-
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 -
The Journal of Biological Chemistry Jul 2021Molnupiravir, a prodrug of the nucleoside derivative β-D-N-hydroxycytidine (NHC), is currently in clinical trials for COVID-19 therapy. However, the biochemical...
Molnupiravir, a prodrug of the nucleoside derivative β-D-N-hydroxycytidine (NHC), is currently in clinical trials for COVID-19 therapy. However, the biochemical mechanisms involved in molnupiravir-induced mutagenesis had not been explored. In a recent study, Gordon et al. demonstrated that NHC can be incorporated into viral RNA and subsequently extended and used as template for RNA-dependent RNA synthesis, proposing a mutagenesis model consistent with available virological evidence. Their study uncovers molecular mechanisms by which molnupiravir drives SARS-CoV-2 into error catastrophe.
Topics: Antiviral Agents; COVID-19; Cytidine; Humans; Hydroxylamines; Point Mutation; RNA, Viral; SARS-CoV-2
PubMed: 34118236
DOI: 10.1016/j.jbc.2021.100867 -
Gan To Kagaku Ryoho. Cancer &... May 2021Epidermal growth factor receptor(EGFR)is a transmembrane receptor tyrosine kinase the signaling of which is important for growth and progression of cancer. An exon 19... (Review)
Review
Epidermal growth factor receptor(EGFR)is a transmembrane receptor tyrosine kinase the signaling of which is important for growth and progression of cancer. An exon 19 deletion mutation and an exon 21 L858R point mutation are frequently detected as EGFR mutations in patients with non‒small cell lung cancer. This review summarizes the differences in epidemiological, nonclinical, and clinical characteristics between the exon 19 deletion mutation and the exon 21 L858R point mutation.
Topics: Carcinoma, Non-Small-Cell Lung; ErbB Receptors; Exons; Humans; Lung Neoplasms; Mutation; Point Mutation; Protein Kinase Inhibitors
PubMed: 34006711
DOI: No ID Found -
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 -
Clinical & Translational Oncology :... Jan 2021About 5-10% of incidences of breast cancers have been reported as a result of germline mutations of BRCA genes. However, the mutational spectrum of BRCA1 and BRCA2 genes...
BACKGROUND
About 5-10% of incidences of breast cancers have been reported as a result of germline mutations of BRCA genes. However, the mutational spectrum of BRCA1 and BRCA2 genes among breast cancer Saudi women patients is inadequate at present. Therefore, the present study aimed to report the specific germinal mutation of BRCA1 and BRCA2 in the entire coding regions, to investigate the prevalence rate of BRCA1 & BRCA2 mutations among Saudi women and the effect of these mutations, both benign and malignant tumors.
METHODOLOGY
A total of 270 tissue samples of benign and malignant breast tumors were collected from Saudi women patients, Riyadh, Saudi Arabia. Examination of BRCA1 and BRCA2 germline mutations was performed using heteroduplex DNA analysis (HDA) or single-stranded conformation analysis (SSCA). 177 breast cancer women with malignant tumors and 93 with benign tumors were enrolled in the study. A total of 62 out of 177 breast cancer patients carried a BRCA1 or BRCA2 mutation (54 BRCA1 and 8 BRCA2). The analysis was done using the Sanger sequence assay.
RESULTS
Point and frameshift mutations through the entire coding area of the two genes indicated that all the mutations were germline alterations and of early-onset breast cancers. The mean ages of diagnosed breast cancer women for BRCA1 and BRCA2 mutation carriers were 36.3 (± 3.5) and 37.9 (± 3.7) years, whereas that of benign control was 35(± 2.5) years.
CONCLUSION
Point and frameshift mutations across the entire coding region of BRCA1 and BRCA2 are responsible for many breast cancers cases.
Topics: Adult; Breast Neoplasms; Female; Frameshift Mutation; Genes, BRCA1; Genes, BRCA2; Genetic Code; Germ-Line Mutation; Humans; Point Mutation; Saudi Arabia
PubMed: 32451972
DOI: 10.1007/s12094-020-02385-9 -
Journal of Bioscience and Bioengineering May 2021The previously reported Streptomyces coelicolor M1146 is commonly used as a host strain for engineering of secondary metabolite production. In this study, absolute...
The previously reported Streptomyces coelicolor M1146 is commonly used as a host strain for engineering of secondary metabolite production. In this study, absolute quantification of intracellular and extracellular metabolites of M1146 was performed in mid-log phase and stationary phase to observe major metabolites and the changes that occurred during growth. Decreased levels of central carbon metabolites (glycolysis, TCA cycle, and pentose phosphate pathway) and increased levels of amino acids were observed in stationary phase compared to mid-log phase. Furthermore, comparative metabolome analyses of M1146 upon expression of the actinorhodin biosynthetic gene cluster (M1146+ACT), a point mutation on the rpoB gene encoding RNA polymerase beta-subunit (M1152), and both expression of actinorhodin biosynthetic gene cluster and a rpoB point mutation (M1152+ACT) were performed. M1146+ACT showed higher levels of important cofactors, such as ATP, NADPH, and FMN while M1152 led to higher levels of intracellular S-adenosyl-methionine, acyl-CoAs, and extracellular nucleosides compared to M1146. M1152+ACT exhibited the highest levels of actinorhodin with elevated bases, nucleosides, and nucleotides, such as intracellular PRPP (phosphoribosyl phosphate), ATP, along with extracellular inosine, uridine, and guanine compared to the other three strains, which were considered to be combined effects of actinorhodin gene cluster expression and a rpoB point mutation. Metabolites analysis by means of absolute quantification demonstrated changes in precursors of secondary metabolites before and after phosphate depletion in M1146. Comparative metabolome analysis provided further insights into the effects of actinorhodin gene cluster expression along with a rpoB point mutation on the metabolome of S. coelicolor.
Topics: Anthraquinones; DNA-Directed RNA Polymerases; Metabolome; Multigene Family; Point Mutation; Streptomyces coelicolor
PubMed: 33549493
DOI: 10.1016/j.jbiosc.2021.01.002 -
Brain : a Journal of Neurology Oct 2022This scientific commentary refers to ‘The impact of phosphorylated PTEN at threonine 366 on cortical connectivity and behaviour’ by Ledderose...
This scientific commentary refers to ‘The impact of phosphorylated PTEN at threonine 366 on cortical connectivity and behaviour’ by Ledderose (https://doi.org/10.1093/brain/awac188).
Topics: Humans; Phosphorylation; Point Mutation; Threonine; PTEN Phosphohydrolase
PubMed: 36148582
DOI: 10.1093/brain/awac350 -
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