Repeat Mutagenesis Research Articles

Effectiveness of repeated mutagenesis of sesame crosses for enhancing polygenic variability in F2M2 generation.

The value of combining hybridization and mutagenesis in sesame was examined to determine if treating hybrid sesame plant material with mutagens generated greater genetic variability in four key productivity traits than either the separate hybridization or mutation of plant material. In a randomized block design with three replications, six F2M2 varieties, three F2varieties, and three parental varieties were assessed at Odisha University of Agriculture and Technology, Bhubaneswar, Odisha, India. The plant characteristics height, number of seed capsules per plant, and seed yield per plant had greater variability in the F2M2 generation than their respective controls (F2), however, the number of primary branches per plant varied less than in the control population. The chances for trait selection to be operative were high for all the characteristics examined except the number of primary branches per plant, as indicated by heritability estimates. Increases in the mean and variability of the characteristics examined indicted a greater incidence of beneficial mutations and the breakdown of undesirable linkages with increased recombination. At both phenotypic and genotypic levels strong positive correlations between both primary branch number and capsule number with seed yield suggest that these traits are important for indirect improvement in sesame seed yield. As a result of the association analysis, sesame seed yield and its component traits improved significantly, which may be attributed to the independent polygenic mutations and enlarged recombination of the polygenes controlling the examined characteristics. Compared to the corresponding control treatment or to one cycle of mutagenic treatment, two cycles of mutagenic treatment resulted in increased variability, higher transgressive segregates, PTS mean and average transgression for sesame seed yield. These findings highlight the value of implementing two EMS treatment cycles to generate improved sesame lines. Furthermore, the extra variability created through hybridization may have potential in subsequent breeding research and improved seed yield segregants may be further advanced to develop ever-superior sesame varieties.

Enhancing photosynthesis at high light levels by adaptive laboratory evolution.

Photosynthesis is readily impaired by high light (HL) levels. Photosynthetic organisms have therefore evolved various mechanisms to cope with the problem. Here, we have dramatically enhanced the light tolerance of the cyanobacterium Synechocystis by adaptive laboratory evolution (ALE). By combining repeated mutagenesis and exposure to increasing light intensities, we generated strains that grow under extremely HL intensities. HL tolerance was associated with more than 100 mutations in proteins involved in various cellular functions, including gene expression, photosynthesis and metabolism. Co-evolved mutations were grouped into five haplotypes, and putative epistatic interactions were identified. Two representative mutations, introduced into non-adapted cells, each confer enhanced HL tolerance, but they affect photosynthesis and respiration in different ways. Mutations identified by ALE that allow photosynthetic microorganisms to cope with altered light conditions could be employed in assisted evolution approaches and could strengthen the robustness of photosynthesis in crop plants.

Breeding of High-yield Alkaline Protease Producing Strain by Atmospheric and Room Temperature Plasma Mutagenesis

To obtain alkaline protease producing strain with high-yield, the strain Bacillus subtilis was treated by atmospheric and room temperature plasma (ARTP) mutagenesis. The results showed that the strain had a higher positive mutation rate when the mutagenesis time was 50s. Finally, a high-yield mutant strain A59 was obtained after repeated mutagenesis by ARTP and enzyme activity increased by 23.38% from 6835U/mL to 8433U/mL. Then fermentation conditions of mutant strain were optimized by single-factor method and response surface method, and the optimum fermentation conditions were as follows: 3.6% soybean meal, 5.2% corn meal, 0.9115% Na2HPO4, 0.1593% Mg2+, 0.09% Tween-80 and 5% inoculation volume. Under the conditions of the verification experiment, the enzyme activity of the A59 strain reached 14026 U/mL, which was 2.05 times that of the original strain.

Environmental Stress Induces Trinucleotide Repeat Mutagenesis in Human Cells by Alt-Nonhomologous End Joining Repair

Multiple pathways modulate the dynamic mutability of trinucleotide repeats (TNRs), which are implicated in neurodegenerative disease and evolution. Recently, we reported that environmental stresses induce TNR mutagenesis via stress responses and rereplication, with more than 50% of mutants carrying deletions or insertions—molecular signatures of DNA double-strand break repair. We now show that knockdown of alt-nonhomologous end joining (alt-NHEJ) components—XRCC1, LIG3, and PARP1—suppresses stress-induced TNR mutagenesis, in contrast to the components of homologous recombination and NHEJ, which have no effect. Thus, alt-NHEJ, which contributes to genetic mutability in cancer cells, also plays a novel role in environmental stress-induced TNR mutagenesis.

Environmental stress induces trinucleotide repeat mutagenesis in human cells

The dynamic mutability of microsatellite repeats is implicated in the modification of gene function and disease phenotype. Studies of the enhanced instability of long trinucleotide repeats (TNRs)-the cause of multiple human diseases-have revealed a remarkable complexity of mutagenic mechanisms. Here, we show that cold, heat, hypoxic, and oxidative stresses induce mutagenesis of a long CAG repeat tract in human cells. We show that stress-response factors mediate the stress-induced mutagenesis (SIM) of CAG repeats. We show further that SIM of CAG repeats does not involve mismatch repair, nucleotide excision repair, or transcription, processes that are known to promote TNR mutagenesis in other pathways of instability. Instead, we find that these stresses stimulate DNA rereplication, increasing the proportion of cells with >4 C-value (C) DNA content. Knockdown of the replication origin-licensing factor CDT1 eliminates both stress-induced rereplication and CAG repeat mutagenesis. In addition, direct induction of rereplication in the absence of stress also increases the proportion of cells with >4C DNA content and promotes repeat mutagenesis. Thus, environmental stress triggers a unique pathway for TNR mutagenesis that likely is mediated by DNA rereplication. This pathway may impact normal cells as they encounter stresses in their environment or during development or abnormal cells as they evolve metastatic potential.

Long-term strain improvements accumulate mutations in regulatory elements responsible for hyper-production of cellulolytic enzymes

Long-term strain improvements through repeated mutagenesis and screening have generated a hyper-producer of cellulases and hemicellulases from Penicillium decumbens 114 which was isolated 30 years ago. Here, the genome of the hyper-producer P. decumbens JU-A10-T was sequenced and compared with that of the wild-type strain 114-2. Further, the transcriptomes and secretomes were compared between the strains. Selective hyper-production of cellulases and hemicellulases but not all the secreted proteins was observed in the mutant, making it a more specific producer of lignocellulolytic enzymes. Functional analysis identified that changes in several transcriptional regulatory elements played crucial roles in the cellulase hyper-producing characteristics of the mutant. Additionally, the mutant showed enhanced supply of amino acids and decreased synthesis of secondary metabolites compared with the wild-type. The results clearly point out that we can target gene regulators and promoters with minimal alterations of the genetic content but maximal effects in genetic engineering.

Examining the potential role of DNA polymerases η and ζ in triplet repeat instability in yeast

Triplet repeats undergo frequent mutations in human families afflicted with certain neurodegenerative diseases and also in model organisms. Although the molecular mechanisms of triplet repeat instability are still being identified, it is likely that aberrant DNA synthesis plays an important role. Many DNA polymerases stall at triplet repeat sequences, probably due to the adoption of unusual DNA secondary structures. One possible mechanism to explain triplet repeat contractions is that a triplet repeat hairpin on the template strand inhibits replicative polymerases and that one or more bypass polymerases are recruited for synthesis past the hairpin. If the translesion synthesis is mutagenic, contractions can be generated. To address this possibility, Saccharomyces cerevisiae strains lacking either pol ζ ( rev7), pol η ( rad30), or both were tested for trinucleotide repeat (TNR) contractions using three separate, sensitive genetic assays. If these bypass polymerases are important for mutagenesis, then the mutants should show a reduction in the contraction rate. Two genetic tests for triplet repeat contractions showed no significant change for the mutants compared to wild type. A third assay showed a five-fold reduction in contraction rates due to pol η ablation. Despite this modest decrease, the overall contraction rate was still high, indicating that many deletions still occur in the absence of both polymerases. Expansion rates were also unaffected in the mutant strains. These results indicate that, in yeast, pol η and pol ζ most likely have little role in triplet repeat mutagenesis.

A C-terminal deletion in Corynebacterium glutamicum homoserine dehydrogenase abolishes allosteric inhibition by l-threonine

In Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum, homoserine dehydrogenase (HD), the enzyme after the branch point of the threonine/methionine and lysine biosynthetic pathways, is allosterically inhibited by l-threonine. To investigate the regulation of the C. glutamicum HD enzyme by l-threonine, the structural gene, hom, was mutated by UV irradiation of whole cells to obtain a deregulated allele, hom dr. l-Threonine inhibits the wild-type (wt) enzyme with a K i of 0.16 mM. The deregulated enzyme remains 80% active in the presence of 50 mM l-threonine. The hom dr gene mutant was isolated and cloned in E. coli. In a C. glutamicum wt host background, but not in E. coli, the cloned hom dr gene is genetically unstable. The cloned hom dr gene is overexpressed tenfold in C. glutamicum and is active in the presence of over 60 mM l-threonine. Sequence analysis revealed that the hom dr mutation is a single nucleotide (G 1964) deletion in codon 429 within the hom reading frame. The resulting frame-shift mutation radically alters the structure of the C terminus, resulting in ten amino acid (aa) changes and a deletion of the last 7 aa relative to the wt protein. These observations suggest that the C terminus may be associated with the l-threonine allosteric response. The hom dr mutation is unstable and probably deleterious to the cell. This may explain why only one mutation was obtained despite repeated mutagenesis.

A novel strategy for the generation of T cell lines lacking expression of endogenous alpha- and/or beta-chain T cell receptor genes.

Mutant T cell lines that do not express the endogenous alpha- and/or beta-chain genes of the TCR were generated from the alpha beta TCR/CD3+ tumor cell line C6VL with a combination of classical mutagenesis methods and selection of somatic hybrid variants. This novel strategy obviated the need for repeated mutagenesis and screening of a large number of individual clones. The loss of either the alpha- or the beta-chain expression in the mutant cells was associated with the loss of surface TCR/CD3 complex, which could be rescued by the transfection of appropriate exogenous alpha- and/or beta-chain gene constructs. Because these cells express a single TCR molecule on the cell surface, they are useful for the study of the assembly and function of the alpha beta TCR. This strategy is also generally applicable for the generation of homozygous mutant cell lines lacking other gene products.

Production of isoprenoid compounds in the facultative methylotroph Protomonas extorquens

Screening of a strain which contained a large amount of ubiquinone Q-10 and a variety of isoprenoid compounds using different culture conditions and mutations was carried out. Protomonas extorquens TK 0045, which was found to contain carotenoid pigments, Hop-22(29)-ene, and Hopan-22-ol, was selected on the basis of cell yield and the content of ubiquinone Q-10. The contents of ubiquinone and sterols increased as the age of the culture increased, and reached a maximum level during the stationary phase. The contents of ubiquinone, sterols and carotenoid pigments, and ubiquinone homologs produced by P. extorquens TK 0045 were varied using mutagenesis. Mutants that had increased or decreased contents of carotenoid pigments were obtained with a high frequency. Most mutants had varying contents of other isoprenoid compounds. The ubiquinone homologs obtained by mutagenesis varied with a high frequency, and mutants which possessed increased levels of ubiquinone Q-9, Q-11 or Q-12 were isolated. However, the major ubiquinone component in these mutants was Q-10 the same as that in the wild strain. The production of ubiquinone was increased considerablyby repeated mutagenesis, with the content of ubiquinone produced by the third generation mutant (strains HB-5) being approximately 3.3 mg·g dry cell −1 (2.5 times that of the wild strain). The acquisition of mutants exhibiting altered synthesis of carotenoid pigments would be useful for increasing the content of ubiquinone Q-10 in bacterial cells.

Repeated mutagenesis and the selection of recessive and dominant mutations in cultured mammalian cells

Repeated mutagenesis and the selection of recessive and dominant mutations in cultured mammalian cells

Isolation of cadmium- and mercury-sensitive mutants of Escherichia coli and some factors influencing their sensitivities.

Several Cd2+ hypersensitive and Hg2+ sensitive mutants of Escherichia coli K-12 were isolated after repeated mutagenesis with nitrosoguanidine. The Cd-hypersensitive mutant, CD17P, could not grow in a chemically defined liquid medium containing 0.5 μm Cd2+, and the growth of Hg-sensitive mutant, HG17P-52, was inhibited by 0.9 μm Hg2+. Thus, CD 17P and HG17P-52 were respectively about 1000 fold and 4 fold more sensitive than the parental strain.Both mutants were also considerably more sensitive to Zn2+ than their parent, but they did not show any appreciable change in sensitivity to other metals tested. Among the various metabolites and chemicals tested, reducing agents such as cysteine, dithiothreitol and vitamin C showed a protective effect against metal toxicity. Reduced glutathione was effective only against Hg2+. EDTA specifically and efficiently reversed only Cd2+ toxicity.