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Methods in Molecular Biology (Clifton,... 2023After DNAs are damaged, DNA repair proteins accumulate and are activated at the DNA damaged site. These accumulated proteins are visualized as foci by fluorescent...
After DNAs are damaged, DNA repair proteins accumulate and are activated at the DNA damaged site. These accumulated proteins are visualized as foci by 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 on chromosomes 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.
Topics: Chromosomes; DNA; DNA Damage; Interphase; Metaphase
PubMed: 36066713
DOI: 10.1007/978-1-0716-2433-3_10 -
MicroPublication Biology 2022Human retinal pigment epithelium RPE-1 cells are immortalized diploid wild-type cells. RPE-1 is increasingly used for studies of spindle assembly dynamics and chromosome...
Human retinal pigment epithelium RPE-1 cells are immortalized diploid wild-type cells. RPE-1 is increasingly used for studies of spindle assembly dynamics and chromosome segregation. Here, we imaged living RPE-1 cells using the spinning disk confocal microscope and report their complete spindle assembly dynamic parameters. Live-cell experiments enabled ascribing precise timing of function of the kinesin-5 Eg5 and kinesin-14 HSET throughout different phases of mitosis. Eg5 functions at prophase and metaphase, to assemble and maintain spindle bipolarity, respectively. Eg5 inhibition results in spindle collapse during prophase and metaphase, resulting in monoastral/monopolar spindles. HSET functions throughout mitosis to maintain spindle length. HSET degradation results in shorter spindles through all phases of mitosis. Double-inhibition of Eg5 and HSET produces only monoastral/monopolar spindles, indicating that Eg5 and HSET may not be antagonistic in wild-type RPE-1 cells, contrary to previous studies using cancer cells. In the context of spindle assembly, our results highlight potential important differences between RPE-1 and other cancer-derived cell lines.
PubMed: 36004005
DOI: 10.17912/micropub.biology.000623 -
Methods in Molecular Biology (Clifton,... 2023Flow cytometry offers a unique way of analyzing and manipulating plant chromosomes. During a rapid movement in a liquid stream, large populations can be classified in a...
Flow cytometry offers a unique way of analyzing and manipulating plant chromosomes. During a rapid movement in a liquid stream, large populations can be classified in a short time according to their fluorescence and light scatter properties. Chromosomes whose optical properties differ from other chromosomes in a karyotype can be purified by flow sorting and used in a range of applications in cytogenetics, molecular biology, genomics, and proteomics. As the samples for flow cytometry must be liquid suspensions of single particles, intact chromosomes must be released from mitotic cells. This protocol describes a procedure for preparation of suspensions of mitotic metaphase chromosomes from meristem root tips and their flow cytometric analysis and sorting for various downstream applications.
Topics: Chromosomes, Plant; Flow Cytometry; Suspensions; Chromosomes; Cytogenetics; Karyotyping
PubMed: 37335476
DOI: 10.1007/978-1-0716-3226-0_10 -
BioRxiv : the Preprint Server For... May 2024The eukaryotic cell division machinery must rapidly and reproducibly duplicate and partition the cell's chromosomes in a carefully coordinated process. However,...
The eukaryotic cell division machinery must rapidly and reproducibly duplicate and partition the cell's chromosomes in a carefully coordinated process. However, chromosome number varies dramatically between genomes, even on short evolutionary timescales. We sought to understand how the mitotic machinery senses and responds to karyotypic changes by using a series of budding yeast strains in which the native chromosomes have been successively fused. Using a combination of cell biological profiling, genetic engineering, and experimental evolution, we show that chromosome fusions are well tolerated up until a critical point. Cells with fewer than five centromeres lack the necessary number of kinetochore-microtubule attachments needed to counter outward forces in the metaphase spindle, triggering the spindle assembly checkpoint and prolonging metaphase. Our findings demonstrate that spindle architecture is a constraining factor for karyotype evolution.
PubMed: 37961714
DOI: 10.1101/2023.10.25.563899 -
Methods in Molecular Biology (Clifton,... 2023Recently 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...
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.).
Topics: Chromosome Painting; In Situ Hybridization, Fluorescence; Chromosomes, Plant; Karyotype; Karyotyping
PubMed: 37335493
DOI: 10.1007/978-1-0716-3226-0_27 -
Cytogenetic and Genome Research 2022Mitotic chromosomes of butterflies, which look like dots or short filaments in most published data, are generally considered to lack localised centromeres and thus to be...
Mitotic chromosomes of butterflies, which look like dots or short filaments in most published data, are generally considered to lack localised centromeres and thus to be holokinetic. This particularity, observed in a number of other invertebrates, is associated with meiotic particularities known as "inverted meiosis," in which the first division is equational, i.e., centromere splitting-up and segregation of sister chromatids instead of homologous chromosomes. However, the accurate analysis of butterfly chromosomes is difficult because (1) their size is very small, equivalent to 2 bands of a mammalian metaphase chromosome, and (2) they lack satellite DNA/heterochromatin in putative centromere regions and therefore marked primary constrictions. Our improved conditions for basic chromosome preparations, here applied to 6 butterfly species belonging to families Nymphalidae and Pieridae challenges the holocentricity of their chromosomes: in spite of the absence of primary constrictions, sister chromatids are recurrently held together at definite positions during mitotic metaphase, which makes possible to establish karyotypes composed of acrocentric and submetacentric chromosomes. The total number of chromosomes per karyotype is roughly inversely proportional to that of non-acrocentric chromosomes, which suggests the occurrence of frequent robertsonian-like fusions or fissions during evolution. Furthermore, the behaviour and morphological changes of chromosomes along the various phases of meiosis do not seem to differ much from those of canonical meiosis. In particular, at metaphase II chromosomes clearly have 2 sister chromatids, which refutes that anaphase I was equational. Thus, we propose an alternative mechanism to holocentricity for explaining the large variations in chromosome numbers in butterflies: (1) in the ancestral karyotype, composed of about 62 mostly acrocentric chromosomes, the centromeres, devoid of centromeric heterochromatin/satellite DNA, were located at contact with telomeric heterochromatin; (2) the instability of telomeric heterochromatin largely contributed to drive the multiple rearrangements, principally chromosome fusions, which occurred during butterfly evolution.
Topics: Humans; Animals; Butterflies; Heterochromatin; DNA, Satellite; Chromosomes; Centromere; Meiosis; Chromatids; Karyotyping; Mammals
PubMed: 36689925
DOI: 10.1159/000526034 -
Genome Jun 2021exhibits high variation in chromosome number both within and among species. The L. karyotype was analyzed in detail using fluorescence in situ hybridization (FISH)...
exhibits high variation in chromosome number both within and among species. The L. karyotype was analyzed in detail using fluorescence in situ hybridization (FISH) with oligonucleotide probes for (AGT) and 5S rDNA, which were tested here for the first time. In total, 90 chromosomes were counted in prometaphase and metaphase, and all exhibited similarly intense (AGT) signals at both ends. (AGT) showed little variation and thus did not allow discrimination among chromosomes, but its location at both ends confirmed the integrity of each chromosome, thus contributing to accurate counting of the numerous, small chromosomes. Oligo-5S rDNA marked the proximal/distal regions of six chromosomes: weak signals on chromosomes 7 and 8, slightly stronger signals on chromosomes 15 and 16, and very strong signals on chromosomes 17 and 18. Therefore, 5S rDNA could assist in chromosome identification in . Metaphase chromosome lengths ranged from 3.00 to 1.18 μm, indicating small chromosomes. The ratios of longest to shortest chromosome length in prometaphase and metaphase were 2.58 and 2.54, respectively, indicating karyotype asymmetry in . These results provide an exact chromosome number and a physical map, which will be useful for genome assembly and contribute to molecular cytogenetics in the genus .
Topics: Chromosome Mapping; Chromosomes, Plant; DNA, Plant; DNA, Ribosomal; Hibiscus; In Situ Hybridization, Fluorescence; Karyotype; Karyotyping; Meiosis; Metaphase; Oligonucleotide Probes; RNA, Ribosomal, 5S
PubMed: 33797299
DOI: 10.1139/gen-2019-0142 -
3 Biotech Mar 2023An efficient in vitro protocol for high-frequency polyploidization for the first time in gerbera hybrid (BGC-2019-01) was developed in the present study. Two-week-old in...
An efficient in vitro protocol for high-frequency polyploidization for the first time in gerbera hybrid (BGC-2019-01) was developed in the present study. Two-week-old in vitro-developed shoots (tips) were treated individually with 0.1%, 0.25% and 0.5% (/) colchicine solutions for 4, 6, 8, and 12 h. The colchicine-treated shoot tips were then inoculated on Murashige and Skoog (MS) medium fortified with 1.5 mg/l -Topolin for multiple shoot proliferation and later transferred into 1.5 mg/l indole-3-acetic acid-fortified MS medium for rooting of shoots. The ploidy levels of the colchicine-treated and regenerated plantlets along with the non-treated ones were confirmed via flow cytometry analysis and metaphasic chromosome count. The highest frequency of tetraploid plantlets (50%) were obtained when shoot tips were treated with 0.1% colchicine for 4 h. Morphological observations revealed that induced tetraploid plantlets exhibited delayed fresh shoot initiation, fewer but longer shoots, as well as fewer but broader leaves. Likewise, the study of stomata revealed that in comparison to their diploid counterparts, the tetraploid plantlets exhibited less frequent yet significantly larger stomata, and higher number of chloroplasts. The tetraploids were recorded with significantly higher chlorophyll, carotenoid, and anthocyanin content during the photosynthetic pigment analyses. During ex vitro acclimatization and field growth, the tetraploid plants exhibited delayed proliferation but with higher vigor and thickened broad leaves. The genetic uniformity among the diploid and the tetraploid plants was confirmed using conserved DNA-derived polymorphism (CDDP), directed amplification of minisatellite-region DNA (DAMD), inter simple sequence repeats (ISSR), and start codon targeted (SCoT) polymorphism marker systems. The tetraploids developed in the present study would be of immense importance for the genetic improvement of gerbera as far as its ornamental values are concerned.
PubMed: 36748015
DOI: 10.1007/s13205-022-03457-z -
Bio-protocol Aug 2023Maintenance of genome integrity requires efficient and faithful resolution of DNA breaks and DNA replication obstacles. Dysfunctions in any of the processes...
Maintenance of genome integrity requires efficient and faithful resolution of DNA breaks and DNA replication obstacles. Dysfunctions in any of the processes orchestrating such resolution can lead to chromosomal instability, which appears as numerical and structural chromosome aberrations. Conventional cytogenetics remains as the golden standard method to detect naturally occurring chromosomal aberrations or those resulting from the treatment with genotoxic drugs. However, the success of cytogenetic studies depends on having high-quality chromosome spreads, which has been proven to be particularly challenging. Moreover, a lack of scoring guidelines and standardized methods for treating cells with genotoxic agents contribute to significant variability amongst different studies. Here, we report a simple and effective method for obtaining well-spread chromosomes from mammalian cells for the analysis of chromosomal aberrations. In this method, cells are (1) arrested in metaphase (when chromosome morphology is clearest), (2) swollen in hypotonic solution, (3) fixed before being dropped onto microscope slides, and (4) stained with DNA dyes to visualize the chromosomes. Metaphase chromosomes are then analyzed using high-resolution microscopy. We also provide examples, representative images, and useful guidelines to facilitate the scoring of the different chromosomal aberrations. This method can be used for the diagnosis of genetic diseases, as well as for cancer studies, by identifying chromosomal defects and providing insight into the cellular processes that influence chromosome integrity.
PubMed: 37638291
DOI: 10.21769/BioProtoc.4739 -
Acta Histochemica Apr 2023Maximising the number of cells arrested at metaphase and their resolution is fundamentally important for molecular cytogenetic investigations, particularly in fish,...
Maximising the number of cells arrested at metaphase and their resolution is fundamentally important for molecular cytogenetic investigations, particularly in fish, which typically yield low mitotic index and have highly condensed chromosomes. To overcome these limitations, fish were injected with a mitotic stimulator (the yeast, Saccharomyces cerevisiae) to improve the mitotic index, and the intercalating agent ethidium bromide to produce elongated chromosomes. Specifically, adults were injected with activated yeast and then Colcemid (0.025 µg/µl solution, 10 µl per 1 g of body weight) at 24-96 h post yeast injections, followed by chromosome preparations from multiple tissues. Results showed that gill tissue had the highest number of dividing cells at 72 h post yeast exposure with no significant (p > 0.05) differences between the sexes. Nonetheless, sex-specific differences in the mitotic index were observed in spleen, kidney, and liver, which may be attributed to sex-specific differences in immune responses. For elongation of mitotic chromosomes, individuals (both sexes) were first injected with activated yeast and after 48 h with ethidium bromide (2 or 4 µg/ml) and Colcemid (0.05 µg/µl solution, 10 µl per 1 g of body weight). Following which, animals were sampled at three time points (1, 4 and 8 h) for chromosome preparations. The results show that the optimum elongation of metaphase chromosomes of males and females was achieved by using 2 µg/ml and 4 µg/ml, respectively, for 1 h. Interestingly, the average mitotic chromosome length (μm) of males and females post-ethidium bromide exposure was significantly different (p < 0.05) for both concentrations, except at 1 h exposure for 2 µg/ml EtBr. Such differences can be attributed to overall chromosomal condensation differences between sexes. Regardless, the increased mitotic index and chromosome resolution could benefit cytogenetic studies in other fish species.
Topics: Male; Animals; Female; Ethidium; Saccharomyces cerevisiae; Demecolcine; Chromosomes; Cytogenetic Analysis; Cyprinodontiformes; Body Weight
PubMed: 37062122
DOI: 10.1016/j.acthis.2023.152029