scholarly journals Characterization of gamma irradiation-induced mutations in Arabidopsis mutants deficient in non-homologous end joining

2020 ◽  
Vol 61 (5) ◽  
pp. 639-647
Author(s):  
Yan Du ◽  
Yoshihiro Hase ◽  
Katsuya Satoh ◽  
Naoya Shikazono
Keyword(s):  
Gamma Rays ◽  
Wild Type ◽  
End Joining ◽  
Complex Type ◽  
Single Base ◽  
In The Wild ◽  
Non Homologous End Joining ◽  
Dna Ends ◽  
Wild Type Control

Abstract To investigate the involvement of the non-homologous end joining (NHEJ) pathway in plant mutagenesis by ionizing radiation, we conducted a genome-wide characterization of the mutations induced by gamma rays in NHEJ-deficient Arabidopsis mutants (AtKu70−/− and AtLig4−/−). Although both mutants were more sensitive to gamma rays than the wild-type control, the AtKu70−/− mutant was slightly more sensitive than the AtLig4−/− mutant. Single-base substitutions (SBSs) were the predominant mutations in the wild-type control, whereas deletions (≥2 bp) and complex-type mutations [i.e. more than two SBSs or short insertion and deletions (InDels) separated by fewer than 10 bp] were frequently induced in the mutants. Single-base deletions were the most frequent deletions in the wild-type control, whereas the most common deletions in the mutants were 11–30 bp. The apparent microhomology at the rejoined sites of deletions peaked at 2 bp in the wild-type control, but was 3–4 bp in the mutants. This suggests the involvement of alternative end joining and single-strand annealing pathways involving increased microhomology for rejoining DNA ends. Complex-type mutations comprising short InDels were frequently detected in the mutants, but not in the wild-type control. Accordingly, NHEJ is more precise than the backup pathways, and is the main pathway for rejoining the broken DNA ends induced by ionizing radiation in plants.

scholarly journals Functional analysis of XRCC4 mutations in reported microcephaly and growth defect patients in terms of radiosensitivity

2021 ◽  
Author(s):  
Anie Day D C Asa ◽  
Rujira Wanotayan ◽  
Mukesh Kumar Sharma ◽  
Kaima Tsukada ◽  
Mikio Shimada ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Strand Break ◽  
Growth Defect ◽  
Wild Type ◽  
End Joining ◽  
Dsb Repair ◽  
Stable Transfectants ◽  
In The Wild ◽  
Non Homologous End Joining ◽  
Green Fluorescent

Abstract Non-homologous end joining is one of the main pathways for DNA double-strand break (DSB) repair and is also implicated in V(D)J recombination in immune system. Therefore, mutations in non-homologous end-joining (NHEJ) proteins were found to be associated with immunodeficiency in human as well as in model animals. Several human patients with mutations in XRCC4 were reported to exhibit microcephaly and growth defects, but unexpectedly showed normal immune function. Here, to evaluate the functionality of these disease-associated mutations of XRCC4 in terms of radiosensitivity, we generated stable transfectants expressing these mutants in XRCC4-deficient murine M10 cells and measured their radiosensitivity by colony formation assay. V83_S105del, R225X and D254Mfs*68 were expressed at a similar level to wild-type XRCC4, while W43R, R161Q and R275X were expressed at even higher level than wild-type XRCC4. The expression levels of DNA ligase IV in the transfectants with these mutants were comparable to that in the wild-type XRCC4 transfectant. The V83S_S105del transfectant and, to a lesser extent, D254Mfs*68 transfectant, showed substantially increased radiosensitivity compared to the wild-type XRCC4 transfectant. The W43R, R161Q, R225X and R275X transfectants showed a slight but statistically significant increase in radiosensitivity compared to the wild-type XRCC4 transfectant. When expressed as fusion proteins with Green fluorescent protein (GFP), R225X, R275X and D254Mfs*68 localized to the cytoplasm, whereas other mutants localized to the nucleus. These results collectively indicated that the defects of XRCC4 in patients might be mainly due to insufficiency in protein quantity and impaired functionality, underscoring the importance of XRCC4’s DSB repair function in normal development.

Ultrastructural characterization of a pigment mutant of the red alga Palmaria palmata

1984 ◽  
Vol 62 (6) ◽  
pp. 1101-1107 ◽  
Author(s):  
C. M. Pueschel ◽  
J. P. van der Meer
Keyword(s):  
Red Alga ◽  
Palmaria Palmata ◽  
Wild Type ◽  
Ultrastructural Examination ◽  
Dark Treatment ◽  
Prolamellar Bodies ◽  
Ultrastructural Characterization ◽  
Wild Type Control

Ultrastructural examination of a green-pigmented mutant of the red alga Palmaria palmata (L.) O. Kuntze revealed unusual features of the chloroplasts. Encircling peripheral thylakoids, characteristic of the wild-type plastids and florideophyte plastids generally, were lacking. Parallel evenly spaced thylakoids occurred in groups, leaving large volumes of thylakoid-free stroma. Irregularly shaped, electron-dense inclusions with an amorphous substructure and diameters up to 3 μm occurred in some plastids. Cells of the sporeling holdfasts contained structures resembling prolamellar bodies. Attempts to induce formation of prolamellar bodies in blades by dark treatment for 5 weeks were unsuccessful. However, some plastids did develop highly corrugated thylakoids with the crests of one thylakoid apposed to the troughs of the adjacent thylakoid. Thylakoid morphology of the wild-type control was not altered by the absence of light.

Characterization of Enterococcus faecium mutants resistant to mundticin KS, a class IIa bacteriocin

Microbiology ◽  
2003 ◽  
Vol 149 (10) ◽  
pp. 2901-2908 ◽  
Author(s):  
Youko Sakayori ◽  
Mizuho Muramatsu ◽  
Satoshi Hanada ◽  
Yoichi Kamagata ◽  
Shinichi Kawamoto ◽  
...  
Keyword(s):  
Enterococcus Faecium ◽  
Antimicrobial Agents ◽  
Wild Type Strain ◽  
Unsaturated Fatty Acids ◽  
Class I ◽  
Wild Type ◽  
Food Preservatives ◽  
In The Wild ◽  
Resistant Mutants

The emergence and spread of mutants resistant to bacteriocins would threaten the safety of using bacteriocins as food preservatives. To determine the physiological characteristics of resistant mutants, mutants of Enterococcus faecium resistant to mundticin KS, a class IIa bacteriocin, were isolated. Two types of mutant were found that had different sensitivities to other antimicrobial agents such as nisin (class I) and kanamycin. Both mutants were resistant to mundticin KS even in the absence of Mg2+ ions. The composition of unsaturated fatty acids in the resistant mutants was significantly increased in the presence of mundticin KS. The composition of the phospholipids in the two resistant mutants also differed from those in the wild-type strain. The putative zwitterionic amino-containing phospholipid in both mutants significantly increased, whereas amounts of phosphatidylglycerol and cardiolipin decreased. These changes in membrane structure may influence resistance of enterococci to class IIa and class I bacteriocins.

scholarly journals Activation of DNA-PK by hairpinned DNA ends reveals a stepwise mechanism of kinase activation

2020 ◽  
Vol 48 (16) ◽  
pp. 9098-9108 ◽  
Author(s):  
Katheryn Meek
Keyword(s):  
Protein Kinase ◽  
End Joining ◽  
Dependent Protein Kinase ◽  
Kinase Activation ◽  
Stepwise Mechanism ◽  
Dependent Protein ◽  
Enzymatic Activation ◽  
Non Homologous End Joining ◽  
Dna Ends ◽  
Step Mechanism

Abstract As its name implies, the DNA dependent protein kinase (DNA-PK) requires DNA double-stranded ends for enzymatic activation. Here, I demonstrate that hairpinned DNA ends are ineffective for activating the kinase toward many of its well-studied substrates (p53, XRCC4, XLF, HSP90). However, hairpinned DNA ends robustly stimulate certain DNA-PK autophosphorylations. Specifically, autophosphorylation sites within the ABCDE cluster are robustly phosphorylated when DNA-PK is activated by hairpinned DNA ends. Of note, phosphorylation of the ABCDE sites is requisite for activation of the Artemis nuclease that associates with DNA-PK to mediate hairpin opening. This finding suggests a multi-step mechanism of kinase activation. Finally, I find that all non-homologous end joining (NHEJ) defective cells (whether deficient in components of the DNA-PK complex or components of the ligase complex) are similarly deficient in joining DNA double-stranded breaks (DSBs) with hairpinned termini.

scholarly journals Isolation and Characterization of Mutations in theEscherichia coli Regulatory Protein XapR

1999 ◽  
Vol 181 (14) ◽  
pp. 4397-4403 ◽  
Author(s):  
Casper Jørgensen ◽  
Gert Dandanell
Keyword(s):  
Amino Acids ◽  
Purine Nucleoside Phosphorylase ◽  
Mutational Analysis ◽  
Regulatory Protein ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Wild Type ◽  
Isolation And Characterization ◽  
In The Wild

ABSTRACT In this work, the LysR-type protein XapR has been subjected to a mutational analysis. XapR regulates the expression of xanthosine phosphorylase (XapA), a purine nucleoside phosphorylase inEscherichia coli. In the wild type, full expression of XapA requires both a functional XapR protein and the inducer xanthosine. Here we show that deoxyinosine can also function as an inducer in the wild type, although not to the same extent as xanthosine. We have isolated and characterized in detail the mutants that can be induced by other nucleosides as well as xanthosine. Sequencing of the mutants has revealed that two regions in XapR are important for correct interactions between the inducer and XapR. One region is defined by amino acids 104 and 132, and the other region, containing most of the isolated mutations, is found between amino acids 203 and 210. These regions, when modelled into the three-dimensional structure of CysB from Klebsiella aerogenes, are placed close together and are most probably directly involved in binding the inducer xanthosine.

scholarly journals Non-homologous End Joining Requires That the DNA-PK Complex Undergo an Autophosphorylation-dependent Rearrangement at DNA Ends

2004 ◽  
Vol 279 (38) ◽  
pp. 39408-39413 ◽  
Author(s):  
Yeturu V. R. Reddy ◽  
Qi Ding ◽  
Susan P. Lees-Miller ◽  
Katheryn Meek ◽  
Dale A. Ramsden
Keyword(s):  
End Joining ◽  
Non Homologous End Joining ◽  
Dna Ends

scholarly journals Estrogen receptor-α mediates estrogen-inducible abnormalities in the developing penis

Reproduction ◽  
2007 ◽  
Vol 133 (5) ◽  
pp. 1057-1067 ◽  
Author(s):  
H O Goyal ◽  
T D Braden ◽  
P S Cooke ◽  
M A Szewczykowski ◽  
C S Williams ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Levator Ani ◽  
Estrogen Receptor Α ◽  
Control Group ◽  
Wild Type ◽  
Penile Length ◽  
In The Wild ◽  
Os Penis ◽  
Harmful Effects ◽  
Wild Type Control

Previously, we reported an association between estrogen receptor-α (ERα) upregulation and detrimental effects of neonatal diethylstilbestrol (DES) exposure in the rat penis. The objective of this study was to employ the ERα knockout (ERαKO) mouse model to test the hypothesis that ERα mediates DES effects in the developing penis. ERαKO and wild-type C57BL/6 mice received oil or DES at a dose of 0.2 μg/pup per day (0.1 mg/kg) on alternate days from postnatal days 2 to 12. Fertility was tested at 80–240 days of age and tissues were examined at 96–255 days of age. DES caused malformation of the os penis, significant reductions in penile length, diameter, and weight, accumulation of fat cells in the corpora cavernosa penis, and significant reductions in weight of the bulbospongiosus and levator ani muscles in wild-type mice. Conversely, ERαKO mice treated with DES developed none of the above abnormalities. While nine out of ten male mice sired pups in the wild-type/control group, none did in the wild-type/DES group. ERαKO mice, despite normal penile development, are inherently infertile. Both plasma and intratesticular testosterone levels were unaltered in the DES-treated wild-type or DES-treated ERαKO mice when compared with controls, although testosterone concentration was much higher in the ERαKO mice. Hence, the resistance of ERαKO mice to developing penile abnormalities provides unequivocal evidence of an obligatory role for ERα in mediating the harmful effects of neonatal DES exposure in the developing penis.

scholarly journals Induction and Characterization of Tetraploids from Seeds of Bletilla striata (Thunb.) Reichb.f.

2018 ◽  
Vol 2018 ◽  
pp. 1-8
Author(s):  
Meiya Li ◽  
Bin Ding ◽  
Weipeng Huang ◽  
Jieli Pan ◽  
Zhishan Ding ◽  
...  
Keyword(s):  
Genetic Improvement ◽  
Herbal Remedy ◽  
Morphological Characteristics ◽  
Colchicine Treatment ◽  
Endangered Plants ◽  
Wild Type ◽  
Bletilla Striata ◽  
In The Wild

Bletilla striata (Thunb.), an ornamental and medicinal plant, is on the list of endangered plants in China. Its pseudobulb is abundant in polysaccharide and has been used for centuries as a herbal remedy. However, a recent rise in demand has placed it at risk of extinction, and therefore, research on its propagation and genetic improvement is essential. Since polyploids tend to possess advantageous qualities, we incubated B. striata seeds with colchicine with the aim of creating tetraploid plantlets. Aseptic seeds treated with 0.1% colchicine for 7 days showed the highest tetraploid induction rate of 40.67 ± 0.89%. Compared with the wild-type, the tetraploids could be identified by their morphological characteristics including larger stomata at a lower density, larger leaf blades, and a thicker petiole. Contents of polysaccharide and phenolic compounds were also determined in the tetraploid pseudobulbs, revealing significantly higher values than in the wild-type. In vitro colchicine treatment can therefore be used to successfully produce B. striata tetraploids with superior pseudobulbs.

scholarly journals Programmed genome editing of the omega-1 ribonuclease 1 of the blood fluke,Schistosoma mansoni

2018 ◽  
Author(s):  
Wannaporn Ittiprasert ◽  
Victoria H. Mann ◽  
Shannon E. Karinshak ◽  
Avril Coghlan ◽  
Gabriel Rinaldi ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Genome Editing ◽  
Human Macrophage ◽  
Wild Type ◽  
End Joining ◽  
Chromosomal Breakage ◽  
Knock Out ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Non Homologous End Joining

AbstractCRISPR/Cas9 based genome editing has yet been reported in parasitic or indeed any species of the phylum Platyhelminthes. We tested this approach by targeting omega-1 (ω1) ofSchistosoma mansonias a proof of principle. This secreted ribonuclease is crucial for Th2 priming and granuloma formation, providing informative immuno-pathological readouts for programmed genome editing. Schistosome eggs were either exposed to Cas9 complexed with a synthetic guide RNA (sgRNA) complementary to exon 6 of ω1 by electroporation or transduced with pseudotyped lentivirus encoding Cas9 and the sgRNA. Some eggs were also transduced with a single stranded oligodeoxynucleotide donor transgene that encoded six stop codons, flanked by 50 nt-long 5’-and 3’-microhomology arms matching the predicted Cas9-catalyzed double stranded break (DSB) within ω1. CRISPResso analysis of amplicons spanning the DSB revealed ∼4.5% of the reads were mutated by insertions, deletions and/or substitutions, with an efficiency for homology directed repair of 0.19% insertion of the donor transgene. Transcripts encoding ω1 were reduced >80% and lysates of ω1-edited eggs displayed diminished ribonuclease activity indicative that programmed editing mutated the ω1 gene. Whereas lysates of wild type eggs polarized Th2 cytokine responses including IL-4 and IL-5 in human macrophage/T cell co-cultures, diminished levels of the cytokines followed the exposure to lysates of ω1-mutated schistosome eggs. Following injection of schistosome eggs into the tail vein of mice, the volume of pulmonary granulomas surrounding ω1-mutated eggs was 18-fold smaller than wild type eggs. Programmed genome editing was active in schistosomes, Cas9-catalyzed chromosomal breakage was repaired by homology directed repair and/or non-homologous end joining, and mutation of ω1 impeded the capacity of schistosome eggs both to drive Th2 polarization and to provoke formation of pulmonary circumoval granulomas. Knock-out of ω1 and the impaired immunological phenotype showcase the novel application of programmed gene editing in and functional genomics for schistosomes.

scholarly journals Generation of a Mouse Model Lacking the Non-Homologous End-Joining Factor Mri/Cyren

2019 ◽  
Author(s):  
Sergio Castañeda-Zegarra ◽  
Camilla Huse ◽  
Øystein Røsand ◽  
Antonio Sarno ◽  
Mengtan Xing ◽  
...  
Keyword(s):  
Dna Repair ◽  
Class Switch Recombination ◽  
Physiological Role ◽  
Wild Type ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Mouse Development ◽  
Class Switch ◽  
Accessory Factors ◽  
Non Homologous End Joining

Classical non-homologous end joining (NHEJ) is a molecular pathway that detects, processes and ligates DNA double-strand breaks (DSBs) throughout the cell cycle. Mutations in several NHEJ genes result in neurological abnormalities and immunodeficiency both in humans and mice. The NHEJ pathway is required for the V(D)J recombination in developing B and T lymphocytes, and for class switch recombination in mature B cells. Ku heterodimer formed by Ku70 and Ku80 recognizes DSBs and facilitates the recruitment of accessory factors (e.g., DNA-PKcs, Artemis, Paxx and Mri/Cyren) and downstream core factors subunits XLF, XRCC4 and Lig4. Accessory factors might be dispensable for the process depending on the genetic background and DNA lesion type. To determine the physiological role of Mri in DNA repair and development, we introduced frame-shift mutation in the Mri gene in mice. We then analyzed the development of Mri-deficient mice as well as wild type and immunodeficient controls. Mice lacking Mri possessed reduced levels of class switch recombination in B lymphocytes and slow proliferation of neuronal progenitors when compared to wild type littermates. Human cell lines lacking Mri were as sensitive to DSBs as WT controls. Overall, we concluded that Mri/Cyren is largely dispensable for DNA repair and mouse development.

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