Formation Of Chromosomes Research Articles

Elucidating the toxicity mechanism of AFM2 and the protective role of quercetin in albino mice

Aflatoxin M2 (AFM2) is a type of mycotoxin detected in milk or dairy products from animals consuming contaminated feed. In this study, the toxicity mechanism of AFM2 and the protective effects of quercetin were investigated in albino mice. For this purpose, the mice were divided into 6 groups and the groups were fed with quercetin and AFM2. The toxic effects of AFM2 and the protective properties of quercetin were investigated using physiological, biochemical and cytogenetic parameters. The genotoxic mechanism of AFM2 and the protective role of quercetin were investigated by molecular docking, which is an in silico model. As a result, 16 mg/kg b.w AFM2 administration caused serious changes in body weight, organ index, kidney and liver weight, and deterioration of antioxidant/oxidant balance in liver and kidney organs. The decrease in glutathione levels along with an increase in malondialdehyde (MDA) levels in the liver and kidney after AFM2 administration indicates that oxidative stress is induced. The increases in alanine transaminase (ALT) and aspartat transaminase (AST) levels, which are indicators of liver damage, and the increases in serum levels of blood urea nitrogen (BUN) and creatinine, which are indicators of kidney damage, confirm the damage in both organs. AFM2 also caused genotoxicity by inducing micronucleus (MN) and chromosomal abnormalities (CAs) in bone marrow tissue. It has been determined that AFM2, which exhibits genotoxicity as a result of its clastogenic and aneugenic effects, causes CAs by interacting with DNA. Quercetin provided significant protection by improving liver and kidney tissues, partial normalization in serum parameter levels, and severe reductions in MN and CAs. The highest protection was determined as 74.1% against dicentric chromosome formations in 50 mg/kg b.w quercetin application. The interaction of quercetin with xanthine oxidase and nitric oxide synthase enzymes was determined in silico with an inhibition constant in the range of 283.71–476.17 nM. These interactions cause changes in the activity of enzymes, reducing the oxidative load in the cell, and in this way, quercetin provides protection. All toxic effects induced by AFM2 were decreased with quercetin administration dose-dependently, and this protective effect was associated with quercetin's reduction of oxidative load by inhibiting the free radical-producing enzyme. All toxic effects caused by AFM2 were decreased with quercetin administration in a dose-dependent manner, and this protective effect was associated with quercetin's reduction of oxidative load by inhibiting the enzyme that produces free radicals.

Characterization of a new IN-I-PpoI fusion protein and a homology-arm containing transgene cassette that improve transgene expression persistence and 28S rRNA gene-targeted insertion of lentiviral vectors.

Targeting transgene integration into a safe genomic locus would be very important for gene therapy. We have generated lentivirus vectors containing the ribosomal RNA-recognising I-PpoI endonuclease fused to viral integrase, and transgene cassettes with target site homology arms to enhance insertion targeting. These new vectors were characterised with respect to the persistence of transgene expression, insertion targeting efficiency and chromosomal integrity of the transduced cells. The aim was to find an optimally safe and effective vector for human gene therapy. Fusion protein vectors with high endonuclease activity were the most effective in the accurate targeting of transgene insertion. The homology construct increased the insertion targeting efficiency to 28% in MRC-5 cells. However, karyotyping analysis showed that the high endonuclease activity induced the formation of derivative chromosomes in as many as 24% of the analysed primary T lymphocytes. The persistence of transgene expression was excellent in homology arm-containing fusion protein vectors with reduced endonuclease activity, and these fusion proteins did not cause any detectable chromosomal rearrangements attributable to the endonuclease activity. We thus conclude that instead of the fusion protein vectors that carry a highly active endonuclease, our vectors with the ability to tether the lentivirus preintegration complex to benign loci in the genome without high ribosomal DNA cleavage activity are better suited for lentivirus-based gene therapy applications.

High-density linkage maps and chromosome level genome assemblies unveil direction and frequency of extensive structural rearrangements in wood white butterflies (Leptidea spp.)

Karyotypes are generally conserved between closely related species and large chromosome rearrangements typically have negative fitness consequences in heterozygotes, potentially driving speciation. In the order Lepidoptera, most investigated species have the ancestral karyotype and gene synteny is often conserved across deep divergence, although examples of extensive genome reshuffling have recently been demonstrated. The genus Leptidea has an unusual level of chromosome variation and rearranged sex chromosomes, but the extent of restructuring across the rest of the genome is so far unknown. To explore the genomes of the wood white (Leptidea) species complex, we generated eight genome assemblies using a combination of 10X linked reads and HiC data, and improved them using linkage maps for two populations of the common wood white (L. sinapis) with distinct karyotypes. Synteny analysis revealed an extensive amount of rearrangements, both compared to the ancestral karyotype and between the Leptidea species, where only one of the three Z chromosomes was conserved across all comparisons. Most restructuring was explained by fissions and fusions, while translocations appear relatively rare. We further detected several examples of segregating rearrangement polymorphisms supporting a highly dynamic genome evolution in this clade. Fusion breakpoints were enriched for LINEs and LTR elements, which suggests that ectopic recombination might be an important driver in the formation of new chromosomes. Our results show that chromosome count alone may conceal the extent of genome restructuring and we propose that the amount of genome evolution in Lepidoptera might still be underestimated due to lack of taxonomic sampling.

Maintaining soluble protein homeostasis between nuclear and cytoplasmic compartments across mitosis.

The nuclear envelope (NE) is central to the architecture of eukaryotic cells, both as a physical barrier separating the nucleus from the cytoplasm and as gatekeeper of selective transport between them. However, in open mitosis, the NE fragments to allow for spindle formation and segregation of chromosomes, resulting in intermixing of nuclear and cytoplasmic soluble fractions. Recent studies have shed new light on the mechanisms driving reinstatement of soluble proteome homeostasis following NE reformation in daughter cells. Here, we provide an overview of how mitotic cells confront this challenge to ensure continuity of basic cellular functions across generations and elaborate on the implications for the proteasome - a macromolecular machine that functions in both cytoplasmic and nuclear compartments.

Associação entre refluxo vesicoureteral e anomalia do cromossomo 22 em anel - relato de caso

Vesicoureteral reflux (VUR) is characterized by retrograde urine flow, caused by impaired occlusion of the intramural portion of the ureter by contraction of the detrusor muscle during urination or secondary to altered bladder dynamics, ureteral obstruction and neuromuscular diseases, such as Phelans syndrome -McDermid. This syndrome is a rare and underreported disease, resulting from the formation of chromosome 22 in a ring and has VUR as its main urological manifestation. The objective of this study is to alert the medical community about one of the differential diagnoses in the VUR etiology, based on the case of a female patient, 4 years and 2 months old, referred to the Pediatric Surgery service of the Mário Covas State Hospital with recurrent infection of urinary tract associated with impaired renal function and bilateral VUR evidenced on cystourethrography. During investigation, the phenotypic findings encouraged the performance of a karyotype that confirmed the alterations found in Phelan-McDermid syndrome. VUR early diagnosis was essential to establish the appropriate therapy, which involved clinical and surgical approaches aimed at improving quality of life, preserving the renal parenchyma and preventing further future complications.

ASSOCIAÇÃO ENTRE REFLUXO VESICOURETERAL E ANOMALIA DO CROMOSSOMO 22 EM ANEL - RELATO DE CASO

Vesicoureteral reflux (VUR) is characterized by retrograde urine flow, caused by impaired occlusion of the intramural portion of the ureter by contraction of the detrusor muscle during urination or secondary to altered bladder dynamics, ureteral obstruction and neuromuscular diseases, such as Phelans syndrome - McDermid. This syndrome is a rare and underreported disease, resulting from the formation of chromosome 22 in a ring and has VUR as its main urological manifestation. The objective of this study is to alert the medical community about one of the differential diagnoses in the VUR etiology, based on the case of a female patient, 4 years and 2 months old, referred to the Pediatric Surgery service of the Mário Covas State Hospital with recurrent infection of urinary tract associated with impaired renal function and bilateral VUR evidenced on cystourethrography. During investigation, the phenotypic findings encouraged the performance of a karyotype that confirmed the alterations found in PhelanMcDermid syndrome. VUR early diagnosis was essential to establish the appropriate therapy, which involved clinical and surgical approaches aimed at improving quality of life, preserving the renal parenchyma and preventing further future complications.

Bridge the gap between fixed-length and variable-length evolutionary neural architecture search algorithms

<abstract><p>Evolutionary neural architecture search (ENAS) aims to automate the architecture design of deep neural networks (DNNs). In recent years, various ENAS algorithms have been proposed, and their effectiveness has been demonstrated. In practice, most ENAS methods based on genetic algorithms (GAs) use fixed-length encoding strategies because the generated chromosomes can be directly processed by the standard genetic operators (especially the crossover operator). However, the performance of existing ENAS methods with fixed-length encoding strategies can also be improved because the optimal depth is regarded as a known priori. Although variable-length encoding strategies may alleviate this issue, the standard genetic operators are replaced by the developed operators. In this paper, we proposed a framework to bridge this gap and to improve the performance of existing ENAS methods based on GAs. First, the fixed-length chromosomes were transformed into variable-length chromosomes with the encoding rules of the original ENAS methods. Second, an encoder was proposed to encode variable-length chromosomes into fixed-length representations that can be efficiently processed by standard genetic operators. Third, a decoder cotrained with the encoder was adopted to decode those processed high-dimensional representations which cannot directly describe architectures into original chromosomal forms. Overall, the performances of existing ENAS methods with fixed-length encoding strategies and variable-length encoding strategies have both improved by the proposed framework, and the effectiveness of the framework was justified through experimental results. Moreover, ablation experiments were performed and the results showed that the proposed framework does not negatively affect the original ENAS methods.</p></abstract>

BAC-FISH Based Physical Map of Endangered Catfish Clarias magur for Chromosome Cataloguing and Gene Isolation through Positional Cloning.

Construction of a physical chromosome map of a species is important for positional cloning, targeted marker development, fine mapping of genes, selection of candidate genes for molecular breeding, as well as understanding the genome organization. The genomic libraries in the form of bacterial artificial chromosome (BAC) clones are also a very useful resource for physical mapping and identification and isolation of full-length genes and the related cis acting elements. Some BAC-FISH based studies reported in the past were gene based physical chromosome maps of Clarias magur (magur) to understand the genome organization of the species and to establish the relationships with other species in respect to genes' organization and evolution in the past. In the present study, we generated end sequences of the BAC clones and analyzed those end sequences within the scaffolds of the draft genome of magur to identify and map the genes bioinformatically for each clone. A total of 36 clones mostly possessing genes were identified and used in probe construction and their subsequent hybridization on the metaphase chromosomes of magur. This study successfully mapped all 36 specific clones on 16 chromosome pairs, out of 25 pairs of magur chromosomes. These clones are now recognized as chromosome-specific makers, which are an aid in individual chromosome identification and fine assembly of the genome sequence, and will ultimately help in developing anchored genes' map on the chromosomes of C. magur for understanding their organization, inheritance of important fishery traits and evolution of magur with respect to channel catfish, zebrafish and other species.

Chromosome arm length, and a species-specific determinant, define chromosome arm width.

Mitotic chromosomes in different organisms adopt various dimensions. What defines these dimensions is scarcely understood. Here, we compare mitotic chromosomes in budding and fission yeasts harboring similarly sized genomes distributed among 16 or 3 chromosomes, respectively. Hi-C analyses and superresolution microscopy reveal that budding yeast chromosomes are characterized by shorter-ranging mitotic chromatin contacts and are thinner compared with the thicker fission yeast chromosomes that contain longer-ranging mitotic contacts. These distinctions persist even after budding yeast chromosomes are fused to form three fission-yeast-length entities, revealing a species-specific organizing principle. Species-specific widths correlate with the known binding site intervals of the chromosomal condensin complex. Unexpectedly, within each species, we find that longer chromosome arms are always thicker and harbor longer-ranging contacts, a trend that we also observe with human chromosomes. Arm length as a chromosome width determinant informs mitotic chromosome formation models.

Directed Evolution Detects Supernumerary Centric Chromosomes Conferring Resistance to Azoles in Candida auris.

Candida auris exhibits resistance to multiple antifungal drug classes and sterilization agents, posing threats to the immunocompromised worldwide. Among the four major geographical clades, the East Asian clade 2 isolates of C. auris are mostly drug susceptible. In this study, we experimentally evolved one such drug-susceptible isolate for multiple generations in the presence of the antifungal compound fluconazole and analyzed changes in the karyotype, DNA sequence, and gene expression profiles in three evolved drug-resistant isolates. Next-generation sequencing and electrophoretic karyotyping confirm the presence of segmental aneuploidy as supernumerary chromosomes originating from centromere-inclusive chromosomal duplication events in two such cases. A 638-kb region and a 675-kb region, both of which originated from chromosome 5 and contained its centromere region, are instances of supernumerary chromosome formation identified in two evolved fluconazole-resistant isolates. Loss of the supernumerary chromosomes from the drug-resistant isolates results in a complete reversal of fluconazole susceptibility. Transcriptome analysis of the third isolate identified overexpression of drug efflux pumps as a possible non-aneuploidy-driven mechanism of drug resistance. Together, this study reveals how both aneuploidy-driven and aneuploidy-independent mechanisms may operate in parallel in an evolving population of C. auris in the presence of an antifungal drug, in spite of starting from the same strain grown under similar conditions, to attain various levels of fluconazole resistance. IMPORTANCE Fungal pathogens develop drug resistance through multiple pathways by acquiring gene mutations, increasing the copy number of genes, or altering gene expression. In this study, we attempt to understand the mechanisms of drug resistance in the recently emerged superbug, C. auris. One approach to studying this aspect is identifying various mechanisms operating in drug-resistant clinical isolates. An alternative approach is to evolve a drug-susceptible isolate in the presence of an antifungal compound and trace the changes that result in drug resistance. Here, we evolve a drug-susceptible isolate of C. auris in the laboratory in the presence of a widely used antifungal compound, fluconazole. In addition to the already known changes like overexpression of drug efflux pumps, this study identifies a novel mechanism of azole resistance by the emergence of additional chromosomes through segmental duplication of chromosomal regions, including centromeres. The centric supernumerary chromosome helps stable amplification of a set of genes with an extra copy to confer fluconazole resistance.

Male meiosis, sporad analysis and pollen viability in a threatened medicinal plant Oroxylum indicum (L.) Kurz

Oroxylum indicum (L.) Kurz a well know and pharmaceutically well explored medicinal plant of the family Bignoniaceae but in the context of cytological work, meagre information is available on same plant. During present investigation six accessions of O. indicum were studied for understanding male meiosis and related events using standard techniques. Although, the meiotic course was found to be regular, anomalies such as chromosomal stickiness and unequal distribution of chromosomes existed in trace percentage and when compared, meiosis- II was found to be more disturbed than meiosis-I. The presence of monads, dyads, triads, and the sporads with micronuclei were common, although in low frequency. Significantly, in spite of chromosomal anomalies and abnormal tetrad formation, the pollens were healthy and viable. The variable values of analyzed cytological parameters among different types (morphs) of stamens suggested a prevalence of cytological dimorphism along with morphological dimorphism in O. indicum.

The B. subtilis Rok protein is an atypical H-NS-like protein irresponsive to physico-chemical cues.

Nucleoid-associated proteins (NAPs) play a central role in chromosome organization and environment-responsive transcription regulation. The Bacillus subtilis-encoded NAP Rok binds preferentially AT-rich regions of the genome, which often contain genes of foreign origin that are silenced by Rok binding. Additionally, Rok plays a role in chromosome architecture by binding in genomic clusters and promoting chromosomal loop formation. Based on this, Rok was proposed to be a functional homolog of E. coli H-NS. However, it is largely unclear how Rok binds DNA, how it represses transcription and whether Rok mediates environment-responsive gene regulation. Here, we investigated Rok's DNA binding properties and the effects of physico-chemical conditions thereon. We demonstrate that Rok is a DNA bridging protein similar to prototypical H-NS-like proteins. However, unlike these proteins, the DNA bridging ability of Rok is not affected by changes in physico-chemical conditions. The DNA binding properties of the Rok interaction partner sRok are affected by salt concentration. This suggests that in a minority of Bacillus strains Rok activity can be modulated by sRok, and thus respond indirectly to environmental stimuli. Despite several functional similarities, the absence of a direct response to physico-chemical changes establishes Rok as disparate member of the H-NS family.

High-Throughput DNA Sequencing Identifies a Novel Splice-Site Mutation in Fanconi Anemia Pathway Gene FANCD2 As a Potential Biomarker of Early and Late Phases of Disease Progression in Chronic Myeloid Leukemia

OtherAuthors: Muhammad Absar4, Tanveer Akhtar4, Masood Shammas8, Salman Basit9, Aamer Aleem10, Amer Mahmood10 8Dana Farbar Cancer Institute, University of Harvard , Boston USA, 9Centre for Genetics and Inherited Diseases, Taibah University, Madinah 10College of Medicine, King Saud University, Riyadh, Saudi Arabia. Introduction: Chronic Myeloid Leukemia (CML) results due to t(22;9) that leads to formation of Philadelphia chromosome and subsequently BCR-ABL fusion transcript, the later leading to leukemogenesis(1). This led to identification of tyrosine kinase inhibitors (TKIs) as novel drugs that revolutionised CML treatment in 21st century by improving the survival of CML equal to general population at least in developed countries (2). Nevertheless TKIs lose their effectiveness if CML patients progress to blast crisis (BC-CML). Despite extensive research in CML field, mechanism of its progression is still poorly understood and universal molecular biomarkers of CML progression are unknown (2). There are different mechanisms associated with initiation and progression of various cancers. DNA repair defects mediated through biologically very important Fanconi anemia (FA) pathway genes are associated with many cancers (3). Nevertheless, FA pathway genes have not been reported in myeloid malignancies. In this study, in searching for mutated genes associated with many mechanism, we hereby report mutations in a very important FA-pathway gene associated exclusively with CML progression for the first time in literature. Materials and Methods: Patient selection: Whole-exome sequencing (WES) was employed to find out mutations in novel TF-genes associated with accelerated phase (AP-CML) and blast crisis phase CML (BC-CML) patients (Experimental groups 1 & 2, respectively). Chronic phase treatment-naïve CML patients (CP-CML) were used as control 1, CP-CML CML long-term TKI responders (at least 3 continuous years of MMR) as control group 2 and TK-resistant CML patients as control group 3, along with healthy controls. Sample collection: DNA extraction and Clinical follow-up: 10 ml peripheral blood was collected from all study subjects. DNA was extracted and patient follow-up was carried out during course of this study. All treatment criteria per ENL guidelines were adopted. Whole Exome Sequencing (WES): WES was carried out using Illumina NGS instrument (HiSeq). bcl files were converted to fastq files by using bcl2fastqtool (4). Raw reads were aligned to genome using BWA tools while whole exome variants were annotated using Illumina Variant Studio (4). R package was employed to align gene mutants to disease phenotypes (5). DNA repair genes mutated in all AP/BC-CML patients but not mutated in any of control groups were selected. Variants were confirmed using Sanger sequencing. Results: A novel splice-site substitution (C2022T) in FANCD2 gene (that is a very important member of FA-pathway genes) was detected in all AP/BC-CML but in none of the controls. It shows that mutated FANCD2 can serve as a novel biomarker exclusively for advanced phases of CML. Discussion: It is the first report that a member of FA-pathway genes (FANCD2) has been reported mutated in progression of myeloid malignancies (6). It shows that DNA repair defects and genomic instability mediated through FA-pathway could be one of the important mechanisms in CML progression. Mutations in FANCD2 and such other genes could be reason why CML blast crisis patients have poor outcome of stem cell transplantation. Moreover, anti-cancer drugs targeting DNA repair defects (PARP inhibitors) like Olaparib in combination with TKIs could be effective in patients with mutated FANCD-2 and other such genes. Conflict of interest: The authors declare that they have no conflict of interests.

P029: Are Reed-Sternberg cells stuck in mitosis by short nucleoplasmic bridges as a consequence of centromeric instability?

Background: Multinucleated giant tumor cells are frequently observed in lymph nodes of untreated lymphoma patients. Malignant Hodgkin lymphoma (HL) cells i. e. small Hodgkin and large bi- or multinucleated Reed-Sternberg (HRS) cells are characterized by genomic instability and downregulation of numerous key regulators of e.g. cell cycle, spindle apparatus, cytokinesis and cell lineage differentiation. So far, the formation of HRS cells remains obscure. We have demonstrated previously that telomere instability is involved in the proliferation of small cells into large binucleated cells. Here, we have used a collection of HL cell lines to analyze the successive steps involved in the transformation of the mononucleated into the multinucleated RS-like cells. Materials and Methods: Seven HL cell lines and 30 HL lymph nodes were used. Cytokinesis-block micronucleus assay and telomere and centromere staining followed by the M-FISH technique were employed to analyze chromosomal instability. DNA repair pathways were investigated. Results: We demonstrate that telomere dysfunction in HL cell lines is associated with the formation of dicentric chromosomes with both centromeres in close proximity, but also with a high rate of formation of micronuclei. Two morphologically different types of nucleoplasmic bridges (NPBs) were observed: (1) Long NPBs related to telomere dysfunction and chromosome fusions, and (2) short NPBs related to centromere breakpoints with configurations of cells looking like “stuck” together as binucleated cells. Short NPBs and binucleated cells were seen in the cultures of all HL cell lines. However, each HL cell line was characterized by an individual signature of the proportion of long and short NPBs, which correlated with centrosome amplification and formation of stable binucleated cells. Similar mechanisms were identified in HL patient lymph nodes prior to treatment, thus underscoring the biological significance of our findings in cell cultures. Transcriptome analysis confirmed the variations in the DNA repair pathways regarding the rate of large cells among the different HL cell lines. Conclusion: The formation of dicentric chromosomes related to centromere instability, and short NPBs were associated with the emergence of binucleated cells through different mechanisms. Our findings open a novel route to understanding the transition from mononucleated cells to multinucleated cells in HL and in other B-cell lymphomas.

Development and applications of a collection of single copy gene-based cytogenetic DNA markers in garden asparagus.

Garden asparagus (Asparagus officinalis, 2n = 2x = 20 chromosomes) is an important dioecious vegetable crop and a model species for studying sex chromosome formation and evolution. However, few molecular cytogenetic studies on garden asparagus have been reported because of its small metaphase chromosomes, the scarcity of distinguished cytogenetic markers, and the high content of repetitive sequences. In this study, a set of single copy genes free of repetitive sequences with sizes ranging from 4.3 kb to 8.2 kb were screened and used as probes for fluorescence in situ hybridization (FISH) to identify individual chromosomes of garden asparagus. The chromosome-specific signal distribution patterns of these probes enabled the distinguishment of each pair of chromosomes. The sequence assembly and cytogenetic map were successfully integrated, and the results confirmed that the chromosome 1 representing the sex chromosome in the genome assembly is chromosome 5 in the karyotype analysis. The cytogenetic identification of the male-specific region of the Y chromosome (MSY) was implemented using a mixed probe derived from a number of MSY-specific single copy sequences. In addition, the chromosome orthologous relationship between garden asparagus (A1–A10, karyotypic analysis) and its hermaphrodite close relative, A. setaceus (B1–B10, karyotypic analysis), was analyzed using this collection of chromosome-specific cytological markers. The results showed that B3 is the ortholog of sex chromosome A5 and thus may represent the ancestral autosome of the current sex chromosome in garden asparagus. Chromosomes B5, B4, B1, B8, B7, and B9 are the orthologs of A2, A3, A4, A7, A8, and A10, respectively. The chromosome identification, cytogenetic recognition of MSY, and the orthologous relationship analysis between garden asparagus and A. setaceus are valuable for the further investigation of the sex chromosome emergence and evolutionary mechanism of garden asparagus and genome structure evolution in the Asparagus genus.

Metal chelating and anti-radical activity of Salvia officinalis in the ameliorative effects against uranium toxicity

Uranium is a highly radioactive heavy metal that is toxic to living things. In this study, physiological, cytogenetic, biochemical and anatomical toxicity caused by uranium and the protective role of sage (Salvia officinalis L.) leaf extract against this toxicity were investigated with the help of Allium test. Germination percentage, root length, weight gain, mitotic index (MI), micronucleus (MN) formation, chromosomal aberrations (CAs), superoxide dismutase (SOD) and catalase (CAT) enzyme activities, malondialdehyde (MDA) levels and changes in root meristem cells were used as indicators of toxicity. In the experimental stage, a total of six groups, one of which was the control, were formed. Group I was treated with tap water, while group II and III were treated only with sage (190 mg/L and 380 mg/L). Groups IV, V and VI were germinated with uranyl acetate dihydrate (0.1 mg/mL), uranyl acetate dihydrate + 190 mg/L sage and uranyl acetate dihydrate + 380 mg/L sage, respectively. Allium cepa L. bulbs of each group were germinated for 72 h, and at the end of the period, routine preparation techniques were applied and physiological, cytogenetic, biochemical and anatomical analyzes were performed. As a result, uranium application caused a significant decrease (p < 0.05) in all physiological parameters and MI values. MN, CAs numbers, SOD and CAT enzyme activities and MDA levels increased significantly (p < 0.05) with uranium application. Uranium promoted CAs in the root tip cells in the form of fragment, vagrant chromosome, sticky chromosome, bridge and unequal distribution of chromatin. In addition, it caused anatomical damages such as epidermis cell damage, cortex cell damage and flattened cell nucleus in root tip meristem cells. Sage application together with uranium caused significant (p < 0.05) increases in physiological parameters and MI values and significant decreases in MN, CAs, SOD and CAT activities and MDA levels. In addition, the application of sage resulted in improvement in the severity of anatomical damages induced by uranium. It was determined that the protective role of sage observed for all parameters investigated was even more pronounced at dose of 380 mg/L. The protective role of sage against uranium toxicity is related to its antioxidant activity, and sage has 82.8% metal chelating and 72.9% DPPH removal activity. As a result, uranyl acetate exhibited versatile toxicity in A. cepa, caused cytotoxicity by decreasing the MI rate, and genotoxicity by increasing the frequencies of MN and CAs. And also, Sage acted as a toxicity-reducing agent by displaying a dose-dependent protective role against the toxic effects induced by uranyl acetate.

Visualizing Active Replication Regions in S-Phase Chromosomes.

A basic question of cell biology is how DNA folds to chromosome. A number of recently accumulated evidences have suggested that folding of chromosome proceeds tightly coupled with DNA replication progresses. Drug-induced PCC is a useful tool for visualization of the interphase nuclei, in particular, S-phase, as S-phase prematurely condensed chromosomes (S-phase PCC). Active replicating DNA is labeled directly with Cy3-dUTP by bead loading method, and then S-phase nuclei is immediately condensed prematurely by calyculin A to obtain S-phase PCC. Active replicating regions on S-PCC are observed under a scanning confocal microscope. Cy3-dUTP-labeled S-phase PCCs clearly reveal the drastic transitional change of chromosome formation through S-phase, starting from a "cloudy nebula" to numerous numbers of "beads on a string" and finally to "striped arrays of banding structured chromosome" known as G- or R-banding pattern. The number, distribution, and shape of replication foci were also measured in individual subphase of S-phase; maximally ~1400 foci of 0.35 μm average radius size were scored at the beginning of S-phase, and the number is reduced to ~100 at the end of S-phase. Drug-induced PCC clearly provided the new insight that eukaryote DNA replication is tightly coupled with the chromosome condensation/compaction for construction of eukaryote higher-ordered chromosome structure.

Macromolecular Structure of Linearly Arranged Eukaryotic Chromosomes.

Eukaryotic chromosomes have not been visualized during the interphase. The fact that chromosomes cannot be seen during the interphase of the cell cycle does not mean that there are no means to make them visible. This work provides visual evidence that reversible permeabilization of the cell membrane followed by the regeneration of cell membranes allows getting a glimpse behind the nuclear curtain. Reversibly permeable eukaryotic cells have been used to synthesize nascent DNA, analyze the 5′-end of RNA primers, view individual replicons and visualize interphase chromosomes. Dextran T-150 in a slightly hypotonic buffer prevented cells from disruption. Upon reversal of permeabilization, the nucleus could be opened at any time during the interphase. A broad spectrum of a flexible chromatin folding pattern was revealed through a series of transient geometric forms of chromosomes. Linear attachment of chromosomes was visualized in several mammalian and lower eukaryotic cells. The linear connection of chromosomes is maintained throughout the cell cycle showing that rather than individual chromosomes, a linear array of chromosomes is the functional giant macromolecule. This study proves that not only the prokaryotic genome but also linearly attached eukaryotic chromosomes form a giant macromolecular unit.

The Roles of Histone Post-Translational Modifications in the Formation and Function of a Mitotic Chromosome.

During mitosis, many cellular structures are organized to segregate the replicated genome to the daughter cells. Chromatin is condensed to shape a mitotic chromosome. A multiprotein complex known as kinetochore is organized on a specific region of each chromosome, the centromere, which is defined by the presence of a histone H3 variant called CENP-A. The cytoskeleton is re-arranged to give rise to the mitotic spindle that binds to kinetochores and leads to the movement of chromosomes. How chromatin regulates different activities during mitosis is not well known. The role of histone post-translational modifications (HPTMs) in mitosis has been recently revealed. Specific HPTMs participate in local compaction during chromosome condensation. On the other hand, HPTMs are involved in CENP-A incorporation in the centromere region, an essential activity to maintain centromere identity. HPTMs also participate in the formation of regulatory protein complexes, such as the chromosomal passenger complex (CPC) and the spindle assembly checkpoint (SAC). Finally, we discuss how HPTMs can be modified by environmental factors and the possible consequences on chromosome segregation and genome stability.

A mini-review of the role of condensin in human nervous system diseases.

Mitosis and meiosis are crucial life activities that transmit eukaryotic genetic information to progeny in a stable and orderly manner. The formation and appearance of chromosomes, which are derived from chromatin, are the preconditions and signs of mitosis. When entering mitosis, interphase loose chromatin is highly spiralized and folded to form compact chromosomes. In recent years, it has been found that in addition to the well-known DNA, histones, and topoisomerase, a large protein complex called condensin plays an important role in the process of chromosome formation. Numerous studies have shown that the abnormal function of condensin can lead to incomplete or excessive concentration of chromatin, as well as disorder of genome organization process, abnormal transmission of genetic information, and ultimately lead to various diseases of individual, especially in nervous system diseases. In this review, the biological function of condensin and the potential pathogenic mechanism of condensin in nervous system diseases are briefly summarized. Therefore, the investigation of these mechanisms makes a significant contribution to the understanding of those related diseases and provides new ideas for clinical treatments.