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G3 (Bethesda, Md.) Aug 2020The mutation rate and mutations' effects on fitness are crucial to evolution. Mutation rates are under selection due to linkage between mutation rate modifiers and...
The mutation rate and mutations' effects on fitness are crucial to evolution. Mutation rates are under selection due to linkage between mutation rate modifiers and mutations' effects on fitness. The linkage between a higher mutation rate and more beneficial mutations selects for higher mutation rates, while the linkage between a higher mutation rate and more deleterious mutations selects for lower mutation rates. The net direction of selection on mutations rates depends on the fitness landscape, and a great deal of work has elucidated the fitness landscapes of mutations. However, tests of the effect of varying a mutation rate on evolution in a single organism in a single environment have been difficult. This has been studied using strains of antimutators and mutators, but these strains may differ in additional ways and typically do not allow for continuous variation of the mutation rate. To help investigate the effects of the mutation rate on evolution, we have genetically engineered a strain of with a point mutation rate that can be smoothly varied over two orders of magnitude. We did this by engineering a strain with inducible control of the mismatch repair proteins MutH and MutL. We used this strain in an approximately 350 generation evolution experiment with controlled variation of the mutation rate. We confirmed the construct and the mutation rate were stable over this time. Sequencing evolved strains revealed a higher number of single nucleotide polymorphisms at higher mutations rates, likely due to either the beneficial effects of these mutations or their linkage to beneficial mutations.
Topics: Escherichia coli; Escherichia coli Proteins; Mutation; Mutation Rate; Point Mutation; Selection, Genetic
PubMed: 32503807
DOI: 10.1534/g3.120.401124 -
Methods in Molecular Biology (Clifton,... 2022Genetic manipulation of microbial genomes is highly relevant for studying biological systems and the development of biotechnologies. In E. coli, λ-Red recombineering is...
Genetic manipulation of microbial genomes is highly relevant for studying biological systems and the development of biotechnologies. In E. coli, λ-Red recombineering is one of the most widely used gene-editing methods, enabling site-specific insertions, deletions, and point mutations of any genomic locus. The no-SCAR system combines λ-Red recombineering with CRISPR/Cas9 for programmable selection of recombinant cells. Recombineering results in the transient production of heteroduplex DNA, as only one strand of DNA is initially altered, leaving the mismatched bases susceptible to repair by the host methyl-directed mismatch repair (MMR) system and reduces the efficiency of generating single nucleotide point mutations. Here we describe a method, where expression of cas9 and the MMR-inhibiting mutL variant are independently controlled by anhydrotetracycline- and cumate-inducible promoters from the pCas9CyMutL plasmid. Thus, MMR is selectively inhibited until recombinant cells have undergone replication and the desired mutation is permanently incorporated. By transiently inhibiting MMR, the accumulation of off-target mutations typically associated with MMR-deficient cell types is minimized. Methods for designing the editing template and sgRNA, cloning of the sgRNA, induction of λ-Red and MutL, the transformation of editing oligo, and induction of Cas9 for mutant selection are detailed within.
Topics: CRISPR-Cas Systems; Escherichia coli; Gene Editing; Nucleotides; Point Mutation
PubMed: 35583736
DOI: 10.1007/978-1-0716-2233-9_9 -
Medecine Sciences : M/S Oct 2022An ingenious system for generating thousands of point mutations in yeast genes and measuring their effect on fitness shows convincingly that, for the chosen subset of...
An ingenious system for generating thousands of point mutations in yeast genes and measuring their effect on fitness shows convincingly that, for the chosen subset of representative non-essential genes, silent mutations have as much effect on fitness as missense mutations. In other words, silent mutations are not neutral, at least under these conditions. This result has important implications for evolutionary biology.
Topics: Biological Evolution; Humans; Mutation; Mutation, Missense; Point Mutation
PubMed: 36219087
DOI: 10.1051/medsci/2022126 -
Methods in Molecular Biology (Clifton,... 2020The clustered regularly interspersed short palindromic repeat (CRISPR)/Cas9 system has emerged as an efficient genome engineering method attributed to its high...
The clustered regularly interspersed short palindromic repeat (CRISPR)/Cas9 system has emerged as an efficient genome engineering method attributed to its high efficiency and versatility. By generating a lethal double-strand DNA break in the target genome, the CRISPR/Cas9 system is capable of selecting the separated crossover events occurring in the traditional genetic manipulation methods in one step, therefore enabling rapid and efficient genome editing in Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA). By engineering the fusion of a cytidine deaminase APOBEC1 and a Cas9 nickase, a base editor was further developed as a highly efficient gene inactivation and point mutation tool in S. aureus. Here we describe a detailed protocol for CRISPR/Cas9-based genome editing in S. aureus, including genome modification and base editing. This protocol outlines in detail the design of primers, the construction and transformation of editing plasmids, as well as the verification of sequence-specific CRISPR/Cas9-mediated mutagenesis in S. aureus.
Topics: CRISPR-Cas Systems; Gene Editing; Methicillin-Resistant Staphylococcus aureus; Mutagenesis; Plasmids; Point Mutation
PubMed: 31523770
DOI: 10.1007/978-1-4939-9849-4_9 -
Journal of Integrative Plant Biology Dec 2019Calcineurin B-like interacting protein kinases (CIPKs) play important roles via environmental stress. However, less is known how to sense the stress in molecular...
Calcineurin B-like interacting protein kinases (CIPKs) play important roles via environmental stress. However, less is known how to sense the stress in molecular structure conformation level. Here, an OsCIPK7 mutant via TILLING procedure with a point mutation in the kinase domain showed increased chilling tolerance, which could be potentially used in the molecular breeding. We found that this point mutation of OsCIPK7 led to a conformational change in the activation loop of the kinase domain, subsequently with an increase of protein kinase activity, thus conferred an increased tolerance to chilling stress.
Topics: Adaptation, Physiological; Amino Acid Sequence; Base Sequence; Cold Temperature; Oryza; Plant Proteins; Point Mutation; Protein Conformation; Protein Kinases; Spectroscopy, Fourier Transform Infrared
PubMed: 30912264
DOI: 10.1111/jipb.12800 -
Progress in Molecular Biology and... 2017Prion diseases, or transmissible spongiform encephalopathies, are a group of rare fatal neurodegenerative maladies that affect humans and animals. The main event in... (Review)
Review
Prion diseases, or transmissible spongiform encephalopathies, are a group of rare fatal neurodegenerative maladies that affect humans and animals. The main event in disease progression is the posttranslational conversion of the ubiquitously expressed cellular form of the prion protein (PrP) into its misfolded and pathogenic isoform, known as prion or PrP. In the presence of specific disease-linked mutations, the conversion may occur spontaneously. Molecular simulation studies on human PrP wild-type and variants from several research groups, including ours, have provided a consistent picture of the effect of such mutations. In particular, the calculations have pinpointed "hot spots" for the conversion across several disease-linked variants. They have also identified a region of the protein containing two helices (Helix 2 and Helix 3) as a key structural element most prone to the conversion, consistently with a wealth of experimental data. Some of these findings are summarized here.
Topics: Amino Acid Sequence; Humans; Models, Molecular; Mutant Proteins; Point Mutation; Prion Proteins; Protein Domains
PubMed: 28838657
DOI: 10.1016/bs.pmbts.2017.07.001 -
Journal of Agricultural and Food... Jan 2024Mefentrifluconazole, a triazole fungicide, exhibits remarkable efficacy in combating spp. The mean EC value of mefentrifluconazole against 124 isolates of was...
Mefentrifluconazole, a triazole fungicide, exhibits remarkable efficacy in combating spp. The mean EC value of mefentrifluconazole against 124 isolates of was determined to be 1.06 μg/mL in this study. Fungicide taming produced five mefentrifluconazole-resistant mutants with resistance factors ranging from 19.21 to 111.34. Compared to the original parental isolates, the fitness of three resistant mutants was much lower, while the remaining two mutants displayed enhanced survival fitness. There was evidence of positive cross-resistance between tebuconazole and mefentrifluconazole. Mefentrifluconazole resistance in can be conferred by FpCYP51B, which was identified in four mutants according to molecular docking and site-directed transformation experiments. Overexpression of was also detected in the resistant mutants. In conclusion, mefentrifluconazole has a low-to-medium resistance risk in , and the L144F mutation in FpCYP51B and the increased expression level of may be responsible for mefentrifluconazole resistance in .
Topics: Fusarium; Point Mutation; Fungicides, Industrial; Molecular Docking Simulation; Plant Diseases; Fluconazole
PubMed: 38194482
DOI: 10.1021/acs.jafc.3c08014 -
Journal of Bone and Mineral Research :... Oct 2017Mitochondrial dysfunction is associated with several clinical manifestations including diabetes mellitus (DM), neurological disorders, renal and hepatic diseases, and...
Mitochondrial dysfunction is associated with several clinical manifestations including diabetes mellitus (DM), neurological disorders, renal and hepatic diseases, and myopathy. Although mitochondrial dysfunction is associated with increased bone resorption and decreased bone formation in mouse models, effects of alterations in mitochondrial function on bone remodeling and mass have not been investigated in humans. We recruited 45 carriers (29 females, 16 males) with the m.3243A>G mutation and healthy controls matched for gender, age, height, and menopausal status. DXA and HRpQCT scans were performed, and bone turnover markers (BTMs) P1NP and CTX were measured. Cases and controls were well matched except for body weight, which was lower in cases (63.6 ± 18.1 kg versus 74.6 ± 14.8 kg, p < 0.01), and manifest DM was present in 25 of 45 cases (none in controls). Bone scans showed lower BMD at the lumbar spine, total hip, and femoral neck in cases. Mean lumbar spine, total hip, and femoral neck T-scores were -1.5, -1.3, and -1.6 in cases, respectively, and -0.8, -0.3, and -0.7 in controls (all p < 0.05). The m.3243A>G mutation was associated with lower BMD, cortical but not trabecular density, cortical thickness, and estimated bone strength. Furthermore, BTMs were lower in the m.3243A>G group before but not after adjustment for DM. The mitochondrial point mutation m.3243A>G was associated with decreased bone mass and strength. Although the coexistence of DM may have influenced bone turnover, the bone phenotype observed in m.3243A>G cases appeared to mirror age-related deterioration in bone, suggesting that mitochondrial dysfunction may cause a premature aging of bone. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.
Topics: Absorptiometry, Photon; Biomarkers; Biomechanical Phenomena; Bone Density; Bone Remodeling; Case-Control Studies; Cortical Bone; Diabetes Mellitus; Female; Humans; Male; Middle Aged; Mitochondria; Point Mutation; Tomography, X-Ray Computed
PubMed: 28603900
DOI: 10.1002/jbmr.3193 -
Viruses Jan 2017A point mutation in the DNA polymerase gene in equine herpesvirus type 1 (EHV-1) is one determinant for the development of neurological disease in horses. Three recently...
A point mutation in the DNA polymerase gene in equine herpesvirus type 1 (EHV-1) is one determinant for the development of neurological disease in horses. Three recently conducted infection experiments using domestic horses and ponies failed to detect statistically significant differences in viral shedding between the neuropathogenic and non-neuropathogenic variants. These results were interpreted as suggesting the absence of a consistent selective advantage of the neuropathogenic variant and therefore appeared to be inconsistent with a systematic increase in the prevalence of neuropathogenic strains. To overcome potential problems of low statistical power related to small group sizes in these infection experiments, we integrated raw data from all three experiments into a single statistical analysis. The results of this combined analysis showed that infection with the neuropathogenic EHV-1 variant led to a statistically significant increase in viral shedding. This finding is consistent with the idea that neuropathogenic strains could have a selective advantage and are therefore systematically increasing in prevalence in domestic horse populations. However, further studies are required to determine whether a selective advantage indeed exists for neuropathogenic strains.
Topics: Animals; Herpesvirus 1, Equid; Horses; Point Mutation; Virulence; Virus Shedding
PubMed: 28075374
DOI: 10.3390/v9010006 -
Methods in Molecular Biology (Clifton,... 2020The rational design of enzymes is a challenging research field, which plays an important role in the optimization of a wide series of biotechnological processes....
The rational design of enzymes is a challenging research field, which plays an important role in the optimization of a wide series of biotechnological processes. Computational approaches allow screening all possible amino acid substitutions in a target protein and to identify a subset likely to have the desired properties. They can thus be used to guide and restrict the huge, time-consuming search in sequence space to reach protein optimality. Here we present HoTMuSiC, a tool that predicts the impact of point mutations on the protein melting temperature, which uses the experimental or modeled protein structure as sole input and is available at the dezyme.com website. Its main advantages include accuracy and speed, which makes it a perfect instrument for thermal stability engineering projects aiming at designing new proteins that feature increased heat resistance or remain active and stable in nonphysiological conditions. We set up a HoTMuSiC-based pipeline, which uses additional information to avoid mutations of functionally important residues, identified as being too well conserved among homologous proteins or too close to annotated functional sites. The efficiency of this pipeline is successfully demonstrated on Rhizomucor miehei lipase.
Topics: Amino Acid Substitution; Enzyme Stability; Hot Temperature; Lipase; Point Mutation; Protein Engineering; Protein Stability; Proteins; Rhizomucor; Temperature
PubMed: 32006278
DOI: 10.1007/978-1-0716-0270-6_5