scholarly journals Antibiotic resistance by high-level intrinsic suppression of a frameshift mutation in an essential gene

2020 ◽  
Vol 117 (6) ◽  
pp. 3185-3191 ◽  
Author(s):  
Douglas L. Huseby ◽  
Gerrit Brandis ◽  
Lisa Praski Alzrigat ◽  
Diarmaid Hughes
Keyword(s):  
Antibiotic Resistance ◽  
Messenger Rna ◽  
Frameshift Mutation ◽  
De Novo ◽  
Essential Gene ◽  
Feedback Mechanism ◽  
Essential Genes ◽  
High Rate ◽  
Reading Frame ◽  
Frameshift Mutations

A fundamental feature of life is that ribosomes read the genetic code in messenger RNA (mRNA) as triplets of nucleotides in a single reading frame. Mutations that shift the reading frame generally cause gene inactivation and in essential genes cause loss of viability. Here we report and characterize a +1-nt frameshift mutation, centrally located in rpoB, an essential gene encoding the beta-subunit of RNA polymerase. Mutant Escherichia coli carrying this mutation are viable and highly resistant to rifampicin. Genetic and proteomic experiments reveal a very high rate (5%) of spontaneous frameshift suppression occurring on a heptanucleotide sequence downstream of the mutation. Production of active protein is stimulated to 61–71% of wild-type level by a feedback mechanism increasing translation initiation. The phenomenon described here could have broad significance for predictions of phenotype from genotype. Several frameshift mutations have been reported in rpoB in rifampicin-resistant clinical isolates of Mycobacterium tuberculosis (Mtb). These mutations have never been experimentally validated, and no mechanisms of action have been proposed. This work shows that frameshift mutations in rpoB can be a mutational mechanism generating antibiotic resistance. Our analysis further suggests that genetic elements supporting productive frameshifting could rapidly evolve de novo, even in essential genes.

A novel way to purify recombinant baculoviruses by using bacmid

2009 ◽  
Vol 29 (2) ◽  
pp. 71-75 ◽  
Author(s):  
Wu-Jie Su ◽  
Wei-De Shen ◽  
Bing Li ◽  
Yan Wu ◽  
Guang Gao ◽  
...  
Keyword(s):  
Insect Cells ◽  
Nuclear Polyhedrosis Virus ◽  
Essential Gene ◽  
Essential Genes ◽  
Reading Frame ◽  
Recombinant Viruses ◽  
Recombinant Bacmid ◽  
Bmnpv Bacmid ◽  
Nuclear Polyhedrosis ◽  
Recombinant Bacmids

In the present study, we studied the feasibility of deleting essential genes in insect cells by using bacmid and purifying recombinant bacmid in Escherichia coli DH10B cells. To disrupt the orf4 (open reading frame 4) gene of BmNPV [Bm (Bombyx mori) nuclear polyhedrosis virus], a transfer vector was constructed and co-transfected with BmNPV bacmid into Bm cells. Three passages of viruses were carried out in Bm cells, followed by one round of purification. Subsequently, bacmid DNA was extracted and transformed into competent DH10B cells. A colony harbouring only orf4-disrupted bacmid DNA was identified by PCR. A mixture of recombinant (white colonies) and non-recombinant (blue colonies) bacmids were also transformed into DH10B cells. PCR with M13 primers showed that the recombinant and non-recombinant bacmids were separated after transformation. The result confirmed that purification of recombinant viruses could be carried out simply by transformation and indicated that this method could be used to delete essential genes. Orf4-disrupted bacmid DNA was extracted and transfected into Bm cells. Viable viruses were produced, showing that orf4 was not an essential gene.

scholarly journals Silencing of Essential Genes within a Highly Coordinated Operon in Escherichia coli

2015 ◽  
Vol 81 (16) ◽  
pp. 5650-5659 ◽  
Author(s):  
Shan Goh ◽  
Angela Hohmeier ◽  
Timothy C. Stone ◽  
Victoria Offord ◽  
Francisco Sarabia ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Target Gene ◽  
Essential Gene ◽  
Transcript Level ◽  
Essential Genes ◽  
Content Type ◽  
Reading Frame ◽  
Knockout Mutants ◽  
Bacterial Genes ◽  
Relationship Of

ABSTRACTEssential bacterial genes located within operons are particularly challenging to study independently because of coordinated gene expression and the nonviability of knockout mutants. Essentiality scores for many operon genes remain uncertain. Antisense RNA (asRNA) silencing or in-frame gene disruption of genes may help establish essentiality but can lead to polar effects on genes downstream or upstream of the target gene. Here, theEscherichia coliribF-ileS-lspA-fkpB-ispHoperon was used to evaluate the possibility of independently studying an essential gene using expressed asRNA and target gene overexpression to deregulate coupled expression. The gene requirement for growth in conditional silencing strains was determined by the relationship of target mRNA reduction with growth inhibition as the minimum transcript level required for 50% growth (MTL50). Mupirocin and globomycin, the protein inhibitors of IleS and LspA, respectively, were used in sensitization assays of strains containing both asRNA-expressing and open reading frame-expressing plasmids to examine deregulation of the overlappingileS-lspAgenes. We found upstream and downstream polar silencing effects when eitherileSorlspAwas silenced, indicating coupled expression. Weighted MTL50values (means and standard deviations) ofribF,ileS, andlspAwere 0.65 ± 0.18, 0.64 ± 0.06, and 0.76 ± 0.10, respectively. However, they were not significantly different (P= 0.71 by weighted one-way analysis of variance). The gene requirement forispHcould not be determined due to insufficient growth reduction. Mupirocin and globomycin sensitization experiments indicated thatileS-lspAexpression could not be decoupled. The results highlight the inherent challenges associated with genetic analyses of operons; however, coupling of essential genes may provide opportunities to improve RNA-silencing antimicrobials.

scholarly journals The energy landscape of −1 ribosomal frameshifting

2020 ◽  
Vol 6 (1) ◽  
pp. eaax6969 ◽  
Author(s):  
Junhong Choi ◽  
Sinéad O’Loughlin ◽  
John F. Atkins ◽  
Joseph D. Puglisi
Keyword(s):  
Single Molecule ◽  
Messenger Rna ◽  
Information Transfer ◽  
Mechanistic Model ◽  
High Rate ◽  
Coding Region ◽  
Ribosomal Frameshifting ◽  
Reading Frame

Maintenance of translational reading frame ensures the fidelity of information transfer during protein synthesis. Yet, programmed ribosomal frameshifting sequences within the coding region promote a high rate of reading frame change at predetermined sites thus enriching genomic information density. Frameshifting is typically stimulated by the presence of 3′ messenger RNA (mRNA) structures, but how these mRNA structures enhance −1 frameshifting remains debatable. Here, we apply single-molecule and ensemble approaches to formulate a mechanistic model of ribosomal −1 frameshifting. Our model suggests that the ribosome is intrinsically susceptible to frameshift before its translocation and this transient state is prolonged by the presence of a precisely positioned downstream mRNA structure. We challenged this model using temperature variation in vivo, which followed the prediction made based on in vitro results. Our results provide a quantitative framework for analyzing other frameshifting enhancers and a potential approach to control gene expression dynamically using programmed frameshifting.

scholarly journals An insertional frameshift mutation of the beta-spectrin gene associated with elliptocytosis in spectrin nice (beta 220/216)

Blood ◽  
1991 ◽  
Vol 78 (2) ◽  
pp. 517-523 ◽  
Author(s):  
WT Tse ◽  
PG Gallagher ◽  
B Pothier ◽  
FF Costa ◽  
A Scarpa ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Frameshift Mutation ◽  
De Novo ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
De Novo Mutation ◽  
Molecular Defect ◽  
Beta Chain ◽  
Alpha Chain ◽  
Oligonucleotide Hybridization

Abstract Spectrin Nice (beta 220/216) is a spectrin variant associated with a shortened beta chain found in a patient with elliptocytosis. The shortened beta chain (beta' chain) appeared as an additional band of approximately 216 Kd on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was defective in its ability to be phosphorylated. There were increased amounts of spectrin dimers in crude spectrin extracts from the propositus and the association constant of spectrin dimer self-association was decreased. There was an associated increase of the alpha I 74-Kd fragment from the alpha chain after partial trypic digestion of spectrin. To identify the underlying molecular defect, we analyzed cDNA for beta spectrin obtained by polymerase chain reaction amplification of reverse-transcribed reticulocyte messenger RNA from peripheral blood of the propositus. DNA sequencing of individual as well as pooled subclones showed that two extra bases (GA) are inserted in codon no. 2046 in one allele of the beta-spectrin gene. The insertion results in a frameshift mutation and generates an aberrant C- terminus truncated by about 4 Kd, consistent with the estimated size of the beta' chain observed. By allele-specific oligonucleotide hybridization, the insertion was shown to be present in the propositus and absent in his parents, confirming a previous proposal that it is a de novo mutation. The determination of the location of the mutation in spectrin Nice points to specific regions of the beta-spectrin chain where phosphorylation may occur. A model is proposed to describe the interaction between the alpha- and beta-spectrin chains and to explain the effects of the mutation found in spectrin Nice on the trypsin digestion pattern of its associated alpha chain.

scholarly journals Correction of Frameshift Mutations in the atpB Gene by Translational Recoding in Chloroplasts of Oenothera and Tobacco

2021 ◽  
Author(s):  
Irina Malinova ◽  
Arkadiusz Zupok ◽  
Amid Massouh ◽  
Mark Aurel Schöttler ◽  
Etienne H Meyer ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Coding Region ◽  
Functional Protein ◽  
Ribosomal Frameshifting ◽  
Photoautotrophic Growth ◽  
Reading Frame ◽  
Evening Primrose ◽  
Frameshift Mutations ◽  
Transplastomic Tobacco ◽  
Albino Phenotype

Abstract Translational recoding, also known as ribosomal frameshifting, is a process that causes ribosome slippage along the messenger RNA, thereby changing the amino acid sequence of the synthesized protein. Whether the chloroplast employs recoding is unknown. I-iota, a plastome mutant of Oenothera (evening primrose), carries a single adenine insertion in an oligoA stretch [11A] of the atpB coding region (encoding a β-subunit of the ATP synthase). The mutation is expected to cause synthesis of a truncated, non-functional protein. We report that a full-length AtpB protein is detectable in I-iota leaves, suggesting operation of a recoding mechanism. To characterize the phenomenon, we generated transplastomic tobacco lines in which the atpB reading frame was altered by insertions or deletions in the oligoA motif. We observed that insertion of two adenines was more efficiently corrected than insertion of a single adenine, or deletion of one or two adenines. We further show that homopolymeric composition of the oligoA stretch is essential for recoding, as an additional replacement of AAA lysine codon by AAG resulted in an albino phenotype. Our work provides evidence for the operation of translational recoding in chloroplasts. Recoding enables correction of frameshift mutations and can restore photoautotrophic growth in the presence of mutation that otherwise would be lethal.

scholarly journals An insertional frameshift mutation of the beta-spectrin gene associated with elliptocytosis in spectrin nice (beta 220/216)

Blood ◽  
1991 ◽  
Vol 78 (2) ◽  
pp. 517-523
Author(s):  
WT Tse ◽  
PG Gallagher ◽  
B Pothier ◽  
FF Costa ◽  
A Scarpa ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Frameshift Mutation ◽  
De Novo ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
De Novo Mutation ◽  
Molecular Defect ◽  
Beta Chain ◽  
Alpha Chain ◽  
Oligonucleotide Hybridization

Spectrin Nice (beta 220/216) is a spectrin variant associated with a shortened beta chain found in a patient with elliptocytosis. The shortened beta chain (beta' chain) appeared as an additional band of approximately 216 Kd on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was defective in its ability to be phosphorylated. There were increased amounts of spectrin dimers in crude spectrin extracts from the propositus and the association constant of spectrin dimer self-association was decreased. There was an associated increase of the alpha I 74-Kd fragment from the alpha chain after partial trypic digestion of spectrin. To identify the underlying molecular defect, we analyzed cDNA for beta spectrin obtained by polymerase chain reaction amplification of reverse-transcribed reticulocyte messenger RNA from peripheral blood of the propositus. DNA sequencing of individual as well as pooled subclones showed that two extra bases (GA) are inserted in codon no. 2046 in one allele of the beta-spectrin gene. The insertion results in a frameshift mutation and generates an aberrant C- terminus truncated by about 4 Kd, consistent with the estimated size of the beta' chain observed. By allele-specific oligonucleotide hybridization, the insertion was shown to be present in the propositus and absent in his parents, confirming a previous proposal that it is a de novo mutation. The determination of the location of the mutation in spectrin Nice points to specific regions of the beta-spectrin chain where phosphorylation may occur. A model is proposed to describe the interaction between the alpha- and beta-spectrin chains and to explain the effects of the mutation found in spectrin Nice on the trypsin digestion pattern of its associated alpha chain.

scholarly journals Unusually efficient CUG initiation of an overlapping reading frame in POLG mRNA yields novel protein POLGARF

2020 ◽  
Vol 117 (40) ◽  
pp. 24936-24946 ◽  
Author(s):  
Gary Loughran ◽  
Alexander V. Zhdanov ◽  
Maria S. Mikhaylova ◽  
Fedor N. Rozov ◽  
Petr N. Datskevich ◽  
...  
Keyword(s):  
Messenger Rna ◽  
De Novo ◽  
Gene Evolution ◽  
Protein Coding ◽  
Reading Frame ◽  
Serum Stimulation ◽  
Extracellular Signaling ◽  
Functional Investigation ◽  
Mitochondrial Dna Polymerase ◽  
Orf 5

While near-cognate codons are frequently used for translation initiation in eukaryotes, their efficiencies are usually low (<10% compared to an AUG in optimal context). Here, we describe a rare case of highly efficient near-cognate initiation. A CUG triplet located in the 5′ leader of POLG messenger RNA (mRNA) initiates almost as efficiently (∼60 to 70%) as an AUG in optimal context. This CUG directs translation of a conserved 260-triplet-long overlapping open reading frame (ORF), which we call POLGARF (POLG Alternative Reading Frame). Translation of a short upstream ORF 5′ of this CUG governs the ratio between POLG (the catalytic subunit of mitochondrial DNA polymerase) and POLGARF synthesized from a single POLG mRNA. Functional investigation of POLGARF suggests a role in extracellular signaling. While unprocessed POLGARF localizes to the nucleoli together with its interacting partner C1QBP, serum stimulation results in rapid cleavage and secretion of a POLGARF C-terminal fragment. Phylogenetic analysis shows that POLGARF evolved ∼160 million y ago due to a mammalian-wide interspersed repeat (MIR) transposition into the 5′ leader sequence of the mammalian POLG gene, which became fixed in placental mammals. This discovery of POLGARF unveils a previously undescribed mechanism of de novo protein-coding gene evolution.

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