects on COC function. A role for CC in protecting the oocyte from harmful contaminants is proposed. Perturbation of the bi-directional communication pathway is likely to influence oocyte quality due to alterations in nutrient availability and timing of follicular events, although these may not be associated with negative outcomes. This study provides evidence that exposure to lifestyle factors and environmental contaminants affect COC function.</p>

scholarly journals Retapamulin-assisted ribosome profiling reveals the alternative bacterial proteome

2019 ◽  
Author(s):  
Sezen Meydan ◽  
James Marks ◽  
Dorota Klepacki ◽  
Virag Sharma ◽  
Pavel V. Baranov ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Translation Initiation ◽  
Single Gene ◽  
Ribosome Profiling ◽  
Genome Wide ◽  
Translation Start ◽  
Alternative Translation

SUMMARYThe use of alternative translation initiation sites enables production of more than one protein from a single gene, thereby expanding cellular proteome. Although several such examples have been serendipitously found in bacteria, genome-wide mapping of alternative translation start sites has been unattainable. We found that the antibiotic retapamulin specifically arrests initiating ribosomes at start codons of the genes. Retapamulin-enhanced Ribo-seq analysis (Ribo-RET) not only allowed mapping of conventional initiation sites at the beginning of the genes but, strikingly, it also revealed putative internal start sites in a number of Escherichia coli genes. Experiments demonstrated that the internal start codons can be recognized by the ribosomes and direct translation initiation in vitro and in vivo. Proteins, whose synthesis is initiated at an internal in-frame and out-of-frame start sites, can be functionally important and contribute to the ‘alternative’ bacterial proteome. The internal start sites my also play regulatory roles in gene expression.

Alterations in Protein-DNA Interactions in the γ-Globin Gene Promoter in Response to Butyrate Therapy

Blood ◽  
1998 ◽  
Vol 92 (8) ◽  
pp. 2924-2933
Author(s):  
Tohru Ikuta ◽  
Yuet Wai Kan ◽  
Paul S. Swerdlow ◽  
Douglas V. Faller ◽  
Susan P. Perrine
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Gene Promoter ◽  
Dna Interactions ◽  
Globin Mrna ◽  
Mobility Shift ◽  
Protein Dna Interactions ◽  
American Society ◽  
Globin Gene Expression

The mechanisms by which pharmacologic agents stimulate γ-globin gene expression in β-globin disorders has not been fully established at the molecular level. In studies described here, nucleated erythroblasts were isolated from patients with β-globin disorders before and with butyrate therapy, and globin biosynthesis, mRNA, and protein-DNA interactions were examined. Expression of γ-globin mRNA increased twofold to sixfold above baseline with butyrate therapy in 7 of 8 patients studied. A 15% to 50% increase in γ-globin protein synthetic levels above baseline γ globin ratios and a relative decrease in β-globin biosynthesis were observed in responsive patients. Extensive new in vivo footprints were detected in erythroblasts of responsive patients in four regions of the γ-globin gene promoter, designated butyrate-response elements gamma 1-4 (BRE-G1-4). Electrophoretic mobility shift assays using BRE-G1 sequences as a probe demonstrated that new binding of two erythroid-specific proteins and one ubiquitous protein, CP2, occurred with treatment in the responsive patients and did not occur in the nonresponder. The BRE-G1 sequence conferred butyrate inducibility in reporter gene assays. These in vivo protein-DNA interactions in human erythroblasts in which γ-globin gene expression is being altered strongly suggest that nuclear protein binding, including CP2, to the BRE-G1 region of the γ-globin gene promoter mediates butyrate activity on γ-globin gene expression. © 1998 by The American Society of Hematology.

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