Perturbation of bacteriochlorophyll molecules in Fenna–Matthews–Olson protein complexes through mutagenesis of cysteine residues
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solar (fuels), photosynthesis (natural and artific↗
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Results for “Mutagenesis”
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Mutagenesis of the FAE1 genes significantly changes fatty acid composition in seeds of Camelina sativa
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Ozseyhan, Mehmet E.↗
Natural selection underlies apparent stress-induced mutagenesis in a bacteriophage infection model
We present that the emergence of mutations following growth-limiting conditions underlies bacterial drug resistance, viral escape from the immune system and fundamental evolution-driven events. Intriguingly, whether mutations are induced by growth limitation conditions or are randomly generated during growth and then selected by growth limitation conditions remains an open question. Here, we show that bacteriophage T7 undergoes apparent stress-induced mutagenesis when selected for improved recognition of its host's receptor. In our unique experimental set-up, the growth limitation condition is physically and temporally separated from mutagenesis: growth limitation occurs while phage DNA is outside the host, and spontaneous mutations occur during phage DNA replication inside the host. We show that the selected beneficial mutations are not pre-existing and that the initial slow phage growth is enabled by the phage particle's low-efficiency DNA injection into the host. Thus, the phage particle allows phage populations to initially extend their host range without mutagenesis by virtue of residual recognition of the host receptor. Finally, mutations appear during non-selective intracellular replication, and the frequency of mutant phages increases by natural selection acting on free phages, which are not capable of mutagenesis.
59 BASIC BIOLOGICAL SCIENCES↗
Combining Random Mutagenesis and Metabolic Engineering for Enhanced Tryptophan Production in Synechocystis sp. Strain PCC 6803
Tryptophan (Trp) is an essential aromatic amino acid that has value as an animal feed supplement, as the amount found in plant-based sources is insufficient. An alternative to production by engineered microbial fermentation is to have tryptophan biosynthesized by a photosynthetic microorganism that could replace or supplement both the plant and industrially used microbes. We selected Synechocystis sp. strain PCC 6803, a model cyanobacterium, as the host and studied metabolic engineering and random mutagenesis approaches. Previous work on engineering heterotrophic microbes for improved Trp titers has targeted allosteric feedback regulation in enzymes 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase (DAHPS) and anthranilate synthase (AS) as major bottlenecks in the shikimate pathway. In this work, the genes encoding feedback-resistant enzymes from Escherichia coli, aroG fbr and trpE fbr , were overexpressed in the host wild-type (WT) strain. Separately, the WT strain was subjected to random mutagenesis and selection using an amino acid analog to isolate tryptophan-overproducing strains. The randomly mutagenized strains were sequenced in order to identify the mutations that resulted in the desirable phenotypes. Interestingly, the tryptophan overproducers had mutations in the gene encoding chorismate mutase (CM), which catalyzes the conversion of chorismate to prephenate. The best tryptophan overproducer from random mutagenesis was selected as a host for metabolic engineering where aroG fbr and trpE fbr were overexpressed. The best strain developed produced 212 ± 23 mg/liter of tryptophan after 10 days of photoautotrophic growth under 3% (vol/vol) CO2. We demonstrated that a combination of random mutagenesis and metabolic engineering was superior to either individual approach.
59 BASIC BIOLOGICAL SCIENCES↗
Gamma-ray mutagenesis studies in a new human-hamster hybrid, A(L)CD59(+/-), which has two human chromosomes 11 but is hemizygous for the CD59 gene
Kraemer, S. M., Vannais, D. B., Kronenberg, A., Ueno, A. and Waldren, C. A. Gamma-Ray Mutagenesis Studies in a New Human-Hamster Hybrid, A(L)CD59(+/-), which has Two Human Chromosomes 11 but is Hemizygous for the CD59 Gene. Radiat. Res. 156, 10-19 (2001).We have developed a human-CHO hybrid cell line, named A(L)CD59(+/-), which has two copies of human chromosome 11 but is hemizygous for the CD59 gene and the CD59 cell surface antigen that it encodes. Our previous studies used the A(L) and A(L)C hybrids that respectively contain one or two sets of CHO chromosomes plus a single copy of human chromosome 11. The CD59 gene at 11p13.5 and the CD59 antigen encoded by it are the principal markers used in our mutagenesis studies. The hybrid A(L)CD59(+/-) contains two copies of human chromosome 11, only one of which carries the CD59 gene. The incidence of CD59 (-) mutants (formerly called S1(-)) induced by (137)Cs gamma rays is about fivefold greater in A(L)CD59(+/-) cells than in A(L) cells. Evidence is presented that this increase in mutant yield is due to the increased induction of certain classes of large chromosomal mutations that are lethal to A(L) cells but are tolerated in the A(L)CD59(+/-) hybrid. In addition, significantly more of the CD59 (-) mutants induced by (137)Cs gamma rays in A(L)CD59(+/-) cells display chromosomal instability than in A(L) cells. On the other hand, the yield of gamma-ray-induced CD59 (-) mutants in A(L)CD59(+/-) cells is half that of the A(L)C hybrid, which also tolerates very large mutations but has only one copy of human chromosome 11. We interpret the difference in mutability as evidence that repair processes involving the homologous chromosomes 11 play a role in determining mutant yields. The A(L)CD59(+/-) hybrid provides a useful new tool for quantifying mutagenesis and shedding light on mechanisms of genetic instability and mutagenesis.
NASA Discipline Radiation Health↗
Construction of a Compact Array of Microplasma Jet Devices and Its Application for Random Mutagenesis of Rhodosporidium toruloides
A small and efficient DNA mutation-inducing machine was constructed with an array of microplasma jet devices (7 × 1) that can be operated at atmospheric pressure for microbial mutagenesis. Using this machine, we report disruption of a plasmid DNA and generation of mutants of an oleaginous yeast Rhodosporidium toruloides. Specifically, a compact-sized microplasma channel (25 × 20 × 2 mm 3 ) capable of generating an electron density of greater than 10 13 cm –3 was constructed to produce reactive species (N 2 *, N 2 +, O, OH, and H α ) under helium atmospheric conditions to induce DNA mutagenesis. The length of microplasma channels in the device played a critical role in augmenting both the volume of plasma and the concentration of reactive species. First, we confirmed that microplasma treatment can linearize a plasmid by creating nicks in vitro. Second, we treated R. toruloides cells with a jet device containing 7 microchannels for 5 min; 94.8% of the treated cells were killed, and 0.44% of surviving cells showed different colony colors as compared to their parental colony. Microplasma-based DNA mutation is energy-efficient and can be a safe alternative for inducing mutations compared to conventional methods using toxic mutagens. In conclusion, this compact and scalable device is amenable for industrial strain improvement involving large-scale mutagenesis.
59 BASIC BIOLOGICAL SCIENCES↗
Rev1 overexpression accelerates N -methyl- N -nitrosourea (MNU)-induced thymic lymphoma by increasing mutagenesis
Rev1 has two important functions in the translesion synthesis pathway, including dCMP transferase activity, and acts as a scaffolding protein for other polymerases involved in translesion synthesis. However, the role of Rev1 in mutagenesis and tumorigenesis in vivo remains unclear. We previously generated Rev1-overexpressing (Rev1-Tg) mice and reported that they exhibited a significantly increased incidence of intestinal adenoma and thymic lymphoma (TL) after N-methyl-N-nitrosourea (MNU) treatment. In this study, we investigated mutagenesis of MNU-induced TL tumorigenesis in wild-type (WT) and Rev1-Tg mice using diverse approaches, including whole-exome sequencing (WES). In Rev1-Tg TLs, the mutation frequency was higher than that in WT TL in most cases. However, no difference in the number of nonsynonymous mutations in the Catalogue of Somatic Mutations in Cancer (COSMIC) genes was observed, and mutations involved in Notch1 and MAPK signaling were similarly detected in both TLs. Mutational signature analysis of WT and Rev1-Tg TLs revealed cosine similarity with COSMIC mutational SBS5 (aging-related) and SBS11 (alkylation-related). Interestingly, the total number of mutations, but not the genotypes of WT and Rev1-Tg, was positively correlated with the relative contribution of SBS5 in individual TLs, suggesting that genetic instability could be accelerated in Rev1-Tg TLs. Finally, we demonstrated that preleukemic cells could be detected earlier in Rev1-Tg mice than in WT mice, following MNU treatment. In conclusion, Rev1 overexpression accelerates mutagenesis and increases the incidence of MNU-induced TL by shortening the latency period, which may be associated with more frequent DNA damage-induced genetic instability.
60 APPLIED LIFE SCIENCES↗
Targeted mutagenesis with sequence–specific nucleases for accelerated improvement of polyploid crops: Progress, challenges, and prospects
Many of the world's most important crops are polyploid. The presence of more than two sets of chromosomes within their nuclei and frequently aberrant reproductive biology in polyploids present obstacles to conventional breeding. The presence of a larger number of homoeologous copies of each gene makes random mutation breeding a daunting task for polyploids. Genome editing has revolutionized improvement of polyploid crops as multiple gene copies and/or alleles can be edited simultaneously while preserving the key attributes of elite cultivars. Most genome–editing platforms employ sequence–specific nucleases (SSNs) to generate DNA double–stranded breaks at their target gene. Such DNA breaks are typically repaired via the error–prone nonhomologous end–joining process, which often leads to frame shift mutations, causing loss of gene function. Genome editing has enhanced the disease resistance, yield components, and end–use quality of polyploid crops. However, identification of candidate targets, genotyping, and requirement of high mutagenesis efficiency remain bottlenecks for targeted mutagenesis in polyploids. In this review, we will survey the tremendous progress of SSN–mediated targeted mutagenesis in polyploid crop improvement, discuss its challenges, and identify optimizations needed to sustain further progress.
60 APPLIED LIFE SCIENCES↗
Investigation of TRPV1 loss-of-function phenotypes in TRPV1 Leu206Stop mice generated by N-ethyl-N-nitrosourea mutagenesis
Highlights: • N-ethyl-N-nitrosourea mutagenesis can generate mouse models for pain research. • TRPV1 Leu206Stop mice closely resemble the phenotype of TRPV1 knockout mice. • Mechanical hypersensitivity is not impaired in TRPV1 Leu206Stop mice. N-ethyl-N-nitrosourea (ENU) random mutagenesis was used to generate a mouse model for the analysis of the transient receptor potential vanilloid 1 (TRPV1) cation channel. A transversion from T→A in exon 4 led to a Leu206Stop mutation generating a loss-of-function mutant. The TRPV1 agonist capsaicin was used to analyze functional and nociceptive parameters in vitro and in vivo in TRPV1 Leu206Stop mice and congenic C3HeB/FeJ controls. Capsaicin-induced [Ca{sup 2+}]{sub i} changes in small diameter DRG neurons were significantly diminished in TRPV1 Leu206Stop mice and administration of capsaicin induced neither hypothermia nor nocifensive behaviour in vivo. TRPV1 Leu206Stop mice were tested in the spinal nerve ligation of mononeuropathic pain and developed mechanical hypersensitivity two weeks after nerve injury. In the open field test, a significant increase in spontaneous locomotion was detected in TRPV1 Leu206Stop mice as compared to wildtype controls. TRPV1 knockout mice have been reported to carry a similar phenotype regarding capsaicin-evoked responses in vitro and in vivo. However, in contrast to TRPV1 Leu206Stop mice, TRPV1 knockout mice did not differ in spontaneous locomotion as compared to congenic C57BL/6 mice, suggesting subtle ENU-dependent or independent strain differences between TRPV1 Leu206Stop mice and their wildtype controls. In summary, these data revealed a target-related (i.e. capsaicin-evoked) phenotype of TRPV1 Leu206Stop mice closely resembling that of published TRPV1 knockout mice. However, since ENU-mutant mice are congenic with the mouse strain initially used in random mutagenesis, direct phenotypic comparison with the respective wildtype controls is possible, and the time-consuming backcrossing in lines with targeted mutations is avoided.
60 APPLIED LIFE SCIENCES↗
Data for Construction of a Compact Array of Microplasma Jet Devices and Its Application for Random Mutagenesis of Rhodosporidium toruloides
A small and efficient DNA mutation-inducing machine was constructed with an array of microplasma jet devices (7 × 1) that can be operated at atmospheric pressure for microbial mutagenesis. Using this machine, we report disruption of a plasmid DNA and generation of mutants of an oleaginous yeast Rhodosporidium toruloides . Specifically, a compact-sized microplasma channel (25 × 20 × 2 mm3) capable of generating an electron density of greater than 1013 cm–3 was constructed to produce reactive species (N2*, N2+, O, OH, and Hα) under helium atmospheric conditions to induce DNA mutagenesis. The length of microplasma channels in the device played a critical role in augmenting both the volume of plasma and the concentration of reactive species. First, we confirmed that microplasma treatment can linearize a plasmid by creating nicks in vitro. Second, we treated R. toruloides cells with a jet device containing 7 microchannels for 5 min; 94.8% of the treated cells were killed, and 0.44% of surviving cells showed different colony colors as compared to their parental colony. Microplasma-based DNA mutation is energy-efficient and can be a safe alternative for inducing mutations compared to conventional methods using toxic mutagens. This compact and scalable device is amenable for industrial strain improvement involving large-scale mutagenesis.
Conversion↗
Effect of foot-and-mouth disease virus 3C protease B 2 β-strand proline mutagenesis on expression and processing of the P1 polypeptide using a plasmid expression vector
The production of experimental molecular vaccines against foot-and-mouth disease virus utilizes the viral encoded 3C protease for processing of the P1 polyprotein. Expression of wild type 3C protease is detrimental to host cells. The molecular vaccine constructs containing the 3C protease L127P mutant significantly reduce adverse effects associated with protease expression while retaining the ability to process and assemble virus-like particles. In published 3C protease crystal structures, the L127 residue is contained within the B 2 β-strand as part of the A 2 –B 2 β-sheet. To provide insight into the mechanism by which the L127P mutant alters the properties of the 3C protease, we performed scanning proline mutagenesis of residues 123–128 of the B 2 β-strand and monitored expression and P1 processing. Simultaneously, we utilized random mutagenesis of the full 3C sequence to identify additional mutations presenting a phenotype similar to the L127P mutation. Six of the tested mutants enhanced expression over wild type, and the I22P, T100P and V124P mutations surpassed the L127P mutation in certain cell lines. These data are interpreted in conjunction with published 3C protease crystal structures to provide insight into the mechanism by which these mutations enhance expression.
3C↗
Development of a Markerless Deletion Mutagenesis System in Nitrate-Reducing Bacterium Rhodanobacter denitrificans
Rhodanobacter has been found as the dominant genus in aquifers contaminated with high concentrations of nitrate and uranium in Oak Ridge, TN, USA. The in situ stimulation of denitrification has been proposed as a potential method to remediate nitrate and uranium contamination. Among the Rhodanobacter species, Rhodanobacter denitrificans strains have been reported to be capable of denitrification and contain abundant metal resistance genes. However, due to the lack of a mutagenesis system in these strains, our understanding of the mechanisms underlying low-pH resistance and the ability to dominate in the contaminated environment remains limited. Here, we developed an in-frame markerless deletion system in two R. denitrificans strains. First, we optimized the growth conditions, tested antibiotic resistance, and determined appropriate transformation parameters in 10 Rhodanobacter strains. We then deleted the upp gene, which encodes uracil phosphoribosyltransferase, in R. denitrificans strains FW104-R3 and FW104-R5. The resulting strains were designated R3_Δupp and R5_Δupp and used as host strains for mutagenesis with 5-fluorouracil (5-FU) resistance as the counterselection marker to generate markerless deletion mutants. Further, to test the developed protocol, the narG gene encoding nitrate reductase was knocked out in the R3_Δupp and R5_Δupp host strains. As expected, the narG mutants could not grow in anoxic medium with nitrate as the electron acceptor. Overall, these results show that the in-frame markerless deletion system is effective in two R. denitrificans strains, which will allow for future functional genomic studies in these strains furthering our understanding of the metabolic and resistance mechanisms present in Rhodanobacter species.
59 BASIC BIOLOGICAL SCIENCES↗
Ionizing radiation-induced mutagenesis: radiation studies in Neurospora predictive for results in mammalian cells
Ionizing radiation was the first mutagen discovered and was used to develop the first mutagenicity assay. In the ensuing 70+ years, ionizing radiation became a fundamental tool in understanding mutagenesis and is still a subject of intensive research. Frederick de Serres et al. developed and used the Neurospora crassa ad-3 system initially to explore the mutagenic effects of ionizing radiation. Using this system, de Serres et al. demonstrated the dependence of the frequency and spectra of mutations induced by ionizing radiation on the dose, dose rate, radiation quality, repair capabilities of the cells, and the target gene employed. This work in Neurospora predicted the subsequent observations of the mutagenic effects of ionizing radiation in mammalian cells. Modeled originally on the mouse specific-locus system developed by William L. Russell, the N. crassa ad-3 system developed by de Serres has itself served as a model for interpreting the results in subsequent systems in mammalian cells. This review describes the primary findings on the nature of ionizing radiation-induced mutagenesis in the N. crassa ad-3 system and the parallel observations made years later in mammalian cells.
NASA Discipline Radiation Health↗
Targeted seed EMS mutagenesis reveals a basic helix–loop–helix transcription factor underlying male sterility in sorghum
Abstract Forward genetic screens of mutant populations are fundamental for functional genomics studies. However, isolating independent mutant alleles to molecularly identify causal genes is challenging in species recalcitrant to genetic manipulation. Here, we demonstrate that classic seed ethyl methanesulfonate (EMS) mutagenesis coupled with genome sequencing can overcome this limitation in sorghum. We used this method to generate new mutant alleles of sorghum MALE STERILE 8 (MS8) and identified the causal locus for the ms8 phenotype as Sobic.004G270900, which encodes the sorghum ortholog of maize bhlh122, a basic helix–loop–helix (bHLH) transcription factor required for male fertility in maize. Bulked segregant analysis mapped ms8-1 to a region on chromosome 4 containing Sobic.004G270900. Seeds from heterozygous MS8/ms8-1 plants were mutagenized and screened for chimeric inflorescences containing sectors with white, sterile anthers resembling the ms8-1 homozygous phenotype. DNA sequencing of sterile and fertile sectors from a single chimeric inflorescence revealed two mutations in Sobic.004G270900 within the sterile sector, but not the fertile sector. Isolation of this loss-of-function allele (ms8-2) established Sobic.004G270900 as the causative locus for male sterility in the ms8 mutant. We generated additional alleles of MS8 in a different genetic background using CRISPR/Cas9-based gene editing, where deletions in Sobic.004G270900 also resulted in male sterility. Our work identified a gene underlying male sterility in sorghum and provides a novel and straightforward genetic tool for researchers who lack access to advanced transformation facilities to validate gene candidates. Unlike gene editing, no prior knowledge of candidate genes is required for targeted seed EMS mutagenesis to aid identification of causal loci.
Genetics & Heredity↗
Data for Comparison of Genotyping Assays for Detection of Targeted CRISPR/Cas Mutagenesis in Highly Polyploid Sugarcane
Sugarcane ( Saccharum spp.) is an important biofuel feedstock and a leading source of global table sugar. Saccharum hybrid cultivars are highly polyploid (2n = 100–130), containing large numbers of functionally redundant hom(e)ologs in their genomes. Genome editing with sequence-specific nucleases holds tremendous promise for sugarcane breeding. However, identification of plants with the desired level of co-editing within a pool of primary transformants can be difficult. While DNA sequencing provides direct evidence of targeted mutagenesis, it is cost-prohibitive as a primary screening method in sugarcane and most other methods of identifying mutant lines have not been optimized for use in highly polyploid species. In this study, non-sequencing methods of mutant screening, including capillary electrophoresis (CE), Cas9 RNP assay, and high-resolution melt analysis (HRMA), were compared to assess their potential for CRISPR/Cas9-mediated mutant screening in sugarcane. These assays were used to analyze sugarcane lines containing mutations at one or more of six sgRNA target sites. All three methods distinguished edited lines from wild type, with co-mutation frequencies ranging from 2% to 100%. Cas9 RNP assays were able to identify mutant sugarcane lines with as low as 3.2% co-mutation frequency, and samples could be scored based on undigested band intensity. CE was highlighted as the most comprehensive assay, delivering precise information on both mutagenesis frequency and indel size to a 1 bp resolution across all six targets. This represents an economical and comprehensive alternative to sequencing-based genotyping methods which could be applied in other polyploid species.
Genomics↗
Comparison of genotyping assays for detection of targeted CRISPR/Cas mutagenesis in highly polyploid sugarcane
Sugarcane (Saccharum spp.) is an important biofuel feedstock and a leading source of global table sugar. Saccharum hybrid cultivars are highly polyploid (2n = 100–130), containing large numbers of functionally redundant hom(e)ologs in their genomes. Genome editing with sequence-specific nucleases holds tremendous promise for sugarcane breeding. However, identification of plants with the desired level of co-editing within a pool of primary transformants can be difficult. While DNA sequencing provides direct evidence of targeted mutagenesis, it is cost-prohibitive as a primary screening method in sugarcane and most other methods of identifying mutant lines have not been optimized for use in highly polyploid species. In this study, non-sequencing methods of mutant screening, including capillary electrophoresis (CE), Cas9 RNP assay, and high-resolution melt analysis (HRMA), were compared to assess their potential for CRISPR/Cas9-mediated mutant screening in sugarcane. These assays were used to analyze sugarcane lines containing mutations at one or more of six sgRNA target sites. All three methods distinguished edited lines from wild type, with co-mutation frequencies ranging from 2% to 100%. Cas9 RNP assays were able to identify mutant sugarcane lines with as low as 3.2% co-mutation frequency, and samples could be scored based on undigested band intensity. CE was highlighted as the most comprehensive assay, delivering precise information on both mutagenesis frequency and indel size to a 1 bp resolution across all six targets. This represents an economical and comprehensive alternative to sequencing-based genotyping methods which could be applied in other polyploid species.
60 APPLIED LIFE SCIENCES↗
Highly efficient libraries design for saturation mutagenesis
Abstract Saturation mutagenesis is a semi-rational approach for protein engineering where sites are saturated either entirely or partially to include amino acids of interest. We previously reported on a codon compression algorithm, where a set of minimal degenerate codons are selected according to user-defined parameters such as the target organism, type of saturation and usage levels. Here, we communicate an addition to our web tool that considers the distance between the wild-type codon and the library, depending on its purpose. These forms of restricted collections further reduce library size, lowering downstream screening efforts or, in turn, allowing more comprehensive saturation of multiple sites. The library design tool can be accessed via http://www.dynamcc.com/dynamcc_d/. Graphical Abstract
59 BASIC BIOLOGICAL SCIENCES↗