Synopsis
Almost diploid nuclei (as judged from the microdensitometric evaluation of the Feulgen positive material) of granular and Purkinje cells of the rat cerebellar cortex, were submitted toin situ DNA denaturation and renaturation experiments.
We assessed the double-strandedness of DNA, by Methyl Green staining according to Scott (1967). Under these conditions a stoichiometric ratio between bound dye and DNA exists, suitable for quantitative micrdensitometric measurments. Our data show that the DNA in the interphasic chromatin is never completely denatured after the treatments we used. Furthermore, the renaturation takes place in a different way in the two cell types.
Owing to the unlike chromatin packing of granular and Purkinje nuclei, we suggest that nuclear proteins must interfere differently on thein situ denaturation and renaturation processes.
Similar content being viewed by others
References
Bernocchi, G. (1975). Contenuto in DNA e area nucleare dei neuroni durante l'istogenesi cerebellare del ratto.Rend. Ist Lomb. Sci. Lett. 109, 143–61.
Bernocchi, G. &De Stefano, G. F. (1976b). Analisi della cinetico de eobrolisidella reasione di Feulgen in cellule a diverso rapporto di eu-erd eterocrometino.Riv. Istoch. Norm. Pat. 20, 120.
Bernocchi, G., De Vivo, M. L. &De Stefano, G. F. (1976). Sull'esistenza di due aspetti morfofunzionali di versi delle cellule di Purkinje nel cervelletto di ratto adulto.Riv. Istoch. Norm. Pat. 20, 119.
Bradbury, E. M. (1975). Histones in chromosomal structure and control of cell division. In:The structure and function of chromatin.Ciba Foundation Symposium.28, pp. 131–55. Amsterdam: Associated Scientific Publishers.
Britten, R. J., Graham, D. E. &Neufeld, B. R. (1974). Analysis of repeating DNA sequences by reassociation. In:Methods in enzymology, Vol. XXIX, Part E. pp. 363–418. New York and London: Academic Press.
Britten, R. J. &Kohne, D. E. (1968). Repeated sequences in DNA.Science 11, 529–40.
Brown, D. M. &Todd, A. R. (1955). Evidence of the nature of the chemical bonds in nucleic acids. In:The nucleic acids (eds. E. Chagraff & J. N. Davidson), Vol 1, pp. 409–45. New York: Academic Press.
Darzynkiewicz, Z., Traganos, F., Sharpless, T. &Melamed, M. R. (1975). Thermal denaturation of DNAin situ as studied by acridine orange staining and automated cytofluorometry.Expl. Cell Res. 90, 411–428.
Davidson, J. N. (1973)Biochimica degli acidi nucleici. Padova: Ed. Piccin.
De La Chapelle, A., Schroeder, J., Selander, R. K. (1973).In situ localization and characterization of different classes of chromosomal DNA: acridine orange and quinacrine mustard fluorescence.Chromosoma 40, 347–60.
Diaz, M. (1972). Methyl green staining and highly repetitive DNA in polytene chromosomes.Chromosoma 37, 131–8.
Engelhardt, W. A. (1975). Location of chromatin components. In:The structure and function of chromatin.Ciba Foundation Symposium, Vol. 28, pp. 337–51. Amsterdam: Elsevier Excerpta Medica. North Holland.
Fraschini, A., De Stefano, G. F., Pellicciari, C. &Bernocchi, G. (1975). Evoluzione strutteusle della cromatina durante la maturazione delle cellule di Purkinje nel cervelletto di ratto.Boll. Zool. 42, 457–8. XLIII Congr. U.Z.I., Siena.
Fraschini, A. &Pellicciari, C. (1976). II verde de metile come indicatore dello stato chimico-fisico del DNA nella cromatina interfasica.Riv. Istoch. Norm. Pat. 20, 95–6.
Frenster, J. H. (1974). Ultrastructure and function of heterochromatin and euchromatin. In:The cell nucleus, Vol. 1, pp. 565–80. New York, London: Academic Press.
Gurr, E. (1971).Synthetic dyes in biology Medicine and Chemistry, pp. 64–87. London: Academic Press.
Kurnick, N. B. (1949). Methyl green-pyronin. I: Bases of selective staining of nucleic acids.J. Gen. Physiol. 33, 265–74.
Kurnick, N. B. (1949). Methyl green-pyronin. II: stoichiometry of reaction with nucleic acids.J. Gen. Physiol. 33, 365–74.
Kurnick, N. B. (1950). The quantitative estimation of deoxyribonucleic acid based on methyl green staining.Expl. Cell Res. 1, 151–8.
Kurnick, N. B. (1952). The bases for the specificity of methyl green staining.Expl. Cell Res. 3, 649–51.
Marmur, J. &Ts'o, P. O. P. (1961). Denaturation of deoxyribonucleic acid by formamide.Biochim. biophys. Acta 51, 32–6.
More, J. A. R. &Paul, J. (1973). Template activity and electron microscopic appearance of salt-extracted chromatin.Expl. Cell. Res. 76, 79–86.
Palay, S. L. &Chan Palay, V. (1974).Cerebellar cortex. Berlin: Springer Verlag.
Pollister, A. W. &Leuchtenberger, C. (1949). The nature of the specificity of methyl green for chromatin.Proc. Natn. Acad. Sci. 35, 111–6.
Rigler, R. (1966). Microfluorometric characterization of intracellular nucleic acids and nucleoproteins by acridine orange.Acta physiol. scand.,67, 7–123.
Rigler, R. (1968). Microfluorometric determination of nucleic acids and nucleo-proteins in single cells by acridine orange. In:Macromolecules and the function of the neuron, (eds. Z. Lodin & S. P. R. Rose), pp. 3–12. Amsterdam: Excerpta Medica Found.
Rigler, R. (1969). Acridine orange in nucleic acid analysis.Ann. N.Y. Acad. Sci. 157, 211–24.
Scott, J. E. (1967). On the mechanism of the methyl green-pyronine stain for nucleic acids.Histochemie 9, 30–46.
Senior, M. B., Olins, A. L. &Olins, D. E. (1975). Chromatin fragments resembling tov bodies.Science 187, 173–5.
Smart, J. E. &Bonner, J. (1971). Selective dissociation of histones from chromatin by sodium deoxycholate.J. Molec. Biol. 58, 651–9.
Stockert, J. C. &Lisanti, J. A. (1972). Acridine orange differential fluorescence of fast- and slow-reassociating chromosomal DNA afterin situ DNA denaturation and reassociation.Chromosoma,37, 117–30.
Sumner, A. T., Evans, H. J. &Buckland, R. A. (1973). Mechanisms involved in the banding of chromosomes with quinacrine and Giemsa.Expl. Cell Res. 81, 214–22.
Wertmur, J. G. &Davidson, N. (1968). Kinetics of renaturation of DNA.J. Molec. Biol. 31, 349–70.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Pellicciari, C., Fraschini, A. Methods of denaturation and renaturation of DNA in interphasic chromatin: cytochemical quantitative analysis by Methyl Green staining. Histochem J 10, 213–222 (1978). https://doi.org/10.1007/BF01003306
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF01003306