Breakage of CRISPR/Cas9-Induced Chromosome Bridges in Mitotic Cells

Chromosomal instability, the most frequent form of plasticity in cancer cells, often proceeds through the formation of chromosome bridges. Despite the importance of these bridges in tumor initiation and progression, debate remains over how and when they are resolved. In this study, we investigated the behavior and properties of chromosome bridges to gain insight into the potential mechanisms underlying bridge-induced genome instability. We report that bridges may break during mitosis or may remain unbroken until the next interphase. During mitosis, we frequently observed discontinuities in the bridging chromatin, and our results strongly suggest that a substantial fraction of chromosome bridges are broken during this stage of the cell cycle. This notion is supported by the observation that the chromatin flanking mitotic bridge discontinuities is often decorated with the phosphorylated form of the histone H2AX, a marker of DNA breaks, and by MDC1, an early mediator of the cell response to DNA breaks. Also, free 3′OH DNA ends were detected in more than half of the bridges during the final stages of cell division. However, even if detected, the DNA ends of broken bridges are not repaired in mitosis. To investigate whether mitotic bridge breakage depends on mechanical stress, we used experimental models in which chromosome bridges with defined geometry are formed. Although there was no association between spindle pole separation or the distance among non-bridge kinetochores and bridge breakage, we found a direct correlation between the distance between bridge kinetochores and bridge breakage. Altogether, we conclude that the discontinuities observed in bridges during mitosis frequently reflect a real breakage of the chromatin and that the mechanisms responsible for chromosome bridge breakage during mitosis may depend on the separation between the bridge kinetochores. Considering that previous studies identified mechanical stress or biochemical digestion as possible causes of bridge breakage in interphase cells, a multifactorial model emerges for the breakage of chromosome bridges that, according to our results, can occur at different stages of the cell cycle and can obey different mechanisms.

Optimizing Laccase Production in Auricularia Cornea by Submerged Fermentation With Wheat Bran Extract: Applications in Decolorization of Malachite Green Dye

Wheat bran extract may enhance laccase activity of Auricularia cornea in submerged fermentation. The laccase activity of A. cornea was maximal (768.78 U/mL) at 26.9 ℃, pH 5, a time point of 6 d, 22 h, and an inducer concentration of 10%. Laccase from A. cornea AC5 decolorized 83.27% of 75 mg/L malachite green within 12 h. High performance liquid chromatography (HPLC) analysis of the extracted metabolites suggested that the decolorization occurred through biodegradation. Malachite green induced cytotoxic damage including formation of a micronucleus and chromosome bridge at anaphase. Degradation products of malachite green proved to be less toxic and had negligible effects on chromosomal aberrations.

Mechanisms generating cancer genome complexity from a single cell division error

The chromosome breakage-fusion-bridge (BFB) cycle is a mutational process that produces gene amplification and genome instability. Signatures of BFB cycles can be observed in cancer genomes alongside chromothripsis, another catastrophic mutational phenomenon. We explain this association by elucidating a mutational cascade that is triggered by a single cell division error—chromosome bridge formation—that rapidly increases genomic complexity. We show that actomyosin forces are required for initial bridge breakage. Chromothripsis accumulates, beginning with aberrant interphase replication of bridge DNA. A subsequent burst of DNA replication in the next mitosis generates extensive DNA damage. During this second cell division, broken bridge chromosomes frequently missegregate and form micronuclei, promoting additional chromothripsis. We propose that iterations of this mutational cascade generate the continuing evolution and subclonal heterogeneity characteristic of many human cancers.

PICH function is required for organization of SUMOylated proteins on mitotic chromosomes

Proper chromosome segregation is essential for faithful cell division and if not maintained results in defective cell function caused by abnormal distribution of genetic information. Polo-like kinase 1 interacting checkpoint helicase (PICH) is a DNA translocase essential in chromosome bridge resolution during mitosis. Its function in resolving chromosome bridges requires both DNA translocase activity and ability to bind chromosomal proteins modified by Small Ubiquitin-like modifier (SUMO). However, it is unclear how these activities are cooperating to resolve chromosome bridges. Here, we show that PICH specifically promotes the organization of SUMOylated proteins like SUMOylated TopoisomeraseIIα (TopoIIα) on mitotic chromosomes. Conditional depletion of PICH using the Auxin Inducible Degron (AID) system resulted in the retention of SUMOylated chromosomal proteins, including TopoIIα, indicating that PICH functions to control proper association of these proteins with chromosomes. Replacement of PICH with its mutants showed that PICH is required for the proper organization of SUMOylated proteins on chromosomes. In vitro assays showed that PICH specifically attenuates SUMOylated TopoIIα activity using its SUMO-binding ability. Taken together, we propose a novel function of PICH in remodeling SUMOylated proteins to ensure faithful chromosome segregation.Summary StatementPolo-like kinase interacting checkpoint helicase (PICH) interacts with SUMOylated proteins to mediate proper chromosome segregation during mitosis. The results demonstrate that PICH controls association of SUMOylated chromosomal proteins, including Topoisomerase IIα, and that function requires PICH translocase activity and SUMO binding ability.

Mechanisms Generating Cancer Genome Complexity From A Single Cell Division Error

ABSTRACTThe chromosome breakage-fusion-bridge (BFB) cycle is a mutational process that produces gene amplification and genome instability. Signatures of BFB cycles can be observed in cancer genomes with chromothripsis, another catastrophic mutational process. Here, we explain this association by identifying a mutational cascade downstream of chromosome bridge formation that generates increasing amounts of chromothripsis. We uncover a new role for actomyosin forces in bridge breakage and mutagenesis. Chromothripsis then accumulates starting with aberrant interphase replication of bridge DNA, followed by an unexpected burst of mitotic DNA replication, generating extensive DNA damage. Bridge formation also disrupts the centromeric epigenetic mark, leading to micronucleus formation that itself promotes chromothripsis. We show that this mutational cascade generates the continuing evolution and sub-clonal heterogeneity characteristic of many human cancers.

Homology bioinformatics analysis of IPA1 gene in higher plants

IPA1 gene controlled paddy has increased in height, sturdy stem, number of tillers decreased, grains per panicle and dry grains weight increased significantly. In this study, the homology of IPA1 gene in 26 types of plant from 15 families was analyzed, and thus constructed its phylogenetic tree. The results of phylogenetic tree construction based on its amino acid sequence showed that the species from the same family, such as Gramineae, Rosaceae and Palmaceae, were well clustered for different branches and had high support rates. Furthermore, we constructed the phylogenetic tree based on its CDS sequence, and the species of Gramineae was well clustered, and the support rate was 100%. Our results indicate that IPA1 has high homology in higher class of plants, especially in the Gramineae, which is of great significance for us to further study the yield of rice and other gramineous species.s with time and concentration effect (3) Achilla glauca extract could make the chromosome bridge, chromosome fragment, chromosome lag in the root tip cells of Vicia faba and increase in micronucleus rate. The results showed that the water extract of Solidago canadensis had different degree of inhibition and damage to silkworm root tip cells; it also had some genetic toxicity and rapid diffusion ability.

Genotoxic Effect of Heavy Metals Cr, Cu, Pb and Zn using Allium cepa L.

ABSTRACT: Angiosperms are recognized as appropriate genetic models to detect heavy metal based environmental mutagens and are used in monitoring studies. Allium cepa (onion) has been used to evaluate DNA damages like chromosome aberrations and abnormalities in the mitotic cycle. The aim of the present study is to analyze the cytotoxic effects of chromium, copper, lead and zinc in A. cepa root tip squash mitotic cell divisions. The root tips were treated with three concentrations, viz. 5, 10 and 20 mg/100 ml of chromium, copper, lead and zinc at room temperature for 24 h. Mitotic indices and chromosomal abnormalities were calculated. It was observed that these heavy metals induced different types of chromosomal abnormalities comprising of Chromosome break, Chromosome bridge, C-mitosis, Vagrant, Delayed Anaphase and Vagrant, Chromosome Loss, Polyploidy and Chromosome Bridge, Chromosome Loss and Loculated Nucles, Stickiness, Multipolarity and Polyploid prophase along with the increasing doses. The effect of chromium and lead at 20 mg/100 ml concentration was found to be more toxic rather than copper and zinc to the root meristem of A. cepa. The ranking of cytotoxic potentials was in the descending order: lead > chromium > copper > zinc.

Stimulatory Effects of Gamma Irradiation on Phytochemical Properties, Mitotic Behaviour, and Nutritional Composition of Sainfoin (Onobrychis viciifoliaScop.)

Sainfoin (Onobrychis viciifoliaScop. Syn.Onobrychis sativaL.) is a bloat-safe forage crop with high levels of tannins, which is renowned for its medicinal qualities in grazing animals. Mutagenesis technique was applied to investigate the influence of gamma irradiation at 30, 60, 90, and 120 Gy on mitotic behavior,in vitrogrowth factors, phytochemical and nutritional constituents of sainfoin. Although a percentage of plant necrosis and non-growing seed were enhanced by irradiation increment, the germination speed was significantly decreased. It was observed that gamma irradiated seeds had higher value of crude protein and dry matter digestibility compared to control seeds. Toxicity of copper was reduced in sainfoin irradiated seeds at different doses of gamma rays. Anthocyanin content also decreased in inverse proportion to irradiation intensity. Accumulation of phenolic and flavonoid compounds was enhanced by gamma irradiation exposure in leaf cells. HPLC profiles differed in peak areas of the two important alkaloids, Berberine and Sanguinarine, in 120 Gy irradiated seeds compared to control seeds. There were positive correlations between irradiation dose and some abnormality divisions such as laggard chromosome, micronucleus, binucleated cells, chromosome bridge, and cytomixis. In reality, radiocytological evaluation was proven to be essential in deducing the effectiveness of gamma irradiation to induce somaclonal variation in sainfoin.

Evaluations of cytogenotoxic and nutrient composition of three commonly consumed vegetables in south-western Nigeria

Consumption of leafy vegetables provides health benefits but sometimes may turn out to be the cause of certain health problems. Thus, adequate toxicological screening is needed to ensure safety of their consumption. Aqueous extracts of Corchorus olitorius L., Celosia argentea L., and Ocimum gratissimum L. were evaluated for cytogenotoxic effects on Allium cepa L. root cells using 0.5, 1.0, 2.5, 5.0, 10.0, 25.0 and 50.0 part per thousand (ppt), each of the vegetable extracts. The levels of macronutrients (moisture, ash, protein, fibre, fat and carbohydrate) in the vegetables and some minerals (cadmium, copper, iron, lead, magnesium, nickel and zinc) of their aqueous extracts were determined. The carbohydrate values were in the order O. gratissimum > C. olitorius > C. argentea with 48.17% as the highest. The values obtained for ash, protein, fat, Mg and Zn contents were in the order C. argentea > C. olitorius > O. gratissimum , with the highest values of 19.98%, 30.79%,, 0.22% ,226.4 mg/L and 2.57mg/L, respectively obtained for C. argentea. The concentration of Cu and Pb were in the order of O.gratissium> C. argentea> C. olitorius with 0.67 and 0.21mg/L obtained for O.gratissium (P<0.05). The aqueous extracts of the three vegetables inhibited root growth and cell division in the A. cepa root tips, with the highest inhibitory effects observed in C. argentea at 50.0 ppt. Induced chromosomal aberrations were significant only at 1.0 and 25 ppt of O. gratissimum, whereas there were no significant differences in aberrant cells in C. olitorius and C. argentea compared to the control. Chromosomal aberrations observed in the treated A. cepa roots include c-mitosis, Chromosome bridge and sticky chromosome. The results showed that the three vegetables have nutritive qualities but with root growth and mitotic inhibitory activities, which were severe in C. argentea. Although the result indicates that chromosomal aberrations might be induced at higher concentrations, the antimitotic potential of the extract of C. argentea may favour its uses in the development of drugs to prevent the uncontrolled proliferation of cancer cells of which investigation is required.