scholarly journals Variation inListeria monocytogenesDose Responses in Relation to Subtypes Encoding a Full-Length or Truncated Internalin A

2010 ◽  
Vol 77 (4) ◽  
pp. 1171-1180 ◽  
Author(s):  
Yuhuan Chen ◽  
William H. Ross ◽  
Richard C. Whiting ◽  
Anna Van Stelten ◽  
Kendra K. Nightingale ◽  
...  
Keyword(s):  
Stop Codon ◽  
Premature Stop Codon ◽  
Intestinal Barrier ◽  
Full Length ◽  
Quantitative Evidence ◽  
Consumption Data ◽  
Genes Encoding ◽  
A Cell ◽  
Dose Responses ◽  
Internalin A

ABSTRACTInternalin A (InlA; encoded byinlA) facilitates the crossing of the intestinal barrier byListeria monocytogenes. Mutations leading to a premature stop codon (PMSC) ininlAand thus attenuated mammalian virulence have been reported. We recently characterized 502L. monocytogenesfood isolates from a retail survey and 507 human clinical isolates from multiple U.S. states with respect to the presence/absence ofinlAmutations. The objective of this study was to investigate the hypothesis that dose responses for human listeriosis vary betweenL. monocytogenesstrains with and those without a PMSC ininlA. Subtype-specific prevalence and concentration distributions in food, along with epidemiologic and consumption data, were input into established dose-response models to generate anrvalue (probability of a cell causing illness). Under the conservative assumption thatL. monocytogeneslevels at retail represent levels consumed, mean log10rvalues were −8.1 and −10.7 forL. monocytogenessubtypes with genes encoding a full-length and a truncated InlA, respectively.L. monocytogenescarrying a 5′ frameshift mutation in a homopolymeric tract showed a mean log10rvalue of −12.1. Confidence intervals for thervalues and their differences varied depending on subtypes. When the increase in concentration ofL. monocytogenessubtypes between retail and consumption was considered, mean log10rvalues were reduced to −10.4, −13.8, and −12.8 for the subtypes with genes encoding a full-length InlA, for the subtypes carrying a PMSC ininlA, and for allL. monocytogenesisolates regardless of subtype, respectively. Our study provides further quantitative evidence thatL. monocytogenessubtypes vary in abilities and relative likelihoods of causing human disease, which were mechanistically related to defined genetic markers.

scholarly journals YPK9 and WHI2 Negatively Interact during Oxidative Stress

2021 ◽  
Vol 9 (12) ◽  
pp. 2584
Author(s):  
Florenal Joseph ◽  
Darach Miller ◽  
Oleg V. Evgrafov ◽  
William J. Chirico
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Genetic Interaction ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Full Length ◽  
Deletion Strain ◽  
Acid Protein ◽  
Insight Into ◽  
Amino Acid Protein

Yeast PARK9 (YPK9) shares homology with human ATP13A2, which encodes a polyamine transporter implicated in juvenile forms of Parkinson’s disease. We used YPK9 to gain insight into how ATP13A2 affects cell growth and sensitivity to oxidative stress. Surprisingly, the YPK9 deletion strain from the Saccharomyces cerevisiae deletion collection (YKO) in wildtype BY4741 (mating type a) grew faster and was more resistant to hydrogen peroxide than a commercial, putative parental BY4741 wildtype strain (BY4741COM). In contrast, deleting YPK9 from BY4741COM rendered it very sensitive to hydrogen peroxide, suggesting its background is different from that of the deletion collection. Whole-genome sequencing revealed that BY4741COM and BY4741COMypk9∆ contain a novel premature stop codon near the 3′ end of WHI2 (WHI2G1324T), whereas the collection’s YPK9 deletion strain contains WHI2, which encodes a 486 amino acid protein, Whi2p. Replacing full-length WHI2 with the sequence coding for the predicted truncation (Whi2pE442*) rendered strains more sensitive to hydrogen peroxide, whereas the converse replacement rendered them more resistant. The sequences of WHI2 in 20 randomly chosen strains from the collection encode the full-length protein, indicating that the putative parental BY4741 WHI2G1324T strain’s genetic background differs from that of the deletion collection. Examination of WHI2 sequences in several commonly used wildtype S. cerevisiae strains and isolates revealed other Whi2p truncations that might yield altered phenotypes. Together, these results demonstrate a novel premature stop codon in WHI2 that renders yeast sensitive to hydrogen peroxide; they also reveal a negative genetic interaction between WHI2 and YPK9 in the presence of hydrogen peroxide in the BY4741 background.

scholarly journals Detection of ALDH3B2 in Human Placenta

2019 ◽  
Vol 20 (24) ◽  
pp. 6292
Author(s):  
Sylwia Michorowska ◽  
Joanna Giebułtowicz ◽  
Renata Wolinowska ◽  
Anna Konopka ◽  
Anna Wilkaniec ◽  
...  
Keyword(s):  
Western Blot ◽  
Human Placenta ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Premature Termination Codon ◽  
Full Length ◽  
Start Codon ◽  
Phosphorylation Sites ◽  
Putative Open Reading Frame ◽  
Healthy Human

Aldehyde dehydrogenase 3B2 (ALDH3B2) gene contains a premature termination codon, which can be skipped or suppressed resulting in full-length protein expression. Alternatively, the longest putative open reading frame starting with the second in-frame start codon would encode short isoform. No unequivocal evidence of ALDH3B2 expression in healthy human tissues is available. The aim of this study was to confirm its expression in human placenta characterized by the highest ALDH3B2 mRNA abundance. ALDH3B2 DNA and mRNA were sequenced. The expression was investigated using western blot. The identity of the protein was confirmed using mass spectrometry (MS). The predicted tertiary and quaternary structures, subcellular localization, and phosphorylation sites were assessed using bioinformatic analyses. All DNA and mRNA isolates contained the premature stop codon. In western blot analyses, bands corresponding to the mass of full-length protein were detected. MS analysis led to the identification of two unique peptides, one of which is encoded by the nucleotide sequence located upstream the second start codon. Bioinformatic analyses suggest cytoplasmic localization and several phosphorylation sites. Despite premature stop codon in DNA and mRNA sequences, full-length ALDH3B2 was found. It can be formed as a result of premature stop codon readthrough, complex phenomenon enabling stop codon circumvention.

scholarly journals ε-Tubulin Is an Essential Component of the Centriole

2002 ◽  
Vol 13 (11) ◽  
pp. 3859-3869 ◽  
Author(s):  
Susan K. Dutcher ◽  
Naomi S. Morrissette ◽  
Andrea M. Preble ◽  
Craig Rackley ◽  
John Stanga
Keyword(s):  
Basal Body ◽  
Positional Cloning ◽  
Stop Codon ◽  
Polyclonal Antibodies ◽  
Premature Stop Codon ◽  
Full Length ◽  
Basal Bodies ◽  
Cleavage Furrow ◽  
Essential Component ◽  
Flagellar Assembly

Centrioles and basal bodies are cylinders composed of nine triplet microtubule blades that play essential roles in the centrosome and in flagellar assembly. Chlamydomonas cells with thebld2-1 mutation fail to assemble doublet and triplet microtubules and have defects in cleavage furrow placement and meiosis. Using positional cloning, we have walked 720 kb and identified a 13.2-kb fragment that contains ε-tubulin and rescues the Bld2 defects. The bld2-1 allele has a premature stop codon and intragenic revertants replace the stop codon with glutamine, glutamate, or lysine. Polyclonal antibodies to ε-tubulin show peripheral labeling of full-length basal bodies and centrioles. Thus, ε-tubulin is encoded by the BLD2 allele and ε-tubulin plays a role in basal body/centriole morphogenesis.

scholarly journals Molecular Characterization of Mycoplasma arthritidis Membrane Lipoprotein MAA1

2000 ◽  
Vol 68 (2) ◽  
pp. 437-442 ◽  
Author(s):  
Leigh Rice Washburn ◽  
Elizabeth J. Miller ◽  
Keith E. Weaver
Keyword(s):  
Amino Acid ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Amino Acid Sequences ◽  
Signal Peptidase ◽  
Cleavage Sites ◽  
Mycoplasma Arthritidis ◽  
Genes Encoding ◽  
Amino Acid Signal

ABSTRACT Genes encoding the Mycoplasma arthritidissurface-exposed lipoprotein MAA1 were cloned and sequenced from MAA1-expressing strains 158p10p9 and PG6, from a low-adherence (LA) variant derived from 158p10p9 that expresses a truncated version of MAA1 (MAA1Δ) and from two MAA1-negative strains, 158 and H39. The deduced amino acid sequences of maa1 from 158p10p9 and PG6 predicted, respectively, 86.5- and 86.4-kDa basic, largely hydrophilic lipoproteins with 29-amino-acid signal peptides and predicted cleavage sites for signal peptidase II (Ala-Ala-Ala↓Cys). The truncation in the LA variant resulted from a G→T substitution at nucleotide 695, which created a premature stop codon. This, in turn, generated a predicted 26.6-kDa prolipoprotein (23.6 kDa after processing), consistent with an M r of ∼24,000 calculated for MAA1Δ. Similarly, absence of MAA1 expression in H39 and 158 resulted from C→A substitutions at nucleotide 208, generating premature stop codons at that site in both strains.

The B-hordein prolamin family of barley

Genome ◽  
2013 ◽  
Vol 56 (3) ◽  
pp. 179-185 ◽  
Author(s):  
Olin D. Anderson
Keyword(s):  
Stop Codon ◽  
Premature Stop Codon ◽  
In Silico Analysis ◽  
Gene Families ◽  
Full Length ◽  
C Terminus ◽  
Est Assembly ◽  
Silico Analysis ◽  
Total Ests ◽  
Relative Gene

The spectrum of B-hordein prolamins and genes in the single barley cultivar Barke is described from an in silico analysis of 1452 B-hordein ESTs and available genomic DNA. Eleven unique B-hordein proteins are derived from EST contigs. Ten contigs encode apparent full-length B-hordeins and the eleventh contains a premature stop codon that will lead to a truncated B-hordein. The 11 sequences are placed within the two previously described classes, i.e., the B1- and B3-type B-hordeins. The number of ESTs assigned to each sequence is used as an estimate of relative gene transcription and expression. Three of the sequences account for 79% of the total ESTs, with one sequence comprises 32% of the total ESTs and has a variant C-terminus caused by an undefined sequence change history near the 3′ coding terminus. The 70× difference in EST distribution among sequences points to the importance of understanding differential rates of expression within closely related gene families. Analysis of available genomic sequences confirms the EST assembly and reveals one full-length and two partial sequences of pseudogenes as evidenced by no matching ESTs for the sequences and premature stop codons and frame shifts.

scholarly journals Significant Shift in Median Guinea Pig Infectious Dose Shown by an Outbreak-AssociatedListeria monocytogenesEpidemic Clone Strain and a Strain Carrying a Premature Stop Codon Mutation ininlA

2011 ◽  
Vol 77 (7) ◽  
pp. 2479-2487 ◽  
Author(s):  
A. Van Stelten ◽  
J. M. Simpson ◽  
Y. Chen ◽  
V. N. Scott ◽  
R. C. Whiting ◽  
...  
Keyword(s):  
Dose Response ◽  
Guinea Pigs ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
The United States ◽  
Risk Assessments ◽  
Significant Shift ◽  
Infectious Dose ◽  
Internalin A ◽  
E Cadherin

ABSTRACTListeria monocytogenescontains (i) epidemic clone (EC) strains, which have been linked to the majority of listeriosis outbreaks worldwide and are overrepresented among sporadic cases in the United States, and (ii) strains commonly isolated from ready-to-eat foods that carry a mutation leading to a premature stop codon (PMSC) ininlA, which encodes the key virulence factor internalin A (InlA). Internalin A binds certain isoforms of the cellular receptor E-cadherin to facilitate crossing the intestinal barrier during the initial stages of anL. monocytogenesinfection. Juvenile guinea pigs, which express the human isoform of E-cadherin that binds InlA, were intragastrically challenged with a range of doses of (i) an EC strain associated with a listeriosis outbreak or (ii) a strain carrying a PMSC mutation ininlA. Recovery ofL. monocytogenesfrom tissues (i.e., liver, spleen, mesenteric lymph nodes, and ileum) was used to develop strain-specific dose-response curves on the basis of individual and combined organ data. Modeling of individual and combined organ data revealed an approximate 1.2 to 1.3 log10increase in the median infectious dose for the strain carrying a PMSC ininlArelative to that for the EC strain. Inclusion of the strain parameter significantly improved the goodness of fit for individual and combined organ models, indicating a significant shift in median infectious dose for guinea pigs challenged with aninlAPMSC strain compared to that for guinea pigs challenged with an EC strain. Results from this work provide evidence that theL. monocytogenesdose-response relationship is strain specific and will provide critical data for enhancement of current risk assessments and development of future risk assessments.

scholarly journals Targeted Gene Mutation in Phytophthora spp.

2006 ◽  
Vol 19 (12) ◽  
pp. 1359-1367 ◽  
Author(s):  
Kurt H. Lamour ◽  
Ledare Finley ◽  
Oscar Hurtado-Gonzales ◽  
Daniel Gobena ◽  
Melinda Tierney ◽  
...  
Keyword(s):  
Plant Pathogens ◽  
Genomic Library ◽  
Stop Codon ◽  
Point Mutations ◽  
Premature Stop Codon ◽  
Phytophthora Sojae ◽  
Phenotypic Effect ◽  
Phytophthora Spp ◽  
Homozygous Mutant ◽  
Genes Encoding

The genus Phytophthora belongs to the oomycetes and is composed of plant pathogens. Currently, there are no strategies to mutate specific genes for members of this genus. Whole genome sequences are available or being prepared for Phytophthora sojae, P. ramorum, P. infestans, and P. capsici and the development of molecular biological techniques for functional genomics is encouraged. This article describes the adaptation of the reverse-genetic strategy of targeting induced local lesions in genomes (TILLING) to isolate gene-specific mutants in Phytophthora spp. A genomic library of 2,400 ethylnitrosourea (ENU) mutants of P. sojae was created and screened for induced point mutations in the genes encoding a necrosis-inducing protein (PsojNIP) and a Phytophthora-specific phospholipase D (PsPXTM-PLD). Mutations were detected in single individuals and included silent, missense, and nonsense changes. Homozygous mutant isolates carrying a potentially deleterious missense mutation in PsojNIP and a premature stop codon in PsPXTM-PLD were identified. No phenotypic effect has yet been found for the homozygous mutant of PsojNIP. For those of PsPXTM-PLD, a reduction in growth rate and an appressed mycelial growth was observed. This demonstrates the feasibility of target-selected gene disruption for Phytophthora spp. and adds an important tool for functional genomic investigation.

scholarly journals G418-mediated ribosomal read-through of a nonsense mutation causing autosomal recessive proximal renal tubular acidosis

2008 ◽  
Vol 295 (3) ◽  
pp. F633-F641 ◽  
Author(s):  
Rustam Azimov ◽  
Natalia Abuladze ◽  
Pakan Sassani ◽  
Debra Newman ◽  
Liyo Kao ◽  
...  
Keyword(s):  
Renal Tubular Acidosis ◽  
Autosomal Recessive ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Immunoblot Analysis ◽  
Full Length ◽  
Aminoglycoside Resistance ◽  
Proximal Renal Tubular Acidosis ◽  
Renal Tubular

Autosomal recessive proximal renal tubular acidosis is caused by mutations in the SLC4A4 gene encoding the electrogenic sodium bicarbonate cotransporter NBCe1-A. The mutations that have been characterized thus far result in premature truncation, mistargeting, or decreased function of the cotransporter. Despite bicarbonate treatment to correct the metabolic acidosis, extrarenal manifestations persist, including glaucoma, cataracts, corneal opacification, and mental retardation. Currently, there are no known therapeutic approaches that can specifically target mutant NBCe1-A proteins. In the present study, we tested the hypothesis that the NBCe1-A-Q29X mutation can be rescued in vitro by treatment with aminoglycoside antibiotics, which are known for their ability to suppress premature stop codons. As a model system, we cloned the NBCe1-A-Q29X mutant into a vector lacking an aminoglycoside resistance gene and transfected the mutant cotransporter in HEK293-H cells. Cells transfected with the NBCe1-A-Q29X mutant failed to express the cotransporter because of the premature stop codon. Treatment of the cells with G418 significantly increased the expression of the full-length cotransporter, as assessed by immunoblot analysis. Furthermore, immunocytochemical studies demonstrated that G418 treatment induced cotransporter expression on the plasma membrane whereas in the absence of G418, NBCe1-A-Q29X was not expressed. In HEK293-H cells transfected with the NBCe1-A-Q29X mutant not treated with G418, NBCe1-A-mediated flux was not detectable. In contrast, in cells transfected with the NBCe1-A-Q29X mutant, G418 treatment induced Na+- and HCO3−-dependent transport that did not differ from wild-type NBCe1-A function. G418 treatment in mock-transfected cells was without effect. In conclusion, G418 induces ribosomal read-through of the NBCe1-A-Q29X mutation in HEK293-H cells. These findings represent the first evidence that in the presence of the NBCe1-A-Q29X mutation that causes proximal renal tubular acidosis, full-length functional NBCe1-A protein can be produced. Our results provide the first demonstration of a mutation in NBCe1-A that has been treated in a targeted and specific manner.

Listeria monocytogenes Isolates Carrying Virulence-Attenuating Mutations in Internalin A Are Commonly Isolated from Ready-to-Eat Food Processing Plant and Retail Environments

2016 ◽  
Vol 79 (10) ◽  
pp. 1733-1740 ◽  
Author(s):  
A. VAN STELTEN ◽  
A. R. ROBERTS ◽  
C. S. MANUEL ◽  
K. K. NIGHTINGALE
Keyword(s):  
Listeria Monocytogenes ◽  
Food Processing ◽  
Food Production ◽  
Stop Codon ◽  
Significant Proportion ◽  
Premature Stop Codon ◽  
Processing Plant ◽  
Contact Surfaces ◽  
Internalin A ◽  
Food Processing Plant

ABSTRACT Listeria monocytogenes is a human foodborne pathogen that may cause an invasive disease known as listeriosis in susceptible individuals. Internalin A (InlA; encoded by inlA) is a virulence factor that facilitates crossing of host cell barriers by L. monocytogenes. At least 19 single nucleotide polymorphisms (SNPs) in inlA that result in a premature stop codon (PMSC) have been described worldwide. SNPs leading to a PMSC in inlA have been shown to be causally associated with attenuated virulence. L. monocytogenes pathogens carrying virulence-attenuating (VA) mutations in inlA have been commonly isolated from ready-to-eat (RTE) foods but rarely have been associated with human disease. This study was conducted to determine the prevalence of VA SNPs in inlA among L. monocytogenes from environments associated with RTE food production and handling. More than 700 L. monocytogenes isolates from RTE food processing plant (n = 409) and retail (n = 319) environments were screened for the presence of VA SNPs in inlA. Overall, 26.4% of isolates from RTE food processing plant and 32.6% of isolates from retail environments carried a VA mutation in inlA. Food contact surfaces sampled at retail establishments were significantly (P < 0.0001) more likely to be contaminated by a L. monocytogenes isolate carrying a VA mutation in inlA (56% of 55 isolates) compared with nonfood contact surfaces (28% of 264 isolates). Overall, a significant proportion of L. monocytogenes isolated from RTE food production and handling environments have reduced virulence. These data will be useful in the revision of current and the development of future risk assessments that incorporate strain-specific virulence parameters.

scholarly journals devIIs an Evolutionarily Young Negative Regulator of Myxococcus xanthus Development

2015 ◽  
Vol 197 (7) ◽  
pp. 1249-1262 ◽  
Author(s):  
Ramya Rajagopalan ◽  
Sébastien Wielgoss ◽  
Gerardo Lippert ◽  
Gregory J. Velicer ◽  
Lee Kroos
Keyword(s):  
Reference Strain ◽  
Stop Codon ◽  
Myxococcus Xanthus ◽  
Premature Stop Codon ◽  
Protein Product ◽  
Negative Regulator ◽  
Content Type ◽  
Genes Encoding ◽  
Natural Isolates ◽  
Cas Proteins

ABSTRACTDuring starvation-induced development ofMyxococcus xanthus, thousands of rod-shaped cells form mounds in which they differentiate into spores. Thedevlocus includes eight genes followed by clustered regularly interspaced short palindromic repeats (CRISPRs), comprising a CRISPR-Cas system (Cas stands for CRISPR associated) typically involved in RNA interference. Mutations indevSordevRof a lab reference strain permit mound formation but impair sporulation. We report that natural isolates ofM. xanthuscapable of normal development are highly polymorphic in the promoter region of thedevoperon. We show that thedevpromoter is predicted to be nonfunctional in most natural isolates and is dispensable for development of a laboratory reference strain. Moreover, deletion of thedevpromoter or the small gene immediately downstream of it, here designateddevI(developmentinhibitor), suppressed the sporulation defect ofdevSordevRmutants in the lab strain. Complementation experiments and the result of introducing a premature stop codon indevIsupport a model in which DevRS proteins negatively autoregulate expression ofdevI, whose 40-residue protein product DevI inhibits sporulation if overexpressed. DevI appears to act in a cell-autonomous manner since experiments with conditioned medium and with cell mixtures gave no indication of extracellular effects. Strikingly, we report thatdevIis entirely absent from mostM. xanthusnatural isolates and was only recently integrated into the developmental programs of some lineages. These results provide important new insights into both the evolutionary history of thedevoperon and its mechanistic role inM. xanthussporulation.IMPORTANCECertain mutations in thedevCRISPR-Cas (clustered regularly interspaced short palindromic repeat-associated) system ofMyxococcus xanthusimpair sporulation. The link between development and a CRISPR-Cas system has been a mystery. Surprisingly, DNA sequencing of natural isolates revealed that many appear to lack a functionaldevpromoter, yet these strains sporulate normally. Deletion of thedevpromoter or the small gene downstream of it suppressed the sporulation defect of a lab strain with mutations indevgenes encoding Cas proteins. The results support a model in which the Cas proteins DevRS prevent overexpression of the small genedevI, which codes for an inhibitor of sporulation. Phylogenetic analysis of natural isolates suggests thatdevIand thedevpromoter were only recently acquired in some lineages.

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