Metaphase Chromosome Spreads Research Articles

Effects of Colchicine and Trypsin Treatments on Metaphase Chromosome Spreading from Larvae and Different Tissues of Adult Female and Male Nile Tilapia

Assessment of results of different biotechnological methods in aquaculture such as ploidy, gynogenesis, and androgenesis are needed cytogenetical techniques. Achieving well-distributed metaphase chromosome spreads is a preeminent step for cytogenetical techniques. The aim of this study was to evaluate the effects of colchicine and trypsin treatments on the yield of countable metaphase chromosome spreads. With this aim, chromosome preparations from gill, spleen, kidney, and caudal fin tissues of adult females and males Nile tilapia, and also whole body of their larvae were carried out. Based on the previous studies reviewed so far, this is the first systematic and extensive study indicated that the effects of colchicine and trypsin on metaphase chromosome spreads regarding gender differences and larvae. In the chromosome preparations, it was designated four treatments as “Trypsin (-) Colchicine (-)”, “Trypsin (-) Colchicine (+)”, “Trypsin (+) Colchicine (-)”, and “Trypsin (+) Colchicine (+)”. Effects of colchicine and trypsin in terms of their usage in chromosome preparations were evaluated by determining the frequencies of metaphase cells. For this purpose, metaphase index were used to reveal differences in detected metaphase cells among the treatments. The highest metaphase index values were calculated in the Trypsin (+) Colchicine (+) treatment group of all tissues. Also, the scores of metaphase index found in spleen and kidney tissues were greater than those found in gill and fin tissues. The present study has also shown that metaphase chromosome spreads could be obtained without colchicine or trypsin treatments.

Discovery of KPT-6566 as STAG1/2 Inhibitor sensitizing PARP and NHEJ Inhibitors to suppress tumor cells growth in vitro

Stromal antigen 1 and 2 (STAG1 and STAG2) are two mutually exclusive components of the cohesin complex that is crucial for centromeric and telomeric cohesion. Beyond its structural role, STAG2 also plays a pivotal role in homologous recombination (HR) repair and has emerged as a promising therapeutic target in cancer treatment. Here, we employed a fluorescence polarization (FP)-based high-throughput screening and identified KPT-6566 as a dual inhibitor of STAG1 and STAG2. Biochemical and biophysical analyses demonstrated that KPT-6566 directly binds to STAG1 and STAG2, disrupting their interactions with SCC1 and double-stranded DNA. A metaphase chromosome spread assay showed that KPT-6566 causes premature chromosome separation and induces chromosome damages in HeLa cells. Furthermore, KPT-6566 also impairs DNA damage repair, leading to the accumulation of double-strand breaks and cell apoptosis. Finally, KPT-6566 can sensitize HeLa and HepG2 cells to PARP inhibitor Olaparib and the NHEJ inhibitor UMI-77, exhibiting a synergistic effect in suppressing cell proliferation. Our findings highlight the potential of STAG1/2 as promising therapeutic targets in cancer treatment, particularly when they are targeted in combination with other DNA damage response inhibitors.

Preliminary cytogenetic study of <em style="mso-bidi-font-style: normal;">Ctenopoma kingsleyae</em> from Esa-Odo water reservoir, Osun State, Nigeria

Ctenopoma kingsleyae belongs to the family Anabantidae covers a wide geographic region. It has been morphologically described by a number of authors, yet it is lacking in adequate cytogenetic information, which is a setback to understanding its karyotypic evolution. This study, therefore, describes the karyotype of C. kingsleyae (n = 10) from Esa-Odo, Reservoir, Osun State, Nigeria. Metaphase chromosome spread was obtained from the gills of fish after intraperitoneal injection with 0.05% colchicines. Slides prepared were stained with Giemsa stain, images of metaphase spread were taken digitally and a karyogram was prepared. The study revealed a karyotypic formula of 2n = 48t with uniarmed chromosomes. This study has provided needful information on C. kingsleyae karyotype and will serve as the basis for further study on its evolution, genetic resource management and production.

Karyotypic analysis of Crucian carp, Carassius carassius (Linnaeus, 1758) from cold waters of Kashmir Himalayas

Carassius carassius (Linnaeus, 1758) is an exotic fish to Kashmir, locally known as “gang gad” and commonly called as “crucian carp”. It belongs to family Cyprinidae. The present study aimed to identify the chromosome number of the Carassius carassius and to optimize the colchicine concentrations (0.01%, 0.025%, and 0.05%) and hypotonic treatment timings (25, 35, and 45 minutes) for the chromosome preparation in Carassius carassius in order to obtain the highest number of clear and identifiable metaphasic chromosomal spreads. Data collected was analyzed and the means of each treatment was compared. The findings of the present study indicated that there was a significant influence of colchicine concentration, hypotonic timings as well as colchicine concentration× hypotonic timings (P<0.01) on the number of metaphase chromosome spreads. Furthermore a significant (P<0.01) strong positive correlation obtained between colchicine concentrations, hypotonic timings and the number of metaphase chromosome spreads. The findings of the present study recommends further research into chromosomal modification techniques such as fish polyploid production, gynogenesis, androgenesis, and inter or intra-species hybridization is needed to generate unique and good inbred lines in aquaculture.

Betulinic acid, a major therapeutic triterpene of Celastrus orbiculatus Thunb., acts as a chemosensitizer of gemcitabine by promoting Chk1 degradation.

Celastrus orbiculatus Thunb., also called as oriental bittersweet vine or climbing spindle berry, a traditional Chinese herbal medicine has been used to treat a spectrum of painful and inflammatory diseases for centuries. Explored for their unique medicinal properties, C.orbiculatus offers additional therapeutic effects on cancerous diseases. The effect of single-agent gemcitabine on survival has not long been encouraging, combination therapies provide patients multiple chances of benefit for improved clinical response. This study aims at expounding the chemopotentiating effects and underlying mechanisms of betulinic acid, a primary therapeutic triterpene of C. orbiculatus in combination with gemcitabine chemotherapy. The preparation of betulinic acid was optimized using ultrasonic-assisted extraction method. Gemcitabine-resistant cell model was established by induction of the cytidine deaminase. MTT, colony formation, EdU incorporation and Annexin V/PI staining assays were used to evaluate cytotoxicity, cell proliferation and apoptosis in BxPC-3 pancreatic cancer cell line and H1299 non-small cell lung carcinoma cell line. Comet assay, metaphase chromosome spread and γH2AX immunostaining were applied for DNA damage assessment. Western blot and co-immunoprecipitation was used to detect the phosphorylation and ubiquitination of Chk1. Mode of action of gemcitabine in combination with betulinic acid was further captured in BxPC-3-derived mouse xenograft model. We noticed that the extraction method had an impact on the thermal stability of C. orbiculatus. Ultrasound-assisted extraction at room temperature in shorter processing time could maximize the overall yields and biological activities of C. orbiculatus. The major constituent was identified as betulinic acid, and the pentacyclic triterpene represented the prominent anticancer activity of C. orbiculatus. Forced expression of cytidine deaminase conferred acquired resistance to gemcitabine, while betulinic acid displayed equivalent cytotoxicity toward gemcitabine-resistant and sensitive cells. A combination therapy of gemcitabine with betulinic acid produced synergistic pharmacologic interaction on cell viability, apoptosis and DNA double-strand breaks. Moreover, betulinic acid abrogated gemcitabine-triggered Chk1 activation by destabilizing Chk1 loading via proteasomal degradation. The combination of gemcitabine and betulinic acid significantly retarded BxPC-3 tumor growth in vivo compared to single-agent gemcitabine treatment alone, accompanied with reduced Chk1 expression. These data provide evidence that betulinic acid is a potential candidate for chemosensitization as a naturally occurring Chk1 inhibitor and warrants further preclinical evaluation.

Application of neural network-based image analysis to detect sister chromatid cohesion defects

Sister chromatid cohesion (SCC) is mediated by the cohesin complex and its regulatory proteins. To evaluate the involvement of a protein in cohesin regulation, preparations of metaphase chromosome spreads and classifications of chromosome shapes after depletion of the target protein are commonly employed. Although this is a convenient and approved method, the evaluation and classification of each chromosome shape has to be performed manually by researchers. Therefore, this method is time consuming, and the results might be affected by the subjectivity of researchers. In this study, we developed neural network-based image recognition models to judge the positional relationship of sister chromatids, and thereby detect SCC defects. Transfer learning models based on SqueeezeNet or ResNet-18 were trained with more than 600 chromosome images labeled with the type of chromosome, which were classified according to the positional relationship between sister chromatids. The SqueezeNet-based trained model achieved a concordance rate of 73.1% with the sample answers given by a researcher. Importantly, the model successfully detected the SCC defect in the CTF18 deficient cell line, which was used as an SCC-defective model. These results indicate that neural-network-based image recognition models are valuable tools for examining SCC defects in different genetic backgrounds.

Bulked Oligo-FISH for Chromosome Painting and Chromosome Barcoding.

Recently developed bulked oligo-FISH is a highly versatile method, which is applicable in any plant species with an assembled genome sequence. This technique allows in situ identification of individual chromosomes, large chromosomal rearrangements, comparative karyotype analysis, or even the reconstruction of the three-dimensional organization of the genome. The method is based on the identification of thousands of short oligonucleotides, unique to specific genome regions, which are synthesized in parallel, fluorescently labeled and used as probes for FISH. In this chapter, we propose a detailed protocol for amplification and labeling of single-stranded oligo-based painting probes from so-called MYtags immortal libraries, the preparation of mitotic metaphase and meiotic pachytene chromosome spreads, and a protocol for the fluorescence in situ hybridization procedure using the synthetic oligo probes. The proposed protocols are demonstrated for banana (Musa spp.).

Tools for Drawing Informative Idiograms.

Each species has a typical karyotype, which represents the phenotypic appearance of the somatic chromosomes including number, size, and morphology. An idiogram is a diagrammatic representation of the chromosomes showing their relative size, homologous groups, and different cytogenetic landmarks. Chromosomal analysis of cytological preparations is an essential component of many investigations, which involves the calculation of karyotypic parameters and the generation of idiograms. Although various tools are available for karyotype analysis, here we demonstrate karyotype analysis using our recently developed tool named KaryoMeasure. KaryoMeasure is a semi-automated free and user-friendly karyotype analysis software that facilitates data collection from different digital images of metaphase chromosome spreads and calculates a wide variety of chromosomal and karyotypic parameters along with the related standard errors. KaryoMeasure draws idiograms of both diploid and allopolyploid species into a vector-based SVG or PDF image file.

Development and applications of chromosome-specific cytogenetic BAC-FISH probes in Larimichthys crocea

Large yellow croaker Larimichthys crocea (Richardson) is an important member in family Sciaenidae, and one of most productive mariculture fishes in China. Fluorescence in situ hybridization is a useful tool for cytogenetics and genomics research. Here, we demonstrated that bacterial artificial chromosome (BAC) clones could be used to identify individual chromosomes in large yellow croaker, and also to study chromosome evolution in the related species. By using BAC paired-end sequencing and sequence alignment, 435 BAC clones were anchored to 24 pseudochromosomes of large yellow croaker genome. Among them, 72 BAC clones with low repeat content were selected and passed PCR test, and then grouped by chromosome for FISH test. As a results, there were 67 BAC clones, 2 to 3 BAC clones per chromosome, generated specific and stable signal at expected position. Then, a dual-color FISH probe cocktail composed of 48 of these BAC clones was formulated and used to hybridize metaphase chromosome spreads, resulting in distinct signal patterns on each chromosome pair, which help to distinguish all chromosomes in the metaphase spreads of large yellow croaker. The chromosome-specific BAC-FISH probes were also applied to a close relative of large yellow croaker, Collichthys lucidus, demonstrating that its Y chromosome originated from the fusion of Chr.1 and Chr.7. Thus, our study provides the first set of chromosome-specific FISH probes in family Sciaenidae, which will play an important role in cytogenetics and genomics research in the family.

Anti-CENP-C Antibody-Based Immunofluorescence Dicentric Assay: Radiation Dose-Response, Validation Studies, and Radiation Dose-Dependency on Sister Centromere Fluorescence.

Dicentric chromosome assay (DCA) is the most accepted cytological technique for the purpose of biological dosimetry in radiological and nuclear accidents, however, it is not always easy to evaluate dicentric chromosomes because of the technical difficulty in identifying dicentric chromosomes on Giemsa-stained metaphase chromosome samples. Here, we applied an antibody recognizing centromere protein (CENP) C, CENP-C, whose antigenicity is resistant to the fixation with Carnoy's solution. Normal human diploid cells were irradiated with various doses of 137Cs γ rays at 1 Gy/ min, treated with hypotonic solution, fixed with Carnoy's fixative, and metaphase chromosome spreads were stained with anti-CENP-C antibody. Dose-dependent induction of dicentric chromosomes was confirmed between 1 and 10 Gy of γ rays, and the results were compatible with those obtained by the conventional Giemsa-stained chromosome samples. The CENP-C assay also uncovered the difference in the fluorescence from the sister centromeres on the same chromosome, which was more pronounced after radiation exposure. Although the underlying mechanism is still to be determined, the result suggests a novel effect of radiation on centromeres. The innovative protocol for CENP-C-based DCA, which enables ideal visualization of centromeres, is simple, effective and reliable. It does not require skilled examiners, so that it may be an alternative method, avoiding uneasiness of the current DCA using Giemsa-stained metaphase chromosome samples.

DNA Damage Foci on Metaphase Chromosomes.

After DNAs are damaged, DNA repair proteins accumulate and areactivated at the DNA damaged site. These accumulated proteins are visualized as fociby fluorescent immunocytochemistry technique. This allows the DNA damage responses in interphase nuclei to be detected; it was earlier times difficult to analyze DNA damage in situ. In order to analyze DNA damage in interphase cells, either DNA is extracted to assay breaks biochemically, or premature chromosome condensation is conducted to observe as chromatin breaks. Although DNA damage-induced foci are typically analyzed in interphase cells, these foci can be also visualized on mitotic chromosomes. The foci where the repair proteins accumulate at the damage site is observed as mitotic chromosome break site. Since mitotic cells attach loosely or not attached to cell culture vessels, it is difficult to analyze foci onchromosomes in culture vessels under a microscope, so metaphase chromosome spread must be prepared for accurate analysis. The cytocentrifuge system is an ideal method to adhere mitotic cells to microscope slides for the fluorescent immunocytochemistry. This chapter introduces cytocentrifuge method to prepare metaphase spread for DNA damage foci analysis.

Metaphase Chromosome Preparation and Classification of Chromosomal Aberrations.

Chromosomal aberrations are changes in structure and number of chromosomes. Metaphase chromosome can be analyzed by a standard light microscope to detect chromosomal aberrations. Recently, detailed analysis or rapid analysis was possible by using fluorescence probes and fluorescent microscope. The origins of chromosomal aberrations can be errors of DNA repair, cell divisions, and DNA synthesis. Analysis of chromosome aberrations can be used for the wide range of analysis. It includes a basic science connecting DNA damage to cellular death and mutagenesis and diagnostic tools for hereditary diseases and biodosimetry following radiation exposure.Specific DNA damages produce unique types of chromosomal aberrations. Analysis of chromosomal aberrations enables us to investigate the mechanisms of genotoxic stress. However, one type of DNA damage provides a variety of changes in chromosome structures. It is often confusing. This chapter introduces the standard technique of metaphase chromosome spread preparation and typical classification of chromosomal aberrations.

Cytogenetic analysis reveals swamp status of the indigenous ‘Bhangor’ buffalo population from Tripura

The domestic water buffalo (Bubalus bubalis) isclassified into the swamp and riverine. However, theirhybrids are also found in some parts of Assam (BrahmaputraValley) in North-east India. Swamp buffaloes have a typicalphenotypic appearance, like the shape of horns, small bodysize as compared to riverine breeds, and body-colour, etc.This study characterizes the indigenous non-descript‘Bhangor’ buffalo population from the Tripura state usingkaryotype analyses. Blood samples were collected fromunrelated animals of both sexes phenotypically identifiedas swamp buffaloes from the breeding tract in Unakotidistrict which has >50% of indigenous buffalo populationin Tripura. The blood leukocytes were cultured, terminated,and harvested using conventional karyotype protocol asstandardised in lab to determine the number ofchromosomes present in the metaphase chromosomespreads after 72 h incubation at 37°C with intermittentshaking in the incubator. Chromosomal spread showedpresence of 2N=48 chromosomes, comprised of 23 pairsof autosomes and a pair of sex chromosomes with clearlydistinct size of fourth pair of metacentric chromosome. Andfor the first time, non-descript indigenous buffalo population‘Bhangor’ from Tripura has been characterized usingkaryotypic analysis.

Effects of carbon dioxide purities on mitotic index in lymphocyte culture and metaphase chromosome quality

Abstract. Purnami S, Wibisari IP, Suvifan VA, Nurhayati S, Ramadhani D. 2021. Effects of carbon dioxide purities on mitotic index in lymphocyte culture and metaphase chromosome quality. Nusantara Bioscience 13: 171-176. The metaphase chromosome spread quality is necessary for a faster individual dose prediction following radiological accidents using dicentric chromosome assay. It is well known that the low-quality metaphase chromosome spreads can lead to false positives of dicentric chromosome identification. Thus, evaluation of the main variable that influences the preparation of high-quality metaphase chromosome spreads is important to perform. Until now, no studies have assessed the effects of CO2 purities on metaphase chromosome spread quality. This study aimed to evaluate the effects of carbon dioxide (CO2) purities on lymphocyte proliferation, and the quality of metaphase chromosome spreads to improve the chromosome aberration assay for cytogenetic biodosimetry purposes. Whole blood samples from three different subjects were cultured and incubated for 48 hours with two different grades of CO2 (high purity and food grades) and without CO2. The mitotic index (MI) from each subject was assessed, and the quality of metaphase chromosome spreads was evaluated by comparing the lengths of chromosomes 1, 2, and 21. Statistical analysis revealed that the difference between manual and automatic MI under three different conditions of CO2 purity was not statistically significant (p = 0.162; p = 0.901). Comparative analysis of the lengths of chromosomes 1, 2, and 21 from 145 metaphases also showed a difference that was not statistically significant (p = 0.745; p = 0.915; p = 0.399). Overall, our findings suggest that CO2 purities do not impair lymphocyte proliferation or metaphase quality. Further investigation should include other technical improvements such as drop-slide optimization.

Loss of BubR1 acetylation provokes replication stress and leads to complex chromosomal rearrangements.

Accurate chromosomal segregation during mitosis is regulated by the spindle assembly checkpoint (SAC). SAC failure results in aneuploidy, a hallmark of cancer. However, many studies have suggested that aneuploidy alone is not oncogenic. We have reported that BubR1 acetylation deficiency in mice (K243R/+) caused spontaneous tumorigenesis via weakened SAC signaling and unstable chromosome-spindle attachment, resulting in massive chromosomal mis-segregation. In addition to aneuploidy, cells derived from K243R/+ mice exhibited moderate genetic instability and chromosomal translocation. Here, we investigated how the loss of BubR1 acetylation led to genetic instability and chromosomal rearrangement. To rescue all chromosomal abnormalities generated by the loss of BubR1 acetylation during development, K243R/+ mice were crossed with p53-deficient mice. Genome-wide sequencing and spectral karyotyping of tumors derived from these double-mutant mice revealed that BubR1 acetylation deficiency was associated with complex chromosomal rearrangements, including Robertsonian-like whole-arm translocations. By analyzing the telomeres and centromeres in metaphase chromosome spreads, we found that BubR1 acetylation deficiency increased the collapse of stalled replication forks, commonly referred to as replication stress, and led to DNA damage and chromosomal rearrangements. BubR1 mutations that are critical in interacting with PCAF acetyltransferase and acetylating K250, L249F and A251P, were found from human cancers. Furthermore, a subset of human cancer cells exhibiting whole-arm translocation also displayed defects in BubR1 acetylation, supporting that defects in BubR1 acetylation in mitosis contributes to tumorigenesis. Collectively, loss of BubR1 acetylation provokes replication stress, particularly at the telomeres, leading to genetic instability and chromosomal rearrangement.

Synergistic Effects of Chronic Restraint-Induced Stress and Low-Dose 56Fe-particle Irradiation on Induction of Chromosomal Aberrations in Trp53-Heterozygous Mice.

Astronauts can develop psychological stress (PS) during space flights due to the enclosed environment, microgravity, altered light-dark cycles, and risks of equipment failure or fatal mishaps. At the same time, they are exposed to cosmic rays including high atomic number and energy (HZE) particles such as iron-56 (Fe) ions. Psychological stress or radiation exposure can cause detrimental effects in humans. An earlier published pioneering study showed that chronic restraint-induced psychological stress (CRIPS) could attenuate Trp53 functions and increase carcinogenesis induced by low-linear energy transfer (LET) γ rays in Trp53-heterozygous (Trp53+/-) mice. To elucidate possible modification effects from CRIPS on high-LET HZE particle-induced health consequences, Trp53+/- mice were received both CRIPS and accelerated Fe ion irradiation. Six-week-old Trp53+/- C57BL/6N male mice were restrained 6 h per day for 28 consecutive days. On day 8, they received total-body Fe-particle irradiation (Fe-TBI, 0.1 or 2 Gy). Metaphase chromosome spreads prepared from splenocytes at the end of the 28-day restraint regimen were painted with the fluorescence in situ hybridization (FISH) probes for chromosomes 1 (green), 2 (red) and 3 (yellow). Induction of psychological stress in our experimental model was confirmed by increase in urinary corticosterone level on day 7 of restraint regimen. Regardless of Fe-TBI, CRIPS reduced splenocyte number per spleen at the end of the 28-day restraint regimen. At 2 Gy, Fe-TBI alone induced many aberrant chromosomes and no modifying effect was detected from CRIPS on induction of aberrant chromosomes. Notably, neither Fe-TBI at 0.1 Gy nor CRIPS alone induced any increase in the frequency of aberrant chromosomes, while simultaneous exposure resulted in a significant increase in the frequency of chromosomal exchanges. These findings clearly showed that CRIPS could enhance the frequency of chromosomal exchanges induced by Fe-TBI at a low dose of 0.1 Gy.

Chromogenic In Situ Hybridization (CISH) as a Method for Detection of C-Myc Amplification in Formalin-Fixed Paraffin-Embedded Tumor Tissue: An Update.

In situ hybridization (ISH) allows evaluation of genetic abnormalities, such as changes in chromosome number, chromosome translocations, or gene amplifications, by hybridization of tagged DNA (or RNA) probes with complementary DNA (or RNA) sequences in interphase nuclei of target tissue. However, chromogenic in situ hybridization (CISH) is also applicable to formalin-fixed, paraffin-embedded (FFPE ) tissues, besides metaphase chromosome spreads. CISH is similar to fluorescent in situ hybridization (FISH) regarding pretreatments and hybridization protocols but differs in the way of visualization. Indeed, CISH signal detection is similar to that used in immunohistochemistry, making use of a peroxidase-based chromogenic reaction instead of fluorescent dyes. In particular, tagged DNA probes are indirectly detected using an enzyme-conjugated antibody targeting the tags. The enzymatic reaction of the chromogenic substrate leads to the formation of strong permanent brown signals that can be visualized by bright-field microscopy at 40× magnification. The advantage of CISH is that it allows the simultaneous observation of gene amplification and tissue morphology, and the slides can be stored for a long time.

Applications of fluorescence in situ hybridization (FISH) in genome research

Fluorescence in situ hybridization (FISH) technique enables the direct detection of DNA sequences inintact cellular materials (e.g. individual chromosomes in metaphase spreads). This review article focuses on theapplications of FISH in genome research, including validation and correction of the genome assembly from thenext-generation sequencing (NGS) projects. DNA probes for specific DNA fragments of the assembly can beobtained from PCR amplicon or cloned products using different vector systems. Localization of these probeson their respective chromosomal regions can be visualized by FISH, providing useful information to crosscheckthe assembly data. Furthermore, the recent refinements in the FISH technology including using smartpooling scheme of differently colored DNA probes, together with consecutive FISH experiments (stripping andreprobing of the same slide) are described. These advances in multicolor FISH can provide crucial linkageinformation on association of linkage groups and assembly scaffolds, resulting in so-called cytogenetic maps.Integration of the cytogenetic maps and assembly sequences assists to resolve the chromosome-level genomeassembly and to reveal new insights in genome architecture and genome evolution. Especially, comparativechromosome painting with pooled DNA probes from one reference species can be used to investigate ancestralrelationships (chromosome homeology and rearrangements) among other not-yet-sequenced species. Inaddition, FISH using DNA probes for certain specific classes of repetitive DNA elements as well as for basicchromosome structures (e.g. centromere or telomere DNA repeats, ribosomal DNA loci) can be used to studythe genome organization and karyotype differentiation. We also discussed about limitations and futureperspectives of the FISH technology.

Loss of AMPKalpha1 Triggers Centrosome Amplification via PLK4 Upregulation in Mouse Embryonic Fibroblasts.

Recent evidence indicates that activation of adenosine monophosphate-activated protein kinase (AMPK), a highly conserved sensor and modulator of cellular energy and redox, regulates cell mitosis. However, the underlying molecular mechanisms for AMPKα subunit regulation of chromosome segregation remain poorly understood. This study aimed to ascertain if AMPKα1 deletion contributes to chromosome missegregation by elevating Polo-like kinase 4 (PLK4) expression. Centrosome proteins and aneuploidy were monitored in cultured mouse embryonic fibroblasts (MEFs) isolated from wild type (WT, C57BL/6J) or AMPKα1 homozygous deficient (AMPKα1−/−) mice by Western blotting and metaphase chromosome spread. Deletion of AMPKα1, the predominant AMPKα isoform in immortalized MEFs, led to centrosome amplification and chromosome missegregation, as well as the consequent aneuploidy (34–66%) and micronucleus. Furthermore, AMPKα1 null cells exhibited a significant induction of PLK4. Knockdown of nuclear factor kappa B2/p52 ameliorated the PLK4 elevation in AMPKα1-deleted MEFs. Finally, PLK4 inhibition by Centrinone reversed centrosome amplification of AMPKα1-deleted MEFs. Taken together, our results suggest that AMPKα1 plays a fundamental role in the maintenance of chromosomal integrity through the control of p52-mediated transcription of PLK4, a trigger of centriole biogenesis.

Development and application of an optimized drop-slide technique for metaphase chromosome spreads in maize

ABSTRACTChromosome spreads are important for complex molecular cytogenetic studies. An adequate chromosome spreading method for identification and isolation of the maize B chromosome, however, has not been reported. We used the maize inbred lines, B73 and Mo17, the hybrid YD08 line and the landrace DP76 to develop an optimized chromosome spreading method. We investigated the effects of four treatment factors on the quality of metaphase chromosome spreads using a factorial analysis of variance. Optimal conditions for metaphase spreading were identified using regression analysis based on multifactor orthogonal design of four treatment factors with five levels for each factor. We developed optimal conditions for metaphase spreading as follows: nitrous oxide treatment for 2 h, glacial acetic acid fixation for 2 h, enzyme hydrolysis for 6.0 h, and a drop height of 35 cm for cell suspension. We obtained high quality metaphase chromosome spreads with large metaphase areas, large numbers of chromosomes, few chromosome overlaps and high frequency of intact metaphases. Our optimized drop-slide procedure was markedly better than the traditional flame smear technique. We identified 487 B chromosomes in three forms from maize landraces from Southwest China. We found no relation between the C-band number and B chromosome. Single B chromosomes also were isolated directly from a metaphase chromosome drop-slide using a micromanipulator. Our optimized method provides a simple, efficient and reproducible procedure for preparing high quality plant chromosome spreads.