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FEBS Open Bio Aug 2023Wolfram syndrome is a monogenic disease mainly caused by mutations in the WFS1 gene. Mutations in the WFS1 gene give rise to diabetes. Here, we characterized mutant WFS1...
Wolfram syndrome is a monogenic disease mainly caused by mutations in the WFS1 gene. Mutations in the WFS1 gene give rise to diabetes. Here, we characterized mutant WFS1 proteins by studying the stability of full-length wild-type (WT) WFS1, a missense mutant P724L, and two C-terminally truncated mutants, W837X and Y652X. We compared their stability by overexpressing them in MIN6 and HEK293T cells. The C-terminally truncated mutants W837X and Y652X are degraded more rapidly than the missense P724L mutant or wild-type WFS1 in MIN6 cells. In contrast, Y652X is more stable than WT or other mutant WFS1 proteins in HEK293T. In conclusion, we found that C-terminally truncated WFS1 mutants are selectively degraded in a cell type-specific manner.
Topics: Humans; HEK293 Cells; Insulin-Secreting Cells; Mutation; Proteasome Endopeptidase Complex; Wolfram Syndrome
PubMed: 37440664
DOI: 10.1002/2211-5463.13674 -
Frontiers in Plant Science 2023has been selected as one of the model legume species for gene functional studies. To elucidate the functions of the very large number of genes present in plant genomes,...
has been selected as one of the model legume species for gene functional studies. To elucidate the functions of the very large number of genes present in plant genomes, genetic mutant resources are very useful and necessary tools. Fast Neutron (FN) mutagenesis is effective in inducing deletion mutations in genomes of diverse species. Through this method, we have generated a large mutant resource in . This mutant resources have been used to screen for different mutant using a forward genetics methods. We have isolated and identified a large amount of symbiotic nitrogen fixation (SNF) deficiency mutants. Here, we describe the detail procedures that are being used to characterize symbiotic mutants in . In recent years, whole genome sequencing has been used to speed up and scale up the deletion identification in the mutant. Using this method, we have successfully isolated a SNF defective mutant FN007 and identified that it has a large segment deletion on chromosome 3. The causal deletion in the mutant was confirmed by tail PCR amplication and sequencing. Our results illustrate the utility of whole genome sequencing analysis in the characterization of FN induced deletion mutants for gene discovery and functional studies in the . It is expected to improve our understanding of molecular mechanisms underlying symbiotic nitrogen fixation in legume plants to a great extent.
PubMed: 37457346
DOI: 10.3389/fpls.2023.1209664 -
Infection and Immunity Jan 2022A variety of eubacteria, plants, and protozoa can modify membrane lipids by cyclopropanation, which is reported to modulate membrane permeability and fluidity. The...
A variety of eubacteria, plants, and protozoa can modify membrane lipids by cyclopropanation, which is reported to modulate membrane permeability and fluidity. The ability to cyclopropanate membrane lipids has been associated with resistance to oxidative stress in Mycobacterium tuberculosis, organic solvent stress in Escherichia coli, and acid stress in E. coli and Salmonella. In bacteria, the gene encoding cyclopropane fatty acid (CFA) synthase is induced during the stationary phase of growth. In the present study, we constructed a mutant of Salmonella enterica serovar Typhimurium 14028s ( Typhimurium) and determined the contribution of CFA-modified lipids to stress resistance and virulence in mice. Cyclopropane fatty acid content was quantified in wild-type and mutant Typhimurium. CFA levels in the mutant were greatly reduced compared to CFA levels in the wild type, indicating that CFA synthase is the major enzyme responsible for cyclopropane modification of lipids in Salmonella. Typhimurium mutants were more sensitive to extreme acid pH, the protonophore CCCP, and hydrogen peroxide compared to the wild type. In addition, mutants exhibited reduced viability in murine macrophages and could be rescued by the addition of the NADPH phagocyte oxidase inhibitor diphenyleneiodonium (DPI) chloride. Typhimurium lacking was also attenuated for virulence in mice. These observations indicate that CFA modification of lipids makes an important contribution to Salmonella virulence.
Topics: Animals; Bacterial Physiological Phenomena; Bacterial Proteins; Biosynthetic Pathways; Cyclopropanes; Disease Models, Animal; Fatty Acids; Hydrogen-Ion Concentration; Macrophages; Mice; Microbial Viability; Mutation; Oxidative Stress; Salmonella Infections; Salmonella typhimurium; Virulence
PubMed: 34662213
DOI: 10.1128/IAI.00479-21 -
Biomolecules Nov 2020The bacterial RNA polymerase (RNAP) is a multi-subunit protein complex (α2ββ'ω σ) containing the smallest subunit, ω. Although identified early in RNAP research,... (Review)
Review
The bacterial RNA polymerase (RNAP) is a multi-subunit protein complex (α2ββ'ω σ) containing the smallest subunit, ω. Although identified early in RNAP research, its function remained ambiguous and shrouded with controversy for a considerable period. It was shown before that the protein has a structural role in maintaining the conformation of the largest subunit, β', and its recruitment in the enzyme assembly. Despite evolutionary conservation of ω and its role in the assembly of RNAP, mutants lacking (codes for ω) are viable due to the association of the global chaperone protein GroEL with RNAP. To get a better insight into the structure and functional role of ω during transcription, several dominant lethal mutants of ω were isolated. The mutants showed higher binding affinity compared to that of native ω to the α2ββ' subassembly. We observed that the interaction between α2ββ' and these lethal mutants is driven by mostly favorable enthalpy and a small but unfavorable negative entropy term. However, during the isolation of these mutants we isolated a silent mutant serendipitously, which showed a lethal phenotype. Silent mutant of a given protein is defined as a protein having the same sequence of amino acids as that of wild type but having mutation in the gene with alteration in base sequence from more frequent code to less frequent one due to codon degeneracy. Eventually, many silent mutants were generated to understand the role of rare codons at various positions in . We observed that the dominant lethal mutants of ω having either point mutation or silent in nature are more structured in comparison to the native ω. However, the silent code's position in the reading frame of plays a role in the structural alteration of the translated protein. This structural alteration in ω makes it more rigid, which affects the plasticity of the interacting domain formed by ω and α2ββ'. Here, we attempted to describe how the conformational flexibility of the ω helps in maintaining the plasticity of the active site of RNA polymerase. The dominant lethal mutant of ω has a suppressor mapped near the catalytic center of the β' subunit, and it is the same for both types of mutants.
Topics: Bacterial Proteins; DNA-Directed RNA Polymerases; Mutant Proteins; Protein Subunits; Structure-Activity Relationship; Transcription Factors
PubMed: 33238579
DOI: 10.3390/biom10111588 -
Proteins Apr 2022Curvature in mammalian fibers, such as wool and human hair, is an important feature of the functional trait of coat structure-it affects mechanical resilience and...
Curvature in mammalian fibers, such as wool and human hair, is an important feature of the functional trait of coat structure-it affects mechanical resilience and thermo-insulation. However, to examine the relationship between fiber curvature, ultrastructure and protein composition fiber diameter variability has to be minimal. To achieve this we utilised the progeny of straight-wool domestic sheep mutant rams (crimp mutants) and wild-type ewes. Proteomic and structural results of the resulting mutant/wild-type twin pairs confirmed that straight crimp mutant wool had a normal cuticle and the same cortical protein and ultrastructural building blocks as wild-type (crimpy) fibers but differed in the layout of its cortical cells and in the relative proportions of keratin (K) and keratin-associated proteins (KAPs). In the case of the crimp mutants (straight fibers), the orthocortex was distributed in a fragmented, annular ring, with some orthocortical cells near the central medulla, a pattern similar to that of straight hairs from humans and other mammals. Crimp mutant fibers were noted for the reduced abundance of some proteins in the high glycine-tyrosine class normally associated with the orthocortex, specifically the KAP6, KAP7, and KAP8 families, while proteins from the KAP16 and KAP19 were found in increased abundance. In addition to this, the type I keratin, K38, which is also associated with the orthocortex, was also found at lower abundance in the mutant fibers. Conversely, proteins from the ultra-high sulfur class normally associated with the paracortex, specifically the KAP4 and KAP9 families, were found in higher abundance.
Topics: Animals; Female; Humans; Keratins; Male; Mammals; Proteomics; Sheep; Sheep, Domestic; Wool; Wool Fiber
PubMed: 34859500
DOI: 10.1002/prot.26289 -
Microbial Cell (Graz, Austria) May 2021In DNA replication is catalyzed by an assembly of proteins, the DNA polymerase III holoenzyme. This complex includes the polymerase and proofreading subunits, the...
In DNA replication is catalyzed by an assembly of proteins, the DNA polymerase III holoenzyme. This complex includes the polymerase and proofreading subunits, the processivity clamp and clamp loader complex. The gene encodes an accessory protein (known as χ) to the core clamp loader complex and is the only protein of the holoenzyme that binds to single-strand DNA binding protein, SSB. HolC is not essential for viability although mutants show growth impairment, genetic instability and sensitivity to DNA damaging agents. In this study we isolate spontaneous suppressor mutants in a Δ strain and identify these by whole genome sequencing. Some suppressors are alleles of RNA polymerase, suggesting that transcription is problematic for mutant strains, and of , stringent starvation protein. Using a conditional plasmid, we examine factors affecting transcription elongation and termination for synergistic or suppressive effects on mutant phenotypes. Alleles of RpoA (α), RpoB (β) and RpoC (β') RNA polymerase holoenzyme can partially suppress loss of HolC. In contrast, mutations in transcription factors DksA and NusA enhanced the inviability of mutants. HolC mutants showed enhanced sensitivity to bicyclomycin, a specific inhibitor of Rho-dependent termination. Bicyclomycin also reverses suppression of by and . An inversion of the highly expressed operon exacerbates the growth defects of mutants. We propose that transcription complexes block replication in mutants and Rho-dependent transcriptional termination and DksA function are particularly important to sustain viability and chromosome integrity.
PubMed: 34055967
DOI: 10.15698/mic2021.06.753 -
Journal of the Royal Society, Interface Aug 2019Understanding how environmental variability (or randomness) affects evolution is of fundamental importance for biology. The presence of temporal or spatial variability...
Understanding how environmental variability (or randomness) affects evolution is of fundamental importance for biology. The presence of temporal or spatial variability significantly affects the competition dynamics in populations, and gives rise to some counterintuitive observations. In this paper, we consider both birth-death (BD) or death-birth (DB) Moran processes, which are set up on a circular or a complete graph. We investigate spatial and temporal variability affecting division and/or death parameters. Assuming that mutant and wild-type fitness parameters are drawn from an identical distribution, we study mutant fixation probability and timing. We demonstrate that temporal and spatial types of variability possess fundamentally different properties. Under temporal randomness, in a completely mixed system, minority mutants experience (i) higher than neutral fixation probability and a higher mean conditional fixation time, if the division rates are affected by randomness and (ii) lower fixation probability and lower mean conditional fixation time if the death rates are affected. Once spatial restrictions are imposed, however, these effects completely disappear, and mutants in a circular graph experience neutral dynamics, but only for the DB update rule in case (i) and for the BD rule in case (ii) above. In contrast to this, in the case of spatially variable environment, both for BD/DB processes, both for complete/circular graph and both for division/death rates affected, minority mutants experience a higher than neutral probability of fixation. Fixation time, however, is increased by randomness on a circle, while it decreases for complete graphs under random division rates. A basic difference between temporal and spatial kinds of variability is the types of correlations that occur in the system. Under temporal randomness, mutants are spatially correlated with each other (they simply have equal fitness values at a given moment of time; the same holds for wild-types). Under spatial randomness, there are subtler, temporal correlations among mutant and wild-type cells, which manifest themselves by cells of each type 'claiming' better spots for themselves. Applications of this theory include cancer generation and biofilm dynamics.
Topics: Animals; Biological Evolution; Ecosystem; Models, Biological; Mutation; Population Dynamics; Time Factors
PubMed: 31409235
DOI: 10.1098/rsif.2018.0781 -
Probiotics and Antimicrobial Proteins Feb 2023The COVID-19 pandemic caused by a novel coronavirus (SARS-CoV-2) is a serious health concern in the twenty-first century for scientists, health workers, and all humans....
The COVID-19 pandemic caused by a novel coronavirus (SARS-CoV-2) is a serious health concern in the twenty-first century for scientists, health workers, and all humans. The absence of specific biotherapeutics requires new strategies to prevent the spread and prophylaxis of the novel virus and its variants. The SARS-CoV-2 virus shows pathogenesis by entering the host cells via spike protein and Angiotensin-Converting Enzyme 2 receptor protein. Thus, the present study aims to compute the binding energies between a wide range of bacteriocins with receptor-binding domain (RBD) on spike proteins of wild type (WT) and beta variant (lineage B.1.351). Molecular docking analyses were performed to evaluate binding energies. Upon achieving the best bio-peptides with the highest docking scores, further molecular dynamics (MD) simulations were performed to validate the structure and interaction stability. Protein-protein docking of the chosen 22 biopeptides with WT-RBD showed docking scores lower than -7.9 kcal/mol. Pediocin PA-1 and salivaricin P showed the lowest (best) docking scores of - 12 kcal/mol. Pediocin PA-1, salivaricin B, and salivaricin P showed a remarkable increase in the double mutant's predicted binding affinity with -13.8 kcal/mol, -13.0 kcal/mol, and -12.5 kcal/mol, respectively. Also, a better predicted binding affinity of pediocin PA-1 and salivaricin B against triple mutant was observed compared to the WT. Thus, pediocin PA-1 binds stronger to mutants of the RBD, particularly to double and triple mutants. Salivaricin B showed a better predicted binding affinity towards triple mutant compared to WT, showing that it might be another bacteriocin with potential activity against the SARS-CoV-2 beta variant. Overall, pediocin PA-1, salivaricin P, and salivaricin B are the most promising candidates for inhibiting SARS-CoV-2 (including lineage B.1.351) entrance into the human cells. These bacteriocins derived from lactic acid bacteria hold promising potential for paving an alternative way for treatment and prophylaxis of WT and beta variants.
Topics: Humans; Bacteriocins; SARS-CoV-2; Lactobacillales; COVID-19; Molecular Docking Simulation; Pandemics
PubMed: 34837166
DOI: 10.1007/s12602-021-09879-0 -
BMC Biology Dec 2020It is widely assumed that all mutant microorganisms present in a culture are able to grow and form colonies, provided that they express the features required for...
BACKGROUND
It is widely assumed that all mutant microorganisms present in a culture are able to grow and form colonies, provided that they express the features required for selection. Unlike wild-type Escherichia coli, PHO-constitutive mutants overexpress alkaline phosphatase and hence can hydrolyze glycerol-2-phosphate (G2P) to glycerol and form colonies on plates having G2P as the sole carbon source. These mutations mostly occur in the pst operon. However, the frequency of PHO-constitutive colonies on the G2P selective plate is exceptionally low.
RESULTS
We show that the rate in which spontaneous PHO-constitutive mutations emerge is about 8.0 × 10/generation, a relatively high rate, but the growth of most existing mutants is inhibited by their neighboring wild-type cells. This inhibition is elicited only by non-mutant viable bacteria that can take up and metabolize glycerol formed by the mutants. Evidence indicates that the few mutants that do form colonies derive from microclusters of mutants on the selective plate. A mathematical model that describes the fate of the wild-type and mutant populations under these circumstances supports these results.
CONCLUSION
This scenario in which neither the wild-type nor the majority of the mutants are able to grow resembles an unavoidable "tragedy of the commons" case which results in the collapse of the majority of the population. Cooperation between rare adjacent mutants enables them to overcome the competition and eventually form mutant colonies. The inhibition of PHO-constitutive mutants provides an example of mutant frequency masked by orders of magnitude due to a competition between mutants and their ancestral wild-type cells. Similar "tragedy of the commons-like" cases may occur in other settings and should be taken into consideration while estimating true mutant frequencies and mutation rates.
Topics: Escherichia coli; Microbial Interactions; Mutation; Nutrients
PubMed: 33317515
DOI: 10.1186/s12915-020-00913-1 -
BMC Plant Biology Aug 2023Induction of mutation through chemical mutagenesis is a novel approach for preparing diverse germplasm. Introduction of functional alleles in the starch biosynthetic...
BACKGROUND
Induction of mutation through chemical mutagenesis is a novel approach for preparing diverse germplasm. Introduction of functional alleles in the starch biosynthetic genes help in the improvement of the quality and yield of cereals.
RESULTS
In the present study, a set of 350 stable mutant lines were used to evaluate dynamic variation of the total starch contents. A megazyme kits were used for measuring the total starch content, resistant starch, amylose, and amylopectin content. Analysis of variance showed significant variation (p < 0.05) in starch content within the population. Furthermore, two high starch mutants (JE0173 and JE0218) and two low starch mutants (JE0089 and JE0418) were selected for studying different traits. A multiple comparison test showed that significant variation in all physiological and morphological traits, with respect to the parent variety (J411) in 2019-2020 and 2020-2021. The quantitative expression of starch metabolic genes revealed that eleven genes of JE0173 and twelve genes of JE0218 had consistent expression in high starch mutant lines. Similarly, in low starch mutant lines, eleven genes of JE0089 and thirteen genes of JE0418 had consistent expression in all stages of seed development. An additional two candidate genes showed over-expression (PHO1, PUL) in the high starch mutant lines, indicating that other starch metabolic genes may also contribute to the starch biosynthesis. The overexpression of SSII, SSIII and SBEI in JE0173 may be due to presence of missense mutations in these genes and SSI also showed overexpression which may be due to 3-primer_UTR variant. These mutations can affect the other starch related genes and help to increase the starch content in this mutant line (JE0173).
CONCLUSIONS
This study screened a large scale of mutant population and identified mutants, could provide useful genetic resources for the study of starch biosynthesis and genetic improvement of wheat in the future. Further study will help to understand new genes which are responsible for the fluctuation of total starch.
Topics: Starch; Triticum; Plant Proteins; Amylose; Amylopectin
PubMed: 37528349
DOI: 10.1186/s12870-023-04389-3