Chromosome Fragments Research Articles

Causes and Consequences of Nuclear Envelope Rupture and DNA Damage in Micronuclei

Micronuclei are small, aberrant nuclear compartments containing mis-segregated chromosomes or chromosomal fragments. During telophase, dysfunctional micronuclear envelope reassembly leaves the micronuclear envelope highly unstable and rupture-prone. Following rupture, micronuclei attempt to repair membrane gaps, but the process is typically unsuccessful and may promote the invasion of ER tubules into the interior of micronuclei. These abnormalities cause ruptured micronuclei to accumulate significant DNA damage in the form of both single-stranded DNA and double-stranded breaks. Because micronuclei are capable of promoting genome instability, it is essential to understand the sources of DNA damage and the mechanism through which it arises in these structures. In this review, I will explore the causes and consequences of micronuclear envelope rupture, beginning with the processes surrounding improper micronuclear envelope reassembly. I will then discuss micronuclear envelope rupture, attempted micronuclear envelope repair and its consequences, and the proposed causes of micronuclear DNA damage.

Methyl Jasmonate Effect on Betulinic Acid Content and Biological Properties of Extract from Senna obtusifolia Transgenic Hairy Roots.

It is known that Senna obtusifolia has been used in medicine since ancient times due to the content of many valuable compounds with a pro-health effect. One of them is betulinic acid, which is a pentacyclic triterpene with antimalarial, antiviral, anti-inflammatory and anticancer properties. In this work, a continuation of our previous research, an attempt was made to increase the level of betulinic acid accumulation by the cultivation of transgenic hairy roots that overexpress the squalene synthase gene in a 10 L sprinkle bioreactor with methyl jasmonate elicitation. We present that the applied strategy allowed us to increase the content of betulinic acid in hairy root cultures to the level of 48 mg/g dry weight. The obtained plant extracts showed a stronger cytotoxic effect on the U87MG glioblastoma cell line than the roots grown without elicitors. Additionally, the induction of apoptosis, reduction of mitochondrial membrane potential, chromosomal DNA fragmentation and activation of caspase cascades are demonstrated. Moreover, the tested extract showed inhibition of topoisomerase I activity.

An endophytic fungus, Penicillium simplicissimum conjugated with C60 fullerene for its potential antimitotic, anti-inflammatory, anticancer and photodegradation activities

ABSTRACT In the present study, endophytic fungus, Penicillium simplicissimum isolated from Loranthus micranthus was used to analyze phytochemical studies by qualitative and GC-MS methods. The endophytic fungus P. simplicissimum yielded novel compound penisimplicissin identified through GC-MS studies. Further, P. simplicissimum was conjugated with C60 fullerene nanoparticles (Ps-FNPs) were verified using UV-vis spectra, XRD, FTIR, DLS, EDX and SEM. Ps-FNPs was confirmed using UV-visible spectra with a peak at 260 nm. The IR bands were recorded at 2085, 1428, 1181, 661, 652, 644, 628, and 604 cm-1. The Ps-FNPs treated cells showed a nucleolar shrinkage and cell arrest atprophase, binuclear and multinucleolar cells, a chromosomal bridge and diversion at anaphase was observed, whereas, chromosomal fragment and abnormal distribution at metaphase stage. The Ps-FNPs exhibited a noteworthy anticancer activity on lung cancer cell line H1975 through cytotoxicity. The cytotoxicity was induced by increasing caspase-3, 7, and 9 activities and also showed highest inhibition in xanthine oxidase and COX-II assay proved good anti-inflammatory activity. Ps-FNPs have been extensively studied for photocatalytic activity test against Rhodamine B, Methylene blue and nigrosine showed potential dye degradation in the presence of sunlight proved to be novel photocatalysts. With all the results recorded, Ps-FNPs also have a synergetic effect having on anti-mitotic, anticancer, anti-inflammation potential and photocatalytic degradation of dyes. Hence, the conjugated Ps-FNPs could be one of the potent nano-drug formulations in future. Thus, the present study gives a clear idea of the multifaceted therapeutic and photocatalytic applications.

Fluctuating Bacteriophage-induced galU Deficiency Region is Involved in Trade-off Effects on the Phage and Fluoroquinolone Sensitivity in Pseudomonas aeruginosa

Pseudomonas aeruginosa, which causes chronic infections, has demonstrated rapid acquisition of antimicrobial resistance (AMR). Therefore, bacteriophages have received significant attention as promising antimicrobial agents; however, previous trials have reported the occurrence of phage-resistant variants. P. aeruginosa has lost large chromosomal fragments via evolutionary selection by MutL. Mutants lacking galU and hmgA, located in close proximity, exhibit phage resistance and brown color phenotype since hmgA encodes a homogentisic acid metabolic enzyme and deletion of galU results in a lack of O-antigen polysaccharide and absence of the phage receptor. In the present study, we evaluated this mechanism for controlling phage resistance in P. aeruginosa veterinary isolate Pa12. Phage-resistant Pa12 brown mutants (brmts) with galU and hmgA deletions were isolated. Whole-genome sequencing of the brmts revealed that regions 148-27 kbp upstream and 261-110 kbp downstream of galU were largely deleted from the Pa12 parental chromosome. Furthermore, all of these fluctuating deleted sequences in Pa12 brmts, tentatively designated bacteriophage-induced galU deficiency (BigD) regions, harbor multi-drug efflux system genes (mexXY). Minimum inhibitory concentration (MIC) assays demonstrated that brmts altered sensitivity to antibiotics and exhibited increased levofloxacin sensitivity compared with the Pa12 parent. Orbifloxacin and enrofloxacin also effectively suppressed growth of the Pa12 brmts, suggesting that MexXY, which mediates quinolone efflux and is located in the BigD region, might be associated with restoration of fluoroquinolone sensitivity. Our findings indicate that AMR-related genes in the BigD region could produce trade-off effects between phages and drug sensitivity and thereby contribute to a potential strategy to control and prevent phage-resistant variants in phage therapy.

Genome-wide association mapping uncovers sex-associated copy number variation markers and female hemizygous regions on the W chromosome in Salix viminalis

BackgroundSex chromosomes are in some species largely undifferentiated (homomorphic) with restricted sex determination regions. Homomorphic but different sex chromosomes are found in the closely related genera Populus and Salix indicating flexible sex determination systems, ideal for studies of processes involved in sex chromosome evolution. We have performed genome-wide association studies of sex and analysed sex chromosomes in a population of 265 wild collected Salix viminalis accessions and studied the sex determining locus.ResultsA total of 19,592 markers were used in association analyses using both Fisher’s exact tests and a single-marker mixed linear model, which resulted in 48 and 41 sex-associated (SA) markers respectively. Across all 48 SA markers, females were much more often heterozygous than males, which is expected if females were the heterogametic sex. The majority of the SA markers were, based on positions in the S. purpurea genome, located on chromosome 15, previously demonstrated to be the sex chromosome. Interestingly, when mapping the genotyping-by-sequencing sequence tag harbouring the two SA markers with the highest significance to the S. viminalis genomic scaffolds, five regions of very high similarity were found: three on a scaffold that represents a part of chromosome 15, one on a scaffold that represents a part of chromosome 9 and one on a scaffold not anchored to the genome. Based on segregation differences of the alleles at the two marker positions and on differences in PCR amplification between females and males we conclude that females had multiple copies of this DNA fragment (chromosome 9 and 15), whereas males only had one (chromosome 9). We therefore postulate that the female specific sequences have been copied from chromosome 9 and inserted on chromosome 15, subsequently developing into a hemizygous W chromosome linked region.ConclusionsOur results support that sex determination in S. viminalis is controlled by one locus on chromosome 15. The segregation patterns observed at the SA markers furthermore confirm that S. viminalis females are the heterogametic sex. We also identified a translocation from chromosome 9 to the W chromosome.

Polygenic basis and biomedical consequences of telomere length variation

Telomeres, the end fragments of chromosomes, play key roles in cellular proliferation and senescence. Here we characterize the genetic architecture of naturally occurring variation in leukocyte telomere length (LTL) and identify causal links between LTL and biomedical phenotypes in 472,174 well-characterized UK Biobank participants. We identified 197 independent sentinel variants associated with LTL at 138 genomic loci (108 new). Genetically determined differences in LTL were associated with multiple biological traits, ranging from height to bone marrow function, as well as several diseases spanning neoplastic, vascular and inflammatory pathologies. Finally, we estimated that, at the age of 40 years, people with an LTL >1 s.d. shorter than the population mean had a 2.5-year-lower life expectancy compared with the group with ≥1 s.d. longer LDL. Overall, we furnish new insights into the genetic regulation of LTL, reveal wide-ranging influences of LTL on physiological traits, diseases and longevity, and provide a powerful resource available to the global research community.

Acclimation of cadmium-induced genotoxicity and oxidative stress in mung bean seedlings by priming effect of phytohormones and proline.

In this research, eight local mung bean (Vigna radiata) varieties were analyzed for their performance against two levels of CdCl2 solution (0.3 and 0.5 mM) alone and priming with gibberellic acid (GA3) (100 μM), salicylic acid (SA) (50 μM) and proline (5 mM) solution prior to Cd exposure. Mung bean seedlings were analyzed for disturbance in cytological, morphological, biochemical and enzymatic parameters under cadmium stress. For cytological studies, 48 h grown mung bean seedlings root tips were used to prepare slides and studied for percent mitotic index (MI%) and to calculate percent C-mitosis, laggard, sticky and fragmented chromosomes, pictures were captured by a Nikon camera (DS-Fi 1 Japan) attached with a microscope. One-week grown mung seedlings were studied for growth traits, malondialdehyde (MDA), protein, proline and antioxidant enzymes. ANOVA and DMR test of this research revealed that all the tested mung bean varieties and treatments were significantly different regarding mitotic index and number of chromosomal aberrations. Both the Cd treatments exhibited increased total chromosomal aberrations with different types and a maximum decrease in MI%. In pretreated samples, GA3, SA and proline serve as mitigating agents that reduce mutagenic effects of Cd in mung bean by increasing MI% and decreasing chromosomal aberrations as compared to non-pretreated samples. Both the Cd treatments showed a decrease in all growth traits. Total proteins were also found to be significantly reduced in a dose-dependent manner in all genotypes. Cd treatment increased the activities of all antioxidant enzymes tested. Cd caused oxidative damage as indicated by elevated levels of MDA content in treated samples in comparison to control. Proline content levels were also high in Cd treated seedlings indicating stress. Results demonstrated that pretreatment with phytohormones and proline before Cd were found to improve all morphological parameters, by altering antioxidant enzymes activities along with a decrease in MDA and proline contents as well. It was further noticed that the performance of GA3 was better at 0.3 mM Cd treatment while SA was found to be a good mitigating agent at 0.5 mM Cd stress in all tested mung bean varieties. This research concluded less deleterious effects of Cd on AZRI-2006 while more sensitivity to NM-51 towards Cd. Priming with phytohormones and proline is a user-friendly, economical, and simple mitigation strategy to reduce Cd toxicity in plants and get better yield from contaminated lands.

Delete and survive: strategies of programmed genetic material elimination in eukaryotes.

ABSTRACTGenome stability is a crucial feature of eukaryotic organisms because its alteration drastically affects the normal development and survival of cells and the organism as a whole. Nevertheless, some organisms can selectively eliminate part of their genomes from certain cell types during specific stages of ontogenesis. This review aims to describe the phenomenon of programmed DNA elimination, which includes chromatin diminution (together with programmed genome rearrangement or DNA rearrangements), B and sex chromosome elimination, paternal genome elimination, parasitically induced genome elimination, and genome elimination in animal and plant hybrids. During programmed DNA elimination, individual chromosomal fragments, whole chromosomes, and even entire parental genomes can be selectively removed. Programmed DNA elimination occurs independently in different organisms, ranging from ciliate protozoa to mammals. Depending on the sequences destined for exclusion, programmed DNA elimination may serve as a radical mechanism of dosage compensation and inactivation of unnecessary or dangerous genetic entities. In hybrids, genome elimination results from competition between parental genomes. Despite the different consequences of DNA elimination, all genetic material destined for elimination must be first recognised, epigenetically marked, separated, and then removed and degraded.

Micronuclei in Kif18a mutant mice form stable micronuclear envelopes and do not promote tumorigenesis.

Micronuclei, whole or fragmented chromosomes spatially separated from the main nucleus, are associated with genomic instability and have been identified as drivers of tumorigenesis. Paradoxically, Kif18a mutant mice produce micronuclei due to asynchronous segregation of unaligned chromosomes in vivo but do not develop spontaneous tumors. We report here that micronuclei in Kif18a mutant mice form stable nuclear envelopes. Challenging Kif18a mutant mice via deletion of the Trp53 gene led to formation of thymic lymphoma with elevated levels of micronuclei. However, loss of Kif18a had modest or no effect on survival of Trp53 homozygotes and heterozygotes, respectively. Micronuclei in cultured KIF18A KO cells form stable nuclear envelopes characterized by increased recruitment of nuclear envelope components and successful expansion of decondensing chromatin compared with those induced by nocodazole washout or radiation. Lagging chromosomes were also positioned closer to the main chromatin masses in KIF18A KO cells. These data suggest that not all micronuclei actively promote tumorigenesis.

Advances in identification methods of alien genomic components in plants

Plants with alien genomic components (alien chromosomes / chromosomal fragments / genes) are important materials for genomic research and crop improvement. To date, four strategies based on trait observation, chromosome analysis, specific proteins, and DNA sequences have been developed for the identification of alien genomic components. Among them, DNA sequence-based molecular markers are mainly used to identify alien genomic components. This review summarized several molecular markers for identification of alien genomic components in wheat, cabbage and other important crops. We also compared the characteristics of nine common molecular markers, such as simple sequence repeat (SSR), insertion-deletion (InDel) and single nucleotide polymorphism (SNP). In general, the accuracy of using a combination of different identification methods is higher than using a single identification method. We analyzed the application of different combination of identification methods, and provided the best combination for wheat, brassica and other crops. High-throughput detection can be easily achieved by using the new generation molecular markers such as InDel and SNP, which can be used to determine the precise localization of alien introgression genes. To increase the identification efficiency, other new identification methods, such as microarray comparative genomic hybridization (array-CGH) and suppression subtractive hybridization (SSH), may also be included.

De novo centromere formation on chromosome fragments with an inactive centromere in maize (Zea mays)

The B chromosome of maize undergoes nondisjunction at the second pollen mitosis as part of its accumulation mechanism. Previous work identified 9-Bic-1 (9-B inactivated centromere-1), which comprises an epigenetically silenced B chromosome centromere that was translocated to the short arm of chromosome 9(9S). This chromosome is stable in isolation, but when normal B chromosomes are added to the genotype, it will attempt to undergo nondisjunction during the second pollen mitosis and usually fractures the chromosome in 9S. These broken chromosomes allow a test of whether the inactive centromere is reactivated or whether a de novo centromere is formed elsewhere on the chromosome to allow recovery of fragments. Breakpoint determination on the B chromosome and chromosome 9 showed that mini chromosome B1104 has the same breakpoint as 9-Bic-1 in the B centromere region and includes a portion of 9S. CENH3 binding was found on the B centromere region and on 9S, suggesting both centromere reactivation and de novo centromere formation. Another mini chromosome, B496, showed evidence of rearrangement, but it also only showed evidence for a de novo centromere. Other mini chromosome fragments recovered were directly derived from the B chromosome with breakpoints concentrated near the centromeric knob region, which suggests that the B chromosome is broken at a low frequency due to the failure of the sister chromatids to separate at the second pollen mitosis. Our results indicate that both reactivation and de novo centromere formation could occur on fragments derived from the progenitor possessing an inactive centromere.

The decreased expression of GW2 homologous genes contributed to the increased grain width and thousand‑grain weight in wheat-Dasypyrum villosum 6VS·6DL translocation lines.

This study demonstrated that the aberrant transcription of DvGW2 contributed to the increased grain width and thousand-grain weight in wheat-Dasypyrum villosum T6VS·6DL translocation lines. Due to the high immunity to powdery mildew, Dasypyrum villosum 6VS has been one of the most successful applications of the wild relatives in modern wheat breeding. Along with the desired traits, side-effects could be brought when large alien chromosome fragments are introduced into wheat, but little is known about effects of 6VS on agronomic traits. Here, we found that T6VS·6DL translocation had significantly positive effects on grain weight, plant heightand spike length, and small negative effects on total spikelet number and spikelet compactness using recipient and wheat-D. villosum T6VS·6DL allohexaploid wheats, Wan7107 and Pm97033. Further analysis showed that the 6VS segment might exert direct genetic effect on grain width, then driving the increase of thousand-grain weight. Furthermore, comparative transcriptome analysis identified 2549 and 1282 differentially expressed genes(DEGs) and 2220 and 1496 specifically expressed genes(SEGs) at 6days after pollination(DAP) grains and 15 DAP endosperms, respectively. Enrichment analysis indicated that the process of cell proliferation category was over-represented in the DEGs. Notably, two homologous genes, TaGW2-D1 and DvGW2, were identified as putative candidate genes associated with grain weight and yield. The expression analysis showed that DvGW2 had an aberrant expression in Pm97033, resulting in significantly lower total expression level of GW2 than Wan7107, which drives the increase of grain weight and width in Pm97033. Collectively, our data indicated that the compromised expression of DvGW2 is critical for increased grain width and weight in T6VS·6DL translocation lines.

Replication protein A large subunit (RPA1a) limits chiasma formation during rice meiosis.

Replication protein A (RPA), a single-stranded DNA-binding protein, plays essential role in homologous recombination. However, because deletion of RPA causes embryonic lethality in mammals, the exact function of RPA in meiosis remains unclear. In this study, we generated an rpa1a mutant using CRISPR/Cas9 technology and explored its function in rice (Oryza sativa) meiosis. In rpa1a, 12 bivalents were formed at metaphase I, just like in wild-type, but chromosome fragmentations were consistently observed at anaphase I. Fluorescence in situ hybridization assays indicated that these fragmentations were due to the failure of the recombination intermediates to resolve. Importantly, the mutant had a highly elevated chiasma number, and loss of RPA1a could completely restore the 12 bivalent formations in the zmm (for ZIP1-4, MSH4/5, and MER3) mutant background. Protein-protein interaction assays showed that RPA1a formed a complex with the methyl methansulfonate and UV sensitive 81 (and the Fanconi anemia complementation group M-Bloom syndrome protein homologs (RECQ4A)-Topoisomerase3α-RecQ-mediated genome instability 1 complex to regulate chiasma formation and processing of the recombination intermediates. Thus, our data establish a pivotal role for RPA1a in promoting the accurate resolution of recombination intermediates and in limiting redundant chiasma formation during rice meiosis.

Loss of smarcad1a accelerates tumorigenesis of malignant peripheral nerve sheath tumors in zebrafish.

Malignant peripheral nerve sheath tumors (MPNSTs) are a type of sarcoma that generally originates from Schwann cells. The prognosis for this type of malignancy is relatively poor due to complicated genetic alterations and the lack of specific targeted therapy. Chromosome fragment 4q22–23 is frequently deleted in MPNSTs and other human tumors, suggesting tumor suppressor genes may reside in this region. Here, we provide evidence that SMARCAD1, a known chromatin remodeler, is a novel tumor suppressor gene located in 4q22–23. We identified two human homologous smarcad1 genes (smarcad1a and smarcad1b) in zebrafish, and both genes share overlapping expression patterns during embryonic development. We demonstrated that two smarcad1a loss-of-function mutants, sa1299 and p403, can accelerate MPNST tumorigenesis in the tp53 mutant background, suggesting smarcad1a is a bona fide tumor suppressor gene for MPNSTs. Moreover, we found that DNA double-strand break (DSB) repair might be compromised in both mutants compared to wildtype zebrafish, as indicated by pH2AX, a DNA DSB marker. In addition, both SMARCAD1 gene knockdown and overexpression in human cells were able to inhibit tumor growth and displayed similar DSB repair responses, suggesting proper SMARCAD1 gene expression level or gene dosage is critical for cell growth. Given that mutations of SMARCAD1 sensitize cells to poly ADP ribose polymerase inhibitors in yeast and the human U2OS osteosarcoma cell line, the identification of SMARCAD1 as a novel tumor suppressor gene might contribute to the development of new cancer therapies for MPNSTs.

P–142 Comparison of the IVF outcome in the course of ICSI vs PICSI treatment continuation in exactly the same group of HBA abnormal patients

Abstract Study question Does the PICSI have a beneficial effect for men with abnormal HBA on the fertilization rate, blastocysts number and clinical pregnancies in the next attempt? Summary answer Patients with HBA <80% choosing to undergo PICSI after ICSI failure see an increase in blastocyst and pregnancy rates. What is known already Hyaluronic acid (HA) is a main component of cervical mucus and the extracellular matrix of cumulus cells. The formation of HA-binding sites in sperm cell membranes is one of the markers of sperm maturation indicating completion the spermatogenic process of remodelling the plasmatic membrane, cytoplasmic extrusion and nuclear maturity. Spermatozoa selected by the HA-binding technique (the physiologically selected intracytoplasmic sperm injection – PICSI) have a potentially reduced risk of chromosomal aneuploidy or DNA fragmentation. Recent evidence do not show significant benefits in using PICSI. However, it has not been analysed in the course of treatment continuation in the same patients. Study design, size, duration This was a retrospective case-control study. It included exactly the same 58 patients with abnormal HBA, who underwent IVF treatment with ICSI initially and later with PICSI, between January 2014 and October 2020 at INVICTA Fertility Centre, Poland. Median female partner age in PICSI group was 36,2±5,34, without PICSI 35,8 ±5,28. Participants/materials, setting, methods 275 cycles (130 ICSI and 145 PICSI) resulted in 793 and 897 MII respectively. Patients were also divided into two groups <80% and ≥80% depending on the obtained HBA score expressed as the percentage of sperm bound with hyaluronan. The analysis covered the fertilization rate (FR), TQ and total blastocyst rate on day 5 and clinical pregnancy rate. Patients with poor response to stimulation were excluded from the study. Main results and the role of chance FR in ICSI and PICSI groups was not significantly difference (57.00%±31.2 vs 59.87%±30.8) even when taking into account the division of patients according to the obtained HBA score. In the <80% group the FR was 57.04%±29.3 vs 59.54%±30.8 in ICSI vs PICSI group respectively. There were no significant differences when comparing the under HBA ≥80% subgroups for all analysed outcomes. Fertilization rate ​​was 56.88% in the ICSI group vs 61.03% in the PICSI group. The percentage of blastocysts was 28.61% vs 34.45% and the percentage of TQ blastocysts on day 5 was 15.32% vs 16.81% with ICSI and PICSI respectively, in the group consisting of the same patients. In the HBA <80% group significant differences were observed in the percentage of obtained blastocysts 37.81% vs 47.61% by comparing the ICSI and PICSI approaches (p < 0.05). Also, percentage of TQ blastocyst on day 5 also was higher in patients with <80% HBA score after PICSI and was statistically significant (17.07% ICSI vs 23.92% PICSI, p < 0.05). We saw statistically significant (p < 0.01) increase in percentage of clinical pregnancies from 29.03% without PICSI to 69.44% in patient’s subsequent procedures involving PICSI. Limitations, reasons for caution More data is required to confirm that improved results of PICSI procedure are consistent and possible to reproduce in a larger group – and as a result could be included as part of the standard treatment process. Wider implications of the findings: The presented results show that in patients with normal HBA score, PICSI does not bring a measurable benefit and this may be important factor to consider in decision-making for couples seeking assistance. Trial registration number Not applicable

Identification and fine mapping of alien fragments associated with enhanced grain weight from Agropyron cristatum chromosome 7P in common wheat backgrounds.

An enhanced grain weight locus from Agropyron cristatum chromosome 7P was verified in two wheat backgrounds, localized to the 7PS1-2 region. Novel translocation lines with this locus were evaluated. Agropyron cristatum is a wild relative of wheat that harbours elite genes for wheat improvement. The wheat-A. cristatum 7P disomic addition line II-5-1 exhibits high grain weight. Here, to dissect the genetic basis of grain weight contributed by A. cristatum chromosome 7P in wheat backgrounds, four segregated populations of the addition line were developed and evaluated in two wheat backgrounds. The results showed that A. cristatum chromosome 7P can stably and significantly increase the grain weight by approximately 2g, mainly by increasing grain length at different grain weight levels of the wheat background. The locus for increased grain weight from chromosome 7P shows dominant inheritance independent of the wheat background. Moreover, two deletion lines and 23 translocation lines were identified by cytological methods and molecular markers, and an enlarged chromosome 7P bin map was constructed with 158 STS markers and 40 bin intervals. With the genetic populations of these deletion and translocation lines, the genetic locus of increased grain weight was narrowed down to bin 7PS1-2. Two translocation lines (7PT-A18 and 7PT-B4) with smaller 7P chromosomal segments exhibited an increase in grain weight, grain length and grain width simultaneously. These translocation lines carrying the 7PS1-2 chromosomal fragment will be valuable genetic resources for wheat grain weight improvement. Collectively, this study uncovers the grain weight locus from chromosome 7P and provides novel pre-breeding lines with enhanced grain weight.

Highly Selective, CRISPR/Cas9-Mediated Isolation of Genes and Genomic Loci from Complex Genomes by TAR Cloning in Yeast.

Here we describe an updated TAR cloning protocol for the selective and efficient isolation of any genomic fragment or gene of interest up to 280 kb in size from genomic DNA. The method exploits the special recombination machinery of the yeast Saccharomyces cerevisiae. TAR cloning is based on the high level of in vivo recombination that occurs between a specific genomic DNA fragment of interest and targeting sequences (hooks) in a TAR vector that are homologous to the 5′ and 3′ ends of the targeted region. Upon co‐transformation into yeast, this results in the isolation of the chromosomal region of interest as a circular YAC molecule, which then propagates and segregates in yeast cells and can be selected for. In the updated TAR cloning protocol described here, the fraction of region‐positive clones typically obtained is increased from 1% up to 35% by pre‐treatment of the genomic DNA with specifically designed CRISPR/Cas9 endonucleases that create double‐strand breaks (DSBs) bracketing the target genomic DNA sequence, thereby making the ends of the chromosomal region of interest highly recombinogenic. In addition, a new TAR vector was constructed that contains YAC and BAC cassettes, permitting direct transfer of a TAR‐cloned DNA from yeast to bacterial cells. Once the TAR vector with the hooks is constructed and genomic DNA is prepared, the entire procedure takes 3 weeks to complete. The updated TAR protocol does not require significant yeast experience or extensively time‐consuming yeast work because screening only about a dozen yeast transformants is typically enough to find a clone with the region of interest. TAR cloning of chromosomal fragments, individual genes, or gene families can be used for functional, structural, and population studies, for comparative genomics, and for long‐range haplotyping, and has potential for gene therapy. Published 2021. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of CRISPR/Cas9‐treated genomic DNA for TAR cloning Basic Protocol 2: Isolation of a gene or genomic locus by TAR cloning Basic Protocol 3: Transfer of TAR/YAC/BAC isolates from yeast to E. coli

Cytotoxicity and Genotoxicity evaluation of municipal wastewater discharged into the head of Blue Nile River using the Allium Cepa test

Municipal wastewaters may contain a variety of genotoxic pollutants that are discharged into rivers and causing deterioration of the environment in downstream reaches. The present study evaluates the hazardous effects of untreated municipal wastewater discharge from the city of Bahir Dar to the head of the Blue Nile River. This was done by collecting wastewater samples from the upper, middle and lower sites of the stream and characterizing its physicochemical qualities. In all cases, tap water was taken as a control and the wastewater qualities were significantly varied among the studied sites. The highest values of EC, TDS, COD, etc. were found at the middle site. Further, the wastewater showed significant differences in terms of cytotoxicity (in root number, root length, mitotic index) and genotoxicity (chromosomal aberrations) in Allium cepa tests after three days of growth at (0, 25, 50, 75 and 100 ml) dilution values of wastewater. The cytotoxic effects of wastewater were significantly varied at different treatment values and collection sites of the stream. The mitotic index was reduced to 1.80 ± 0.03 from 12.59 ± 0.28, 1.26 ± 0.02 from 12.78 ± 0.38 and 2.05 ± 0.04 from 12.81 ± 0.19 at 100 ml of wastewater compared to the control at the upper, middle and lower sites respectively. The EC50 values for root growth inhibition followed the order middle < upper < lower, indicating greater toxicity of a middle site. Moreover, the wastewater incurred significantly more genotoxic effects on root tip cells in comparison to the control and also varied among sampling sites. Most recorded abnormalities were anaphase bridges, chromosome fragments, c-mitosis, binucleated and micronucleated cells. Thus untreated wastewater discharges have a potential threat to the water ecosystem and public health as the river is used for different purposes by downstream societies.

Cytogenetic abnormalities in young cattle during vaccination against salmonelloses

The spectrum and the frequencies of cytogenetic abnormalities in young cattle immunized with a vaccine against salmonellosis of calves were investigated. The study was carried out on the farm of Novosibirsk region on 10 clinically healthy Holstein black-and-white calves of 10-17 days of age. A concentrated formol-alum vaccine against salmonellosis (paratyphoid) of calves was used at a dose of 2 ml (reimmunization at a dose of 2 ml) with an interval of 10 days between injections. The vaccine was made from the culture of bacteria of the Salmonella dublin strain № 373, inactivated with formalin with the addition of potassium alum and calcium chloride. Cytogenetic analysis of peripheral blood in calves was carried out before vaccination (control), 2 and 9 days after vaccination, 2 and 9 days after revaccination. It was found that the spectrum of somatic chromosomal instability in peripheral blood lymphocytes of calves after vaccination and revaccination is represented by polyploidy, hypoploidy and hyperploidy, chromatid and chromosomal breaks, single and paired fragments of chromosomes. It was revealed that the spectrum of somatic chromosomal instability after double immunizations with an inactivated vaccine against salmonellosis did not differ from the spectrum of spontaneously occurring mutations in this species. Vaccination and subsequent revaccination of calves in comparison with the pre-vaccination period did not lead to a significant increase in the frequency of aneuploid and polyploid cells. During double immunization of calves, a wave pattern in the variation of genomic mutation frequencies from maximum to minimum values in the lymphocytic blood cells of animals was noted, similar to prolonged mutagenesis. A tendency was found for the frequency of structural chromosome abnormalities to increase 2 and 9 days after vaccination and 2 days after revaccination. There was a credible 2.9-fold increase in the frequency of cells with chromosomal aberrations in the blood lymphocytes of animals 9 days after their repeated immunization due to breaks and paired fragments of chromosomes. After vaccination and revaccination, chromatid breaks were most often recorded in the medial regions of one of the chromatids, and chromosomal breaks in the medial and telomeric regions of both chromatids.

Chronic exposure to Cytolethal Distending Toxin (CDT) promotes a cGAS-dependent type I interferon response

The Cytolethal Distending Toxin (CDT) is a bacterial genotoxin produced by pathogenic bacteria causing major foodborne diseases worldwide. CDT activates the DNA Damage Response and modulates the host immune response, but the precise relationship between these outcomes has not been addressed so far. Here, we show that chronic exposure to CDT in HeLa cells or mouse embryonic fibroblasts promotes a strong type I interferon (IFN) response that depends on the cytoplasmic DNA sensor cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) through the recognition of micronuclei. Indeed, despite active cell cycle checkpoints and in contrast to other DNA damaging agents, cells exposed to CDT reach mitosis where they accumulate massive DNA damage, resulting in chromosome fragmentation and micronucleus formation in daughter cells. These mitotic phenotypes are observed with CDT from various origins and in cancer or normal cell lines. Finally, we show that CDT exposure in immortalized normal colonic epithelial cells is associated to cGAS protein loss and low type I IFN response, implying that CDT immunomodulatory function may vary depending on tissue and cell type. Thus, our results establish a direct link between CDT-induced DNA damage, genetic instability and the cellular immune response that may be relevant in the context of natural infection associated to chronic inflammation or carcinogenesis.