Skip to main content
Springer Nature Link
Log in

Microfluorometric investigations of chromatin structure

III. Estimation of histones and DNA in thymocyte and hepatocyte nuclei. Effects of extraction at pH 3.0

  • Published:
Save article
View saved research
Histochemistry Aims and scope Submit manuscript

Summary

Experiments were undertaken in which the histones of mouse thymocyte and hepatocyte nuclei were stained by a fluorescent version of the Alfert and Geschwind (1953) procedure and measured by microfluorometry. These measurements were compared with those obtained after treatment of the nuclei with a pH 3.0 phosphate-citrate buffer which is known to displace fraction H1 histones from the chromatin of HeLa cell nuclei. Estimates of DNA with the fluorochromes Hoechst 33258, mithramycin, and proflavine were obtained in the same nuclei, with and without extraction at pH 3.0. In addition, the effects of extracting the nuclei in a phosphate-citrate buffer at pH 2.5 on DNA measurements were also determined. The results of hydrolyzing the nuclei over a period of 30 min to 5 h in 5% TCA at 60° C and staining at pH 8.0 with brilliant sulfaflavine for the demonstration of histones revealed that the fluorescence of 2c hepatocyte nuclei was always much greater than that of thymocyte nuclei. In addition, hepatocyte nuclei responded more slowly than thymocyte nuclei to hydrolysis; and the ratios of 4c:2c, 8c:4c, and 8c:2c hepatocyte nuclei generally exceeded the expected 2:2:4 values. While the fluorescence of thymocyte nuclei extracted at pH 3.0 and stained by the histone procedure was slightly greater than that of control thymocyte nuclei at all times during the hydrolysis period except for the earliest stages, the fluorescence of pH 3.0-extracted hepatocyte nuclei was always less than that of control hepatocyte nuclei; and the 2, 4, and 8c ploidy classes were not resolved clearly. Extraction at pH 3.0 and 2.5 generally produced an increase in fluorescence of both thymocyte and hepatocyte nuclei stained by the various DNA methods; but the absolute amount of change could not be related directly to known dependencies of the dyes on AT or GC base pairs. The results of the fluorescent histone procedure and those obtained with the various DNA methods after extraction at pH 3.0 and 2.5 indicate that these methods function as “probes” of nucleoprotein organization, rather than as absolutely quantitative procedures. It is presently unclear how the measurements are related to specific differences in the organization of the chromatin and/or transcriptional activities of the two types of nuclei.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+
from €37.37 /Month
  • Starting from 10 chapters or articles per month
  • Access and download chapters and articles from more than 300k books and 2,500 journals
  • Cancel anytime
View plans

Buy Now

Price includes VAT (Netherlands)

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Explore related subjects

Discover the latest articles, books and news in related subjects, suggested using machine learning.

References

  • Alfert M, Geschwind II (1953) A selective staining method for basic proteins of cell nuclei. Proc Natl Acad Sci USA 39:991–999

    Google Scholar 

  • Alvarez MR (1974) Early nuclear cytochemical changes in regenerating mammalian liver. Exp Cell Res 83:225–230

    Google Scholar 

  • Arceci RJ, Gross PR (1980) Histone gene expression: progeny of isolated early blastomeres in culture make the same change as the embryo. Science 209:607–609

    Google Scholar 

  • Auer G (1972) Nuclear protein content and DNA-histone interaction. Exp Cell Res 75:231–236

    Google Scholar 

  • Auer G, Zetterberg A, Killander D (1970) Changes in binding between DNA and arginine residues in histone induced by cell crowding. Exp Cell Res 62:32–38

    Google Scholar 

  • Bak AL, Zeuthen J (1978) Higher-order structure of mitotic chromosomes. Cold Spring Harbor Symp Quant Biol 42:367–377

    Google Scholar 

  • Black MM, Ansley HR (1965a) Antigen-induced changes in lymphoid cell histones. I. Thymus. J Cell Biol 26:201–208

    Google Scholar 

  • Black MM, Ansley HR (1965b) Antigen-induced changes in lymphoid cell histones. II. Regional lymph nodes. J Cell Biol 26:797–803

    Google Scholar 

  • Black MM, Ansley HR (1966) Histone specificity revealed by ammoniacal silver staining. J Histochem Cytochem 14:177–181

    Google Scholar 

  • Black MM, Ansley HR (1967) Antigen-induced changes in lymphoid cell histones. III. In vitro and in extract. J Cell Biol 35:619–625

    Google Scholar 

  • Bloch DP (1969) A catalog of sperm histones. Genetics (Suppl) 61:93–111

    Google Scholar 

  • Bloch DP, Hew HYC (1960) Schedule of spermatogenesis in the pulmonate snail Helix aspersa, with special reference to histone transition. J Biophys Biochem Cytol 7:515–532

    Google Scholar 

  • Bloch DP, Hew HYC (1961) Changes in nuclear histones during fertilization and early embryonic development in the pulmonate snail, Helix aspersa. J Biophys Biochem Cytol 8:69–81

    Google Scholar 

  • Bonner J (1979) Expressed and nonexpressed portions of the genome: their separation and their characterization. In: Nicolini CA (ed) Chromatin structure and function. Molecular and cellular biophysical methods, Part A. Plenum Press, New York, pp 15–23

    Google Scholar 

  • Busch H (1965) Histones and other nuclear proteins. Academic Press, New York

    Google Scholar 

  • Cohen LH, Newrock KM, Zweidler A (1975) State-specific switches in histone synthesis during embryogenesis of the sea urchin. Science 190:994–997

    Google Scholar 

  • Cohn NS (1973) A model system analysis of the parameters in histone staining: I Alkaline fast green. Histochem J 5:529–545

    Google Scholar 

  • Cowden RR (1962) Further cytochemical investigations on the growth and development of slug oocytes. Growth 26:209–234

    Google Scholar 

  • Cowden RR, Curtis SK (1981a) Microfluorometric investigations of chromatin structure. I. Evaluation of nine DNA-specific fluorochromes as probes of chromatin organization. Histochemistry 72:11–23

    Google Scholar 

  • Cowden RR, Curtis SK (1981b) Microfluorometric investigations of chromatin structure. II. Mordant fluorochroming with ions that complex with Morin. Histochemistry 72:391–400

    Google Scholar 

  • Curtis SK, Cowden RR (1980) The effects of fixation and preparation on three quantitative fluorescent cytochemical procedures. Histochemistry 68:29–38

    Google Scholar 

  • Dubochet J, Noll M (1978) Nucleosome arcs and helices. Science 202:280–286

    Google Scholar 

  • Geyer G (1960) Zur Eiweissfärbung mit Amidoschwartz 10 B. Acta Histochem 10:286–292

    Google Scholar 

  • Gledhill BL, Gledhill MP, Rigler R, Ringertz NR (1966) Changes in deoxyribonucleoprotein during spermiogenesis in the bull. Exp Cell Res 41:652–665

    Google Scholar 

  • Hentschel CC, Birnstiel ML (1981) The organization and expression of histone gene families. Cell 26:301–313

    Google Scholar 

  • Hnilica LS (1964) The specificity of histones in chicken erythrocytes. Experientia 20:13–14

    Google Scholar 

  • Jackson V (1978) Studies on histone organization in the nucleosome using formaldehyde as a reversible cross-linking agent. Cell 15:945–954

    Google Scholar 

  • Klingholz R, Strätling WH, Schäfer H (1981) Structure of rat liver after depletion and reassociation of histone H1. Exp Cell Res 132:399–409

    Google Scholar 

  • Kornberg RD (1974) Chromatin structure: a repeating unit of histones and DNA. Science 184:868–871

    Google Scholar 

  • Kornberg RD (1977) Structure of chromatin. Annu Rev Biochem 46:931–954

    Google Scholar 

  • Kornberg RD, Klug A (1981) The nucleosome. Sci Am 244:52–64

    Google Scholar 

  • Kornberg RD, Thomas JO (1974) Chromatin structure: oligamers of histones. Science 184:865–868

    Google Scholar 

  • Latt SA, Wholleb JC (1975) Optical studies of the interaction of 33258 Hoechst with DNA, chromatin, and metaphase chromosomes. Chromosoma 52:297–316

    Google Scholar 

  • Lawson GM, Cole RD (1979) Selective displacement of histone H1 from whole HeLa nuclei: effect on chromatin structure in situ as probed by micrococcal nuclease. Biochemistry 18:2160–2166

    Google Scholar 

  • Leemann U, Ruch F (1972) Cytofluorometric determination of basic and total proteins with sulfaflavine. J Histochem Cytochem 20:659–671

    Google Scholar 

  • Lief RC, Easter HN, Warters RL, Thomas RA, Dunlap LA, Austin MF (1971) Centrifugal cytology. I. A quantitative technique for the preparation of glutaraldehyde-fixed cells for the light and scanning electron microscope. J Histochem Cytochem 19:203–215

    Google Scholar 

  • Lillie RD, Fullmer HM (1976) Histopathologic technic and practical histochemistry, 4th ed. McGraw-Hill, New York

    Google Scholar 

  • Macrae EK, Meetz GD (1970) Electron microscopy of the ammoniacal silver reaction for histones in the erythropoietic cells of the chick. J Cell Biol 45:235–245

    Google Scholar 

  • Morris NR (1976) A comparison of the structure of chicken erythrocyte and chicken liver chromatin. Cell 9:627–632

    Google Scholar 

  • Müller W, Gautier F (1975) Interactions of heteroaromatic compounds with nucleic acids. A·T-specific non-intercalating DNA ligands. Eur J Biochem 54:385–394

    Google Scholar 

  • Myśliwski A (1971) Reaction on histones with ammoniacal silver in chromatin in different states of condensation. Acta Histochem 39:248–251

    Google Scholar 

  • Noeske K (1973) Discrepancies between cytophotometric alkaline fast green measurements and nuclear histone protein content. Histochem J 5:303–311

    Google Scholar 

  • Noll M (1974) Subunit structure of chromatin. Nature 251:249–251

    Google Scholar 

  • Olins AL, Olins DE (1974) Spheroid chromatin units (v bodies). Science 183:330–332

    Google Scholar 

  • Olins DE, Olins AL (1978) Nucleosomes: the structural quantum in chromosomes. Am Sci 66:704–711

    Google Scholar 

  • Oudet P, Gross-Bellard M, Chambon P (1975) Electron microscopic and biochemical evidence that chromatin structure is a repeating unit. Cell 4:281–300

    Google Scholar 

  • Petrov P, Raitcheva E, Tsanev R (1980) Nucleosomes and nonribosomal RNA transcription in early mouse embryo. An electron microscope study. Eur J Cell Biol 22:708–713

    Google Scholar 

  • Pipkin JL Jr (1968) Cytological and cytochemical methodology of histones. In: Prescott DM (ed) Methods in cell physiology, vol III. Academic Press, New York, pp 307–346

    Google Scholar 

  • Poccia D, Salik J, Krystal G (1981) Transitions in histone variants of the male pronucleus following fertilization and evidence for a maternal store of cleavage-stage histones in the sea urchin egg. Dev Biol 82:287–296

    Google Scholar 

  • Ringertz NR (1969) Cytochemical properties of nuclear proteins and deoxyribonucleoprotein complexes in relation to nuclear function. In: Lima-De-Faria A (ed) Handbook of molecular cytology. American Elsevier, New York, pp 656–684

    Google Scholar 

  • Ringertz NR, Zetterberg A (1966) Cytochemical demonstration of histones. Mechanism and specificity of the alkaline bromphenol blue binding reaction. Exp Cell Res 42:243–259

    Google Scholar 

  • Ruch F (1970) Principles and some applications of cytofluorometry. In: Wied GL (ed) Introduction to quantitative cytochemistry, vol II. Academic Press, New York, pp 431–450

    Google Scholar 

  • Ruch F, Rosselet A (1970) A cytochemical study of euchromatin and heterochromatin in roots of Rhoeo discolor. Exp Cell Res 62:219–227

    Google Scholar 

  • Schmitz KS, Ramanathan B (1980) Generation of a third-order folded structure for chromatin. J Theor Biol 83:297–311

    Google Scholar 

  • Spicer SS (1962) Histochemically selective acidophilia of basic proteins in chromatin and nucleoli at alkaline pH. J Histochem Cytochem 10:691–703

    Google Scholar 

  • Šponar J, Šormová Z (1972) Complexes of histone F1 with DNA in 0.15 M NaCl. Selectivity of interaction with respect to DNA composition. Eur J Biochem 29:99–103

    Google Scholar 

  • Šonar J, Doskočil J, Bláha K, Zimmer Ch (1976) Basic polypeptides as histone models. Effect of conformation, base composition and methylation of nucleic acids on the interaction with H1 and histone models and on the circular dichroism of complexes. Nucleic Acids Res 3:507–521

    Google Scholar 

  • Swift H (1966) The quantitative cytochemistry of RNA. In: Wied GL (ed) Introduction to quantitative cytochemistry. Academic Press, New York, pp 355–386

    Google Scholar 

  • Thoma F, Koller Th, Klug A (1979) Involvement of histone H1 in the organization of the nucleosome and of the salt-dependent superstructures of chromatin. J Cell Biol 83:403–427

    Google Scholar 

  • Thomes JC, Weill G, Duane M (1969) Fluorescence of proflavine-DNA complexes: Heterogeneity of binding sites. Biopolymers 8:647–659

    Google Scholar 

  • Ward DC, Reich E, Goldberg IH (1969) Base specificity in the interaction of polynucleotides with antibiotic drugs. Science 149:1259–1263

    Google Scholar 

  • Waring M (1970) Variation of the supercoils in closed circular DNA by binding of antibiotics and drugs: evidence for molecular models involving intercalation. J Mol Biol 54:247–279

    Google Scholar 

  • Weisblum B, Haenssler E (1974) Fluorometric properties of the bibenzimidazole derivative Hoechst 33258, a fluorescent probe specific for AT concentration in chromosomal DNA. Chromosoma 46:255–260

    Google Scholar 

  • Woodcock CLF (1973) Ultrastructure of inactive chromatin. J Cell Biol 59:368a

    Google Scholar 

  • Wray W, Conn PM, Wray VP (1977) Isolation of nuclei using hexylene glycol. In: Stein G, Stein J, Kleinsmith LJ (eds) Methods in cell biology, vol XVI. Academic Press, New York, pp 69–86

    Google Scholar 

  • Zachau HG, Igo-Kemenes T (1981) Face to phase with nucleosomes. Cell 24:597–598

    Google Scholar 

  • Zetterberg A, Auer G (1968) Early changes in the binding between DNA and histone in human leucocytes exposed to phytohaemagglutinin. Exp Cell Res 56:122–126

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biophysics, Quillen-Dishner College of Medicine, East Tennessee State University, 37614, Johnson City, TN, USA

    R. R. Cowden & S. K. Curtis

Authors
  1. R. R. Cowden
    View author publications

    Search author on:PubMed Google Scholar

  2. S. K. Curtis
    View author publications

    Search author on:PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Cowden, R.R., Curtis, S.K. Microfluorometric investigations of chromatin structure. Histochemistry 74, 469–480 (1982). https://doi.org/10.1007/BF00496660

Download citation

  • Accepted:

  • Issue date:

  • DOI: https://doi.org/10.1007/BF00496660

Keywords