scholarly journals Evidence that the supE44 Mutation of Escherichia coli Is an Amber Suppressor Allele of glnX and that It Also Suppresses Ochre and Opal Nonsense Mutations

2010 ◽  
Vol 192 (22) ◽  
pp. 6039-6044 ◽  
Author(s):  
B. Singaravelan ◽  
B. R. Roshini ◽  
M. Hussain Munavar
Keyword(s):  
Escherichia Coli ◽  
Growth Phase ◽  
Stationary Growth Phase ◽  
Luciferase Assay ◽  
Logarithmic Phase ◽  
Nonsense Mutations ◽  
Translational Readthrough ◽  
Amber Suppressor ◽  
Nonsense Codons

ABSTRACT Translational readthrough of nonsense codons is seen not only in organisms possessing one or more tRNA suppressors but also in strains lacking suppressors. Amber suppressor tRNAs have been reported to suppress only amber nonsense mutations, unlike ochre suppressors, which can suppress both amber and ochre mutations, essentially due to wobble base pairing. In an Escherichia coli strain carrying the lacZU118 episome (an ochre mutation in the lacZ gene) and harboring the supE44 allele, suppression of the ochre mutation was observed after 7 days of incubation. The presence of the supE44 lesion in the relevant strains was confirmed by sequencing, and it was found to be in the duplicate copy of the glnV tRNA gene, glnX. To investigate this further, an in vivo luciferase assay developed by D. W. Schultz and M. Yarus (J. Bacteriol. 172:595-602, 1990) was employed to evaluate the efficiency of suppression of amber (UAG), ochre (UAA), and opal (UGA) mutations by supE44. We have shown here that supE44 suppresses ochre as well as opal nonsense mutations, with comparable efficiencies. The readthrough of nonsense mutations in a wild-type E. coli strain was much lower than that in a supE44 strain when measured by the luciferase assay. Increased suppression of nonsense mutations, especially ochre and opal, by supE44 was found to be growth phase dependent, as this phenomenon was only observed in stationary phase and not in logarithmic phase. These results have implications for the decoding accuracy of the translational machinery, particularly in stationary growth phase.

scholarly journals Adherence of Staphylococcus aureus to Endothelial Cells: Influence of Capsular Polysaccharide, Global Regulatoragr, and Bacterial Growth Phase

2000 ◽  
Vol 68 (9) ◽  
pp. 4865-4871 ◽  
Author(s):  
Petra Pöhlmann-Dietze ◽  
Martina Ulrich ◽  
Kevin B. Kiser ◽  
Gerd Döring ◽  
Jean C. Lee ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Endothelial Cells ◽  
Bacterial Growth ◽  
Growth Phase ◽  
Parental Strain ◽  
Double Mutant ◽  
Capsular Polysaccharide ◽  
Stationary Growth Phase ◽  
Exponential Growth Phase ◽  
Logarithmic Phase

ABSTRACT The adherence of Staphylococcus aureus to human endothelial cells (EC) is probably an important step in the pathogenesis of systemic staphylococcal infections. We examined the influence of type 5 capsular polysaccharide (CP5) production, the global regulator agr, and the bacterial growth phase onS. aureus adherence to EC. Whereas S. aureusNewman showed maximal adherence to EC in the logarithmic phase of growth, an isogenic agr mutant showed maximal adherence in the stationary growth phase. S. aureus adherence to EC and CP5 expression were negatively correlated: a mutation in theagr locus diminished CP5 production and led to increased adherence. Likewise, induction of CP5 expression by addition of NaCl to the growth medium resulted in reduced staphylococcal adherence to EC.S. aureus Newman cells that adhered to EC did not express CP5. A Newman cap5O mutant was acapsular and showed significantly greater adherence to EC than the parental strain did (P < 0.005). Complementation of the cap5Omutation in trans restored CP5 expression and reduced EC adherence to a level similar to that of the parental strain. The enhanced adherence shown by the cap5O mutant was similar in magnitude to that of the agr mutant or the cap5O agr double mutant. Cells of the cap5O mutant andcap5O agr double mutant harvested from stationary-phase cultures adhered significantly better than did cells harvested in the exponential growth phase. These data are consistent with the postexponential and agr-independent expression by S. aureus of at least one putative EC adhesin, whose binding domain may be masked by CP5.

scholarly journals Suppression of Nonsense Mutations Induced by Expression of an RNA Complementary to a Conserved Segment of 23S rRNA

1999 ◽  
Vol 181 (17) ◽  
pp. 5257-5262 ◽  
Author(s):  
Natalya S. Chernyaeva ◽  
Emanuel J. Murgola ◽  
Alexander S. Mankin
Keyword(s):  
Escherichia Coli ◽  
Reporter Genes ◽  
Chain Termination ◽  
23S Rrna ◽  
Specific Interactions ◽  
Peptidyl Transferase ◽  
Nonsense Mutations ◽  
Peptidyl Transferase Center ◽  
Codon Positions

ABSTRACT We identified a short RNA fragment, complementary to theEscherichia coli 23S rRNA segment comprising nucleotides 735 to 766 (in domain II), which when expressed in vivo results in the suppression of UGA nonsense mutations in two reporter genes. Neither UAA nor UAG mutations, examined at the same codon positions, were suppressed by the expression of this antisense rRNA fragment. Our results suggest that a stable phylogenetically conserved hairpin at nucleotides 736 to 760 in 23S rRNA, which is situated close to the peptidyl transferase center, may participate in one or more specific interactions during peptide chain termination.

Introduction of UAG, UAA, and UGA nonsense mutations at a specific site in the Escherichia coli chloramphenicol acetyltransferase gene: use in measurement of amber, ochre, and opal suppression in mammalian cells

1986 ◽  
Vol 6 (9) ◽  
pp. 3059-3067
Author(s):  
J P Capone ◽  
J M Sedivy ◽  
P A Sharp ◽  
U L RajBhandary
Keyword(s):  
Escherichia Coli ◽  
Enzymatic Activity ◽  
Mammalian Cells ◽  
Chloramphenicol Acetyltransferase ◽  
Nonsense Suppression ◽  
Trna Genes ◽  
Suppressor Activity ◽  
Nonsense Mutations ◽  
Suppressor Trna ◽  
Nonsense Codons

We have used oligonucleotide-directed site-specific mutagenesis to convert serine codon 27 of the Escherichia coli chloramphenicol acetyltransferase (cat) gene to UAG, UAA, and UGA nonsense codons. The mutant cat genes, under transcriptional control of the Rous sarcoma virus long terminal repeat, were then introduced into mammalian cells by DNA transfection along with UAG, UAA, and UGA suppressor tRNA genes derived from a human serine tRNA. Assay for CAT enzymatic activity in extracts from such cells allowed us to detect and quantitate nonsense suppression in monkey CV-1 cells and mouse NIH3T3 cells. Using such an assay, we provide the first direct evidence that an opal suppressor tRNA gene is functional in mammalian cells. The pattern of suppression of the three cat nonsense mutations in bacteria suggests that the serine at position 27 of CAT can be replaced by a wide variety of amino acids without loss of enzymatic activity. Thus, these mutant cat genes should be generally useful for the quantitation of suppressor activity of suppressor tRNA genes introduced into cells and possibly for the detection of naturally occurring nonsense suppressors.

In vivo analyses of constitutive and regulated promoters in halophilic archaea

Microbiology ◽  
2005 ◽  
Vol 151 (1) ◽  
pp. 25-33 ◽  
Author(s):  
Dagmar Gregor ◽  
Felicitas Pfeifer
Keyword(s):  
Growth Phase ◽  
Promoter Activity ◽  
Halophilic Archaea ◽  
Stationary Growth Phase ◽  
Halobacterium Salinarum ◽  
Exponential Growth Phase ◽  
Haloferax Mediterranei ◽  
Basal Promoter Activity ◽  
Transcriptional Activator Protein

The two gvpA promoters PcA and PpA of Halobacterium salinarum, and the PmcA promoter of Haloferax mediterranei were investigated with respect to growth-phase-dependent expression and regulation in Haloferax volcanii transformants using the bgaH reading frame encoding BgaH, an enzyme with β-galactosidase activity, as reporter. For comparison, the Pfdx promoter of the ferredoxin gene of Hbt. salinarum and the PbgaH promoter of Haloferax lucentense (formerly Haloferax alicantei) were analysed. Pfdx , driving the expression of a house-keeping gene, was highly active during the exponential growth phase, whereas PbgaH and the three gvpA promoters yielded the largest activities during the stationary growth phase. Compared to Pfdx , the basal promoter activities of PpA and PmcA were rather low, and larger activities were only detected in the presence of the endogenous transcriptional activator protein GvpE. The PcA promoter does not yield a detectable basal promoter activity and is only active in the presence of the homologous cGvpE. To investigate whether the PcA -TATA box and the BRE element were the reason for the lack of the basal PcA activity, these elements and also sequences further upstream were substituted with the respective sequences of the stronger PpA promoter and investigated in Hfx. volcanii transformants. All these promoter chimera did not yield a detectable basal promoter activity. However, whenever the PpA -BRE element was substituted for the PcA -BRE, an enhanced cGvpE-mediated activation was observed. The promoter chimeras harbouring PpA -BRE plus 5 (or more) bp further upstream also gained activation by the heterologous pGvpE and mcGvpE proteins. The sequence required for the GvpE-mediated activation was determined by a 4 bp scanning mutagenesis with the 45 bp region upstream of PmcA -BRE. None of these alterations influenced the basal promoter activity, but the sequence TGAAACGG-n4-TGAACCAA was important for the GvpE-mediated activation of PmcA .

scholarly journals Aquaporin Z of Escherichia coli: Reassessment of Its Regulation and Physiological Role

2002 ◽  
Vol 184 (15) ◽  
pp. 4304-4307 ◽  
Author(s):  
Eric Soupene ◽  
Natalie King ◽  
Haidy Lee ◽  
Sydney Kustu
Keyword(s):  
Escherichia Coli ◽  
Adverse Effects ◽  
Growth Phase ◽  
Sigma Factor ◽  
Physiological Role ◽  
Single Copy ◽  
Stationary Growth Phase ◽  
Stationary Growth ◽  
Factor Expression ◽  
Aquaporin Z

ABSTRACT Transcription of an aqpZ-lac fusion in a single copy on the Escherichia coli chromosome increased as cells entered the stationary growth phase. This was true in a variety of media, and increased transcription in enriched medium required the RpoS sigma factor. Expression of the aqpZ-lac fusion was not affected by up- or downshifts in osmolality. Disruption of aqpZ had no detectable adverse effects.

scholarly journals CFTR mRNAs with nonsense codons are degraded by the SMG6-mediated endonucleolytic pathway

2021 ◽  
Author(s):  
Edward Sanderlin ◽  
Melissa Keenan ◽  
Martin Mense ◽  
Alexey Revenko ◽  
Brett Monia ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Exon Junction Complex ◽  
Transmembrane Conductance Regulator ◽  
Loss Of Function ◽  
Transmembrane Conductance ◽  
Nonsense Mutations ◽  
Nonsense Mediated Decay ◽  
Nonsense Codon ◽  
Translational Readthrough ◽  
Nonsense Codons

Abstract Cystic fibrosis is caused by loss of function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene resulting in severe lung disease. Nearly 10% of cystic fibrosis patients have at least one CFTR allele with a nonsense mutation that generates a nonsense codon in the mRNA. Nonsense mutations can result in significant reduction of gene expression partially due to rapid mRNA degradation through the nonsense-mediated decay (NMD) pathway. It has not been thoroughly investigated which branch of the NMD pathway governs the decay of CFTR mRNAs containing nonsense codons. Here we utilized antisense oligonucleotides targeting NMD factors to evaluate the regulation of nonsense codon-containing CFTR mRNAs by the NMD pathway. Interestingly, we found that CFTR mRNAs with G542X, R1162X, and W1282X nonsense codons require UPF2, UPF3, and exon junction complex proteins for NMD, whereas CFTR mRNAs with the Y122X nonsense codon do not. Furthermore, we demonstrated that all evaluated CFTR mRNAs harboring nonsense codons were degraded by the SMG6-mediated endonucleolytic pathway rather than the SMG5/SMG7-mediated exonucleolytic pathway. Finally, we found that stabilization of CFTR mRNAs by NMD inhibition alone improved functional W1282X protein production, and improved the efficiency of aminoglycoside translational readthrough of CFTR-Y122X, -G542X, and -R1162X mRNAs.

scholarly journals Gentamicin B1 is a minor gentamicin component with major nonsense mutation suppression activity

2017 ◽  
Vol 114 (13) ◽  
pp. 3479-3484 ◽  
Author(s):  
Alireza Baradaran-Heravi ◽  
Jürgen Niesser ◽  
Aruna D. Balgi ◽  
Kunho Choi ◽  
Carla Zimmerman ◽  
...  
Keyword(s):  
Genetic Disorders ◽  
Xenograft Model ◽  
Minor Component ◽  
Premature Termination Codon ◽  
Tumor Xenograft ◽  
Nonsense Suppression ◽  
Nonsense Mutations ◽  
Nonsense Codons ◽  
Dynamics Simulations

Nonsense mutations underlie about 10% of rare genetic disease cases. They introduce a premature termination codon (PTC) and prevent the formation of full-length protein. Pharmaceutical gentamicin, a mixture of several related aminoglycosides, is a frequently used antibiotic in humans that can induce PTC readthrough and suppress nonsense mutations at high concentrations. However, testing of gentamicin in clinical trials has shown that safe doses of this drug produce weak and variable readthrough activity that is insufficient for use as therapy. In this study we show that the major components of pharmaceutical gentamicin lack PTC readthrough activity but the minor component gentamicin B1 (B1) is a potent readthrough inducer. Molecular dynamics simulations reveal the importance of ring I of B1 in establishing a ribosome configuration that permits pairing of a near-cognate complex at a PTC. B1 induced readthrough at all three nonsense codons in cultured cancer cells with TP53 (tumor protein p53) mutations, in cells from patients with nonsense mutations in the TPP1 (tripeptidyl peptidase 1), DMD (dystrophin), SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like 1), and COL7A1 (collagen type VII alpha 1 chain) genes, and in an in vivo tumor xenograft model. The B1 content of pharmaceutical gentamicin is highly variable and major gentamicins suppress the PTC readthrough activity of B1. Purified B1 provides a consistent and effective source of PTC readthrough activity to study the potential of nonsense suppression for treatment of rare genetic disorders.

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