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Eisuke Gotoh - One of the best experts on this subject based on the ideXlab platform.
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g2 premature Chromosome condensation Chromosome aberration assay drug induced premature Chromosome condensation pcc protocols and cytogenetic approaches in mitotic Chromosome and interphase chromatin for radiation biology
Methods of Molecular Biology, 2019Co-Authors: Eisuke GotohAbstract:Chromosome analysis is a fundamental technique for a wide range of cytogenetic studies. Chromosome aberrations are easily introduced by many kinds of clastogenic agents such as ionizing irradiation, UV, or alkylating agents, and damaged Chromosomes may be prone to cancer. Chromosomes are conventionally prepared from mitotic cells arrested by the colcemid block method. However, obtaining of mitotic Chromosomes is sometimes hampered under several circumstances, for example after high-dose (over several Gys of γ-rays) ionizing irradiation exposure accident. As a result, cytogenetic analysis will be often difficult or even impossible in such cases. Premature Chromosome condensation (PCC) is an alternative technique that has proved to be a unique and useful way in Chromosome analysis. Previously, PCC has been achieved following cell fusion mediated either by fusogenic viruses (for example Sendai virus) or by polyethylene glycol (PEG) (cell-fusion PCC), but the cell-fusion PCC has several drawbacks. The novel drug-induced PCC use of specific inhibitors for serine/threonine protein phosphatase was introduced about 20 years ago. This method is much simple and easy even than the conventional mitotic Chromosome Preparation using colcemid block protocol and the obtained PCC index (equivalent to mitotic index for metaphase Chromosome) is much higher. Furthermore, this method allows the interphase chromatin to be condensed and visualized like mitotic Chromosomes, and thus has been opening the way for Chromosome analysis not only in metaphase Chromosomes but also in interphase chromatin. The drug-induced PCC has therefore proven the usefulness in cytogenetics and other many cell biology fields. Since the first version of drug-induced PCC protocol has been published in 2009 (Gotoh, Methods in molecular biology. Humana Press, New York, 2009), many newer applications of drug-induced PCC in radiation biology and Chromosome science fields in a wide range of species from animal to plant have been reported (Gotoh et al., Biomed Res 16:63-68, 1995; Lamadrid Boada et al., Mutat Res 757:45-51, 2013; Ravi et al., Biochimie 95:124-33, 2013; Ono et al., J Cell Biol 200:429-41, 2013; Vagnarelli, Exp Cell Res 318:1435-41, 2012; Roukos et al., Nat Protoc 9:2476-92, 2014; Miura and Blakely, Cytometry A 79:1016-22, 2013; Zabka et al., J Plant Physiol 174:62-70, 2015; Samaniego et al., Planta 215:195-204, 2002; Rybaczek et al., Folia Histochem Cytobiol 40:51-9, 2002; Gotoh and Durante J Cell Physiol 209:297-304, 2006). Therefore as a new edition, I will write in this chapter the drug-induced PCC technique with newer findings, in particular focused drug-induced PCC protocols in radiation biology with referring updated articles published recently.
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drug induced premature Chromosome condensation pcc protocols cytogenetic approaches in mitotic Chromosome and interphase chromatin
Methods of Molecular Biology, 2015Co-Authors: Eisuke GotohAbstract:Chromosome analysis is a fundamental technique which is used in wide areas of cytogenetic study including karyotyping species, hereditary diseases diagnosis, or Chromosome biology study. Chromosomes are usually prepared from mitotic cells arrested by colcemid block protocol. However, obtaining mitotic Chromosomes is often hampered under several circumstances. As a result, cytogenetic analysis will be sometimes difficult or even impossible in such cases. Premature Chromosome condensation (PCC) (see Note 1) is an alternative method that has proved to be a unique and useful way in Chromosome analysis. Former, PCC has been achieved following cell fusion method (cell-fusion PCC) mediated either by fusogenic viruses (e.g., Sendai virus) or cell fusion chemicals (e.g., polyethylene glycol), but the cell fusion PCC has several drawbacks. The novel drug-induced PCC using protein phosphatase inhibitors was introduced about 20 years ago. This method is much simpler and easier even than the conventional mitotic Chromosome Preparation protocol use with colcemid block and furthermore obtained PCC index (equivalent to mitotic index for metaphase Chromosome) is usually much higher than colcemid block method. Moreover, this method allows the interphase chromatin to be condensed to visualize like mitotic Chromosomes. Therefore drug-induced PCC has opened the way for Chromosome analysis not only in metaphase Chromosomes but also in interphase chromatin. The drug-induced PCC has thus proven the usefulness in cytogenetics and other cell biology fields. For this second edition version, updated modifications/changes are supplemented in Subheadings 2, 3, and 4, and a new section describing the application of PCC in Chromosome science fields is added with citation of updated references.
Kunbo Wang - One of the best experts on this subject based on the ideXlab platform.
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Preparations of Meiotic Pachytene Chromosomes and Extended DNA Fibers from Cotton Suitable for Fluorescence In Situ Hybridization
2013Co-Authors: Renhai Peng, Tao Zhang, Fang Liu, Jian Ling, Chunying Wang, Xiangdi Zhang, Yuhong Wang, Kunbo WangAbstract:Fluorescence in situ hybridization (FISH) has become one of the most important techniques applied in plant molecular cytogenetics. However, the application of this technique in cotton has lagged behind because of difficulties in Chromosome Preparation. The focus of this article was FISH performed not only on cotton pachytene Chromosomes, but also on cotton extended DNA fibers. The cotton pollen mother cells (PMCs) instead of buds or anthers were directly digested in enzyme to completely breakdown the cell wall. Before the routine acetic acid treatment, PMCs were incubated in acetic acid and enzyme mixture to remove the cytoplasm and clear the background. The method of ice-cold Carnoy’s solution spreading Chromosome was adopted instead of nitrogen removed method to avoid Chromosomes losing and fully stretch Chromosome. With the above-improved steps, the high-quality well-differentiated pachytene Chromosomes with clear background were obtained. FISH results demonstrated that a mature protocol of cotton pachytene Chromosomes Preparation was presented. Intact and no debris cotton nuclei were obtained by chopping from etiolation cotyledons instead of the conventional liquid nitrogen grinding method. After incubating the nuclei with nucleus lysis buffer on slide, the parallel and clear background DNA fibers were acquired along the slide. This method overcomes the twist, accumulation and fracture of DNA fibers compared with other methods. The entire process of DNA fibers Preparation requires only 30 min, in contrast, it takes 3 h with routine nitrogen grinding method. The poisonous mercaptoethanol in nucleus lysi
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Preparations of meiotic pachytene Chromosomes and extended dna fibers from cotton suitable for fluorescence in situ hybridization
PLOS ONE, 2012Co-Authors: Renhai Peng, Tao Zhang, Fang Liu, Jian Ling, Chunying Wang, Xiangdi Zhang, Yuhong Wang, Kunbo WangAbstract:Fluorescence in situ hybridization (FISH) has become one of the most important techniques applied in plant molecular cytogenetics. However, the application of this technique in cotton has lagged behind because of difficulties in Chromosome Preparation. The focus of this article was FISH performed not only on cotton pachytene Chromosomes, but also on cotton extended DNA fibers. The cotton pollen mother cells (PMCs) instead of buds or anthers were directly digested in enzyme to completely breakdown the cell wall. Before the routine acetic acid treatment, PMCs were incubated in acetic acid and enzyme mixture to remove the cytoplasm and clear the background. The method of ice-cold Carnoy's solution spreading Chromosome was adopted instead of nitrogen removed method to avoid Chromosomes losing and fully stretch Chromosome. With the above-improved steps, the high-quality well-differentiated pachytene Chromosomes with clear background were obtained. FISH results demonstrated that a mature protocol of cotton pachytene Chromosomes Preparation was presented. Intact and no debris cotton nuclei were obtained by chopping from etiolation cotyledons instead of the conventional liquid nitrogen grinding method. After incubating the nuclei with nucleus lysis buffer on slide, the parallel and clear background DNA fibers were acquired along the slide. This method overcomes the twist, accumulation and fracture of DNA fibers compared with other methods. The entire process of DNA fibers Preparation requires only 30 min, in contrast, it takes 3 h with routine nitrogen grinding method. The poisonous mercaptoethanol in nucleus lysis buffer is replaced by nonpoisonous dithiothreitol. PVP40 in nucleus isolation buffer is used to prevent oxidation. The probability of success in isolating nuclei for DNA fiber Preparation is almost 100% tested with this method in cotton. So a rapid, safe, and efficient method for the Preparation of cotton extended DNA fibers suitable for FISH was established.
Takato Koba - One of the best experts on this subject based on the ideXlab platform.
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an improved method for inducing prometaphase Chromosomes in plants
Molecular Cytogenetics, 2018Co-Authors: Agus Setiawan, Chee How Teo, Shinji Kikuchi, Hidenori Sassa, Takato KobaAbstract:Detailed karyotyping using metaphase Chromosomes in melon (Cucumis melo L.) remains a challenge because of their small Chromosome sizes and poor stainability. Prometaphase Chromosomes, which are two times longer and loosely condensed, provide a significantly better resolution for fluorescence in situ hybridization (FISH) than metaphase Chromosomes. However, suitable method for acquiring prometaphase Chromosomes in melon have been poorly investigated. In this study, a modified Carnoy’s solution II (MC II) [6:3:1 (v/v) ethanol: acetic acid: chloroform] was used as a pretreatment solution to obtain prometaphase Chromosomes. We demonstrated that the prometaphase Chromosomes obtained using the MC II method are excellent for karyotyping and FISH analysis. We also observed that a combination of MC II and the modified air dry (ADI) method provides a satisfactory meiotic pachytene Chromosome Preparation with reduced cytoplasmic background and clear chromatin spreads. Moreover, we demonstrated that pachytene and prometaphase Chromosomes of melon and Abelia × grandiflora generate significantly better FISH images when prepared using the method described. We confirmed, for the first time, that Abelia × grandiflora has pairs of both strong and weak 45S ribosomal DNA signals on the short arms of their metaphase Chromosomes. The MC II and ADI method are simple and effective for acquiring prometaphase and pachytene Chromosomes with reduced cytoplasm background in plants. Our methods provide high-resolution FISH images that can help accelerate molecular cytogenetic research in plants.
Zhigang Zhou - One of the best experts on this subject based on the ideXlab platform.
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Localization of a Female-Specific Marker on the Chromosomes of the Brown Seaweed Saccharina japonica Using Fluorescence In Situ Hybridization
2016Co-Authors: Yu Liu, Zhigang ZhouAbstract:Background: There is a heteromorphic alternative life in the brown seaweed, Saccharina japonica (Aresch.) C. E. Lane, C. Mayes et G. W. Saunders ( = Laminaria japonica Aresch.), with macroscopic monoecious sporophytes and microscopic diecious gametophytes. Female gametophytes are genetically different from males. It is very difficult to identify the parent of a sporophyte using only routine cytological techniques due to homomorphic Chromosomes. A sex-specific marker is one of the best ways to make this determination. Methodology/Principal Findings: To obtain clear images, Chromosome Preparation was improved using maceration enzymes and fluorochrome 49, 6-diamidino-2-phenylindole (DAPI). The Chromosome number of both male and female haploid gametophytes was 31, and there were 62 Chromosomes in diploid sporophytes. Although the female Chromosomes ranged from 0.77 mm to 2.61 mm in size and were larger than the corresponding ones in the males (from 0.57 mm to 2.16 mm), there was not a very large X Chromosome in the females. Based on the known female-related FRML-494 marker, co-electrophoresis and Southern blot profiles demonstrated that it was inheritable and specific to female gametophytes. Using modified fluorescence in situ hybridization (FISH), this marker could be localized on one unique Chromosome of the female gametophytes as well as the sporophytes, whereas no hybridization signal was detected in the male gametophytes. Conclusions/Significance: Our data suggest that this marker was a female Chromosome-specific DNA sequence. This is th
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localization of a female specific marker on the Chromosomes of the brown seaweed saccharina japonica using fluorescence in situ hybridization
PLOS ONE, 2012Co-Authors: Yu Liu, Zhigang ZhouAbstract:Background There is a heteromorphic alternative life in the brown seaweed, Saccharina japonica (Aresch.) C. E. Lane, C. Mayes et G. W. Saunders ( = Laminaria japonica Aresch.), with macroscopic monoecious sporophytes and microscopic diecious gametophytes. Female gametophytes are genetically different from males. It is very difficult to identify the parent of a sporophyte using only routine cytological techniques due to homomorphic Chromosomes. A sex-specific marker is one of the best ways to make this determination. Methodology/Principal Findings To obtain clear images, Chromosome Preparation was improved using maceration enzymes and fluorochrome 4′, 6-diamidino-2-phenylindole (DAPI). The Chromosome number of both male and female haploid gametophytes was 31, and there were 62 Chromosomes in diploid sporophytes. Although the female Chromosomes ranged from 0.77 µm to 2.61 µm in size and were larger than the corresponding ones in the males (from 0.57 µm to 2.16 µm), there was not a very large X Chromosome in the females. Based on the known female-related FRML-494 marker, co-electrophoresis and Southern blot profiles demonstrated that it was inheritable and specific to female gametophytes. Using modified fluorescence in situ hybridization (FISH), this marker could be localized on one unique Chromosome of the female gametophytes as well as the sporophytes, whereas no hybridization signal was detected in the male gametophytes. Conclusions/Significance Our data suggest that this marker was a female Chromosome-specific DNA sequence. This is the first report of molecular marker localization on algal Chromosomes. This research provides evidence for the benefit of using FISH for identifying molecular markers for sex identification, isolation of specific genes linked to this marker in the females, and sex determination of S. japonica gametophytes in the future.
Renhai Peng - One of the best experts on this subject based on the ideXlab platform.
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Preparations of Meiotic Pachytene Chromosomes and Extended DNA Fibers from Cotton Suitable for Fluorescence In Situ Hybridization
2013Co-Authors: Renhai Peng, Tao Zhang, Fang Liu, Jian Ling, Chunying Wang, Xiangdi Zhang, Yuhong Wang, Kunbo WangAbstract:Fluorescence in situ hybridization (FISH) has become one of the most important techniques applied in plant molecular cytogenetics. However, the application of this technique in cotton has lagged behind because of difficulties in Chromosome Preparation. The focus of this article was FISH performed not only on cotton pachytene Chromosomes, but also on cotton extended DNA fibers. The cotton pollen mother cells (PMCs) instead of buds or anthers were directly digested in enzyme to completely breakdown the cell wall. Before the routine acetic acid treatment, PMCs were incubated in acetic acid and enzyme mixture to remove the cytoplasm and clear the background. The method of ice-cold Carnoy’s solution spreading Chromosome was adopted instead of nitrogen removed method to avoid Chromosomes losing and fully stretch Chromosome. With the above-improved steps, the high-quality well-differentiated pachytene Chromosomes with clear background were obtained. FISH results demonstrated that a mature protocol of cotton pachytene Chromosomes Preparation was presented. Intact and no debris cotton nuclei were obtained by chopping from etiolation cotyledons instead of the conventional liquid nitrogen grinding method. After incubating the nuclei with nucleus lysis buffer on slide, the parallel and clear background DNA fibers were acquired along the slide. This method overcomes the twist, accumulation and fracture of DNA fibers compared with other methods. The entire process of DNA fibers Preparation requires only 30 min, in contrast, it takes 3 h with routine nitrogen grinding method. The poisonous mercaptoethanol in nucleus lysi
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Preparations of meiotic pachytene Chromosomes and extended dna fibers from cotton suitable for fluorescence in situ hybridization
PLOS ONE, 2012Co-Authors: Renhai Peng, Tao Zhang, Fang Liu, Jian Ling, Chunying Wang, Xiangdi Zhang, Yuhong Wang, Kunbo WangAbstract:Fluorescence in situ hybridization (FISH) has become one of the most important techniques applied in plant molecular cytogenetics. However, the application of this technique in cotton has lagged behind because of difficulties in Chromosome Preparation. The focus of this article was FISH performed not only on cotton pachytene Chromosomes, but also on cotton extended DNA fibers. The cotton pollen mother cells (PMCs) instead of buds or anthers were directly digested in enzyme to completely breakdown the cell wall. Before the routine acetic acid treatment, PMCs were incubated in acetic acid and enzyme mixture to remove the cytoplasm and clear the background. The method of ice-cold Carnoy's solution spreading Chromosome was adopted instead of nitrogen removed method to avoid Chromosomes losing and fully stretch Chromosome. With the above-improved steps, the high-quality well-differentiated pachytene Chromosomes with clear background were obtained. FISH results demonstrated that a mature protocol of cotton pachytene Chromosomes Preparation was presented. Intact and no debris cotton nuclei were obtained by chopping from etiolation cotyledons instead of the conventional liquid nitrogen grinding method. After incubating the nuclei with nucleus lysis buffer on slide, the parallel and clear background DNA fibers were acquired along the slide. This method overcomes the twist, accumulation and fracture of DNA fibers compared with other methods. The entire process of DNA fibers Preparation requires only 30 min, in contrast, it takes 3 h with routine nitrogen grinding method. The poisonous mercaptoethanol in nucleus lysis buffer is replaced by nonpoisonous dithiothreitol. PVP40 in nucleus isolation buffer is used to prevent oxidation. The probability of success in isolating nuclei for DNA fiber Preparation is almost 100% tested with this method in cotton. So a rapid, safe, and efficient method for the Preparation of cotton extended DNA fibers suitable for FISH was established.