Abstract
Analysis of the formation of extended chromatin fibers (ECFs) in response to the action of gravity following lysis by hypertonic and detergent solutions is a useful technical procedure relevant for studies of the positioning of particular DNA signals on chromatin filaments. Additionally, if toluidine blue molecules are allowed to bind electrostatically to available DNA phosphates on ECFs, the birefringence brightness generated in these filaments, as observed by polarization microscopy, facilitates the description of the frequency of ECF formation and extension of the chromatin filaments generated. Thus, different patterns of DNA-nuclear matrix protein associations related to varying transcriptional activities and chromatin organization in isolated cells can be assessed. A technique for producing ECFs in different isolated cell types under variable physiological and/or pathological conditions is detailed in this chapter.
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References
Heng HHQ, Squire J, Tsui LC (1992) High-resolution mapping of mammalian genes by in situ hybridization to free chromatin. Proc Natl Acad Sci U S A 89:9509–9513
Haaf T, Ward DC (1994) High resolution ordering of YAC contigs using extended chromatin and chromosomes. Human Mol Genet 3:629–633
Haaf T, Ward DC (1994) Structural analysis of α-satellite DNA and centromere proteins using extended chromatin and chromosomes. Human Mol Genet 3:697–709
Vidal BC (2000) Extended chromatin fibres: crystallinity, molecular order and reactivity to concanavalin A. Cell Biol Int 24:723–728
Fidlerova H, Senger G, Kost M et al (1994) Two simple procedures for releasing chromatin from routinely fixed cells for fluorescence in situ hybridization. Cytogenet Cell Genet 65:203–205
Haaf T, Ward DC (1995) Higher order nuclear structure in mammalian sperm revealed by in situ hybridization and extended chromatin fibers. Exp Cell Res 219:604–611
Moraes AS, Mello MLS (2005) Extended chromatin fibers in mouse testicular spermatozoa. Braz J Morphol Sci 22:91–96
Moraes AS, Vidal BC, Guaraldo AMA et al (2005) Chromatin organization and extensibility in mouse hepatocytes following starvation and refeeding. Cytometry Part A 63:94–107
Moraes AS, Guaraldo AMA, Mello MLS (2007) Chromatin supraorganization and extensibility in mouse hepatocytes with development and aging. Cytometry Part A 71:28–37
Moraes AS, Mondin M, Beletti ME et al (2010) Age-related association of rDNA and telomeres with the nuclear matrix in mouse hepatocytes. Cell Biol Int 34:925–931
Mello MLS, Aldrovani M, Moraes AS et al (2009) DNA content, chromatin supraorganization, nuclear glycoproteins and RNA amounts in hepatocytes of mice expressing insulin-dependent diabetes. Micron 40:577–585
Mello MLS, Moraes AS, Vidal BC (2011) Extended chromatin fibers and chromatin organization. Biotech Histochem 88:213–225
Choo KHA (ed) (1994) In situ hybridization protocols. In: Methods in molecular biology, vol 33. Humana Press, Totowa, pp 1–480
Darby IA (ed) (2000) In situ hybridization protocols. In: Methods in molecular biology, vol 123, 2nd edn. Humana Press, Totowa, pp 1–337
Franz PF, Alonso-Blanco C, Liharska TB et al (1996) High-resolution physical mapping in Arabidopsis thaliana and tomato by fluorescence in situ hybridization to extended DNA fibres. Plant J 9:421–430
Yang K, Zhang H, Converse R et al (2011) Fluorescence in situ hybridization on plant extended chromatin DNA fibers for single-copy and repetitive DNA sequences. Plant Cell Rep 30:1779–1786
Peng R, Zhang T, Liu F et al (2012) Preparations of meiotic pachytene chromosomes and extended DNA fibers from corn suitable for fluorescence in situ hybridization. PLoS One 7:e33847
Vidal BC (1972) Doppelbrechungsdispersion und Lineardichroismus von Eu- und Heterochromatin nach Färbung mit Toluidinblau. Nachweis eines Cotton-Effektes. Beitr Pathol 145:269–285
Mello MLS, Vidal BC (1973) Linear dichroism and anomalous dispersion of birefringence on bee sperm heads. Acta Histochem 45:109–114
Bensimon A, Simon A, Chiffaudel A et al (1994) Alignment and sensitive detection of DNA by a moving interface. Science 265:2096–2098
Michalet X, Ekong R, Fougerousse F et al (1997) Dynamic molecular combing: stretching the whole human genome for high-resolution studies. Science 277:1518–1523
Tuduri S, Tourrière H, Pasero P (2010) Defining replication origin efficiency using DNA fiber assays. Chromosome Res 18:91–102
Vidal BC, Moraes AS, Mello MLS (2006) Nucleus image properties assessed by video image analysis in mouse hepatocytes under a short lysis for extended chromatin fiber formation. Cytometry Part A 69:1106–1113
Alvarenga EM, Mondin M, Martins JA et al (2011) Spatial distribution of AT- and GC-rich DNA within interphase cell nuclei of Triatoma infestans Klug. Micron 42:568–578
Stockert JC, López-Arias B, Del Castillo P et al (2012) Replacing xylene with n-heptane for paraffin embedding. Biotech Histochem 87:464–467
Gerdes MG, Carter KC, Moen PT et al (1994) Dynamic changes in the higher-level chromatin organization of specific sequences revealed by in situ hybridization to nuclear halos. J Cell Biol 126:289–304
Iarovaia OV, Akopov SB, Nikolaev LG et al (2005) Induction of transcription within chromosomal DNA loops flanked by MAR elements causes an association of loop DNA with the nuclear matrix. Nucleic Acids Res 33:4157–4163
Acknowledgments
This work was supported by the São Paulo State Research Foundation (FAPESP, grant nos. 2010/50015-6 and 2003/04597-0) and the Brazilian National Council on Development and Research (CNPq).
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Mello, M.L.S., de Campos Vidal, B. (2014). Polarization Microscopy of Extended Chromatin Fibers. In: Stockert, J., Espada, J., Blázquez-Castro, A. (eds) Functional Analysis of DNA and Chromatin. Methods in Molecular Biology, vol 1094. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-706-8_6
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DOI: https://doi.org/10.1007/978-1-62703-706-8_6
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