Download Now Download to read offline and view in fullscreen. Download Now Download Download to read offline. Biology in Focus - Chapter 9. CSI and Evidence Collection. JonBenet Ramsey. Forensic Science Chapter 1 Vocabulary. Perch dissection. Shark basics. Sea star dissection. Squid Dissection Prelab. Related Books Free with a 30 day trial from Scribd. Elizabeth Howell. Ralph Eubanks. Logicomix: An epic search for truth Apostolos Doxiadis. Related Audiobooks Free with a 30 day trial from Scribd.
Jen Gunter. A Wild Idea Jonathan Franklin. Akshat Verma. Sebastian Rodriguez. Yanet Roche-Behar. Mo Grove. Miaya MeMe. Jasmine Al-Amin. Kiana Holland. Selyn Ortiz Flores. Kristina Bulacan. Show More.
Views Total views. Actions Shares. No notes for slide. Biology in Focus - Chapter 10 1. Figure Concept Individual chromosomes line up. Sister chromatids separate. Daughter cells of meiosis II n n n n Daughter cells of meiosis I UN01 Okadaic acid and Calyculin A, both are extracted from marine sponges Halichondria okadai Kadota or Discodermia Calyx, respectively.
Cell culture and maintain exponentially growing condition is a key point to obtain good PCC index. The PCC index depends also on cell types for the experiment use.
Usually 50 nM calyculin A for 30 minutes of incubation is enough to obtain substantial number of PCCs. Note that the optimal condition will be different for individual cell types.
The researcher should fix the best combination of concentration and incubation time of calyculin A in general, PCC index increases as either concentration or incubation time increase, but the shape of chromosome will be more condense.
Thirty-minutes prior to cell harvest, add 50 nM final concentration of calyculin A 1 mM stock solution in ethanol or DMSO to the medium and another culturing. At harvest time, cells usually rounded and loosely attached or even detached from the culture flask and suspended in the medium due to calyculin A effect.
Gently pipetting the medium and harvest the cells with a medium into a centrifuge tube. Fix the cell with same volume of Carnoy's fixative methanol: acetic acid vol: vol , then spin down again the same above condition. The cell pellet is again re-suspended with the same fixative.
In case of cells are too sparse, do the same steps but with less volume of fixative. Usually one slide glass will enough to score and analyze substantial number of chromosome spreads. Chromosome sample is then subject to conventional staining protocol such as Giemsa staining or FISH study if required. PCC is a very interesting phenomenon not only of a biological point of view but also as useful tools for cytogenetic fields.
Chromosomes are usually prepared from mitotic cells using colcemid block method. The obtained chromosomes are therefore mitotic chromosomes only because colcemid simply inhibits assembly of spindle body at mitotic phase, thus the cell's arrest in mitosis with condensed chromosomes.
In contrast, PCC forces the chromosomes to be condensed 'prematurely' not only in mitosis but also in interphase nuclei of any cell cycle G1-, S-, G2-, M-phase Gotoh et al. Due to this unique aspect, PCC has been used for analyze various nuclear events that proceed during interphase, such as chromosome breakage and repair after exposure of ionizing irradiation or mutagens either using fusion-mediated PCC Hittelman and Rao, ; Hittelman and Rao, ; Cornforth and Bedford, ; Hittelman and Pollard, ; Sen and Hittelman, ; Maillie et al.
One of most successful application of PCC technique is on radiation biodosimetry , in particular to estimate the irradiation dose after an exposure accident of large dose irradiation Gotoh and Asakawa, ; IAEA, ; Gotoh and Tanno, ; Gotoh et al.
Estimation of the body irradiation exposure dose by means of cytogenetic approach by scoring chromosomal aberrations is widely used as a standard biodosimetry. Prepare and score chromosomes of peripheral blood lymphocytes, using the colcemid block protocol which is a simple and well established method cytogenetic biodosimetry, see reviews by Lloyd , International Atomic Energy Agency IAEA , However, after human body is irradiated with large doses i.
As a consequence, it is usually impossible to obtain chromosome from severely damaged cells for cytogenetic analysis, which has limited the application of the conventional colcemid block method for cytogenetic biodosimetry to estimate radiation doses above 10 Gy. This dose limitation was first overcame using drug-induced PCC technique Gotoh et al. As clearly seen, all chromosomes are severely damaged and no intact chromosome remains. Such chromosomes, extremely destroyed, have not ever been obtained from the cells exposed to high dose of irradiation.
Figure 2. Newer cytological approaches have been challenged utilizing with PCC technique. Classically, it was thought that chromosomes condense during mitotic phase. However, recently accumulated findings revealed that chromosome condensation is not an independent event proceeding during mitosis, but tightly coupled with DNA replication or chromosome repair process Zink et al. But visualizing approaches for chromosome dynamics coupled with DNA replication was somehow limited, because it should be required to observe chromosomes in S-phase, but chromosomes are usually invisible in S-phase as they are de-condensed.
Visualizing dynamics of chromosome condensation has been accomplished using drug-induced PCC technique Gotoh, , which showed that chromosome condensation is very tightly coupled with DNA replication. Involvement of condensin II in sister chromatid separation during the S-phase has also elucidated by means of drug-induced PCC Ono et al.
However, precise mechanism is still mostly unclear. PCC is partly the similar but not completely the same phenomenon as normal chromosome condensation Ghosh et al.
Acknowledgement: I wish deeply express my thanks to Dr. Jean-Loup Huret for give me an opportunity to write this chapter. Bibliography Chromosome pulverization in human binucleate cells following colcemid treatment. Kato H, Sandberg AA. J Cell Biol.
PMID Chromosome pulverization in human cells with micronuclei. J Natl Cancer Inst. PMID Mammalian cell fusion: induction of premature chromosome condensation in interphase nuclei. PMID Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes. Masui Y, Markert CL. J Exp Zool. PMID Bleomycin-induced damage in prematurely condensed chromosomes and its relationship to cell cycle progression in CHO cells.
Cancer Res. PMID A cytostatic factor in amphibian oocytes: its extraction and partial characterization. Masui Y. PMID The nature of adriamycin-induced cytotoxicity in Chinese hamster cells as revealed by premature chromosome condensation. PMID X-ray--induced breakage and rejoining of human interphase chromosomes. PMID Units of chromosome replication and packing.
J Cell Sci. PMID A simple method for premature chromosome condensation induction in primary human and rodent cells using polyethylene glycol. Somatic Cell Genet. PMID Prematurely condensed chromosomes: a model system for visualizing effects of DNA damage, repair and inhibition at the level of chromosome structure. Hittelman WN. Nucleic Acids Symp Ser.
PMID Premature chromosome condensation studies in human leukemia: 5. Prediction of early relapse. PMID Visualization of chromatin events associated with repair of ultraviolet light-induced damage by premature chromosome condensation. Hittelman WN, Pollard M. PMID An overview of radiation dosimetry by conventional cytogenetic methods. Lloyd DC. Springer, Berlin, p The use of peripheral blood mononuclear cell prematurely condensed chromosomes for biological dosimetry.
Radiat Res. PMID Kinetics and extent of repair of bleomycin-induced chromosome damage in quiescent normal human fibroblasts and human mononuclear blood cells. Sen P, Hittelman WN. PMID Premature chromosome condensation: evidence for in vivo cell fusion in human malignant tumours. Kovacs G. Int J Cancer. International Atomic Energy Agency. Vienna: IAEA. Measuring damage to human lymphocytes in vitro after low-level radiation exposure using prematurely condensed chromosomes.
Health Phys. PMID Maturation-promoting factor and the regulation of the cell cycle. J Cell Sci Suppl. PMID Fission yeast cdc25 is a cell-cycle regulated protein. Biochem Biophys Res Commun. PMID Complementation of the mitotic activator, p80cdc25, by a human protein-tyrosine phosphatase. PMID Regulation of mitosis by cyclic accumulation of p80cdc25 mitotic inducer in fission yeast.
PMID The cdc25 protein contains an intrinsic phosphatase activity. Dunphy WG, Kumagai A. PMID cdc25 is a specific tyrosine phosphatase that directly activates p34cdc2. PMID Cell cycle-dependent behavior of microtubules in hybrids of mouse oocytes and blastomeres. Kubiak JZ. Int J Dev Biol. PMID The cdc25 protein controls tyrosine dephosphorylation of the cdc2 protein in a cell-free system.
Kumagai A, Dunphy WG. PMID Failure of kinetochore development and mitotic spindle formation in okadaic acid-induced premature mitosis in HeLa cells. Exp Cell Res. PMID Periodic changes in phosphorylation of the Xenopus cdc25 phosphatase regulate its activity.
Mol Biol Cell. PMID Kinetochore formation and behaviour following premature chromosome condensation. Rattner JB, Wang T. PMID Negative regulation of mitosis by the fission yeast protein phosphatase ppa2. Genes Dev. Biomedical Research. Chromosome condensation induced by fostriecin does not require p34cdc2 kinase activity and histone H1 hyperphosphorylation, but is associated with enhanced histone H2A and H3 phosphorylation.
EMBO J. PMID Induction and repair of chromosome aberrations in scid cells measured by premature chromosome condensation. PMID Detection and evaluation of chromosomal aberrations induced by high doses of gamma-irradiation using immunogold-silver painting of prematurely condensed chromosomes. Gotoh E, Asakawa Y. Int J Radiat Biol. PMID A method for detecting sister chromatid exchanges using prematurely condensed chromosomes and immunogold-silver staining.
Asakawa Y, Gotoh E. PMID Histone H3 phosphorylation is required for the initiation, but not maintenance, of mammalian chromosome condensation. PMID Structure and dynamics of human interphase chromosome territories in vivo.
Hum Genet. PMID Chromatid break rejoining and exchange aberration formation following gamma-ray exposure: analysis in G2 human fibroblasts by chemically induced premature chromosome condensation.
PMID Cytogenetic analysis for radiation dose assesment. A manual. Epstein-Barr virus nuclear antigen-1 is highly colocalized with interphase chromatin and its newly replicated regions in particular. J Gen Virol. Pflumm MF. Epub Mar PMID Dose response relationships for acute ionizing-radiation lethality.
0コメント