Skip to main content
SpringerLink
Log in

Energy transfer and binding competition between dyes used to enhance staining differentiation in metaphase chromosomes

  • Published:
Chromosoma Aims and scope Submit manuscript

Abstract

The ability of electronic energy transfer and direct binding competition between pairs of dyes to enhance contrast in human or bovine metaphase chromosome staining patterns is illustrated, and the relative effectiveness of these two mechanisms compared. The existence of energy transfer between quinacrine or 33258 Hoechst and 7-amino-actinomycin D in doubly stained chromosomes is demonstrated directly by microfluorometry. The ability of the dyes 7-amino-actinomycin D, methyl green, or netropsin, acting as counterstains, to displace quinacrine, 33258 Hoechst, or chromomycin A3 from chromosomes, is estimated by quantitative analysis of energy transfer data, by photobleaching of the counterstains, or by selective removal of counter-stains by appropriate synthetic polynucleotides. Effects on the fluorescence of soluble 33258 Hoechst-DNA complexes due to energy transfer or binding displacement, by actinomycin D or netropsin, respectively, are further differentiated by nanosecond fluorescence decay measurements. Examples are presented of dye combinations for which (a) energy transfer is the primary mechanism operative, (b) binding competition exists, with consequences reinforcing those due to energy transfer, or (c) binding competition is the most important interaction. These analyses of mechanisms responsible for contrast enhancement in doubly stained chromosomes are used to derive information about the relationship between chromosome composition and banding patterns.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Behr, W., Honikel, K., Hartman, G.: Interaction of the RNA polymerase inhibitor chromomycin with DNA. European J. Biochem. 9, 82–92 (1969)

    Google Scholar 

  • Bittman, R.: Studies of the binding of eithdium bromide to transfer ribonucleic acid: Absorption fluorescence, ultracentrifugation and kinetic investigations. J. molec. Biol. 46, 25–268 (1969)

    Google Scholar 

  • Caspersson, T., Lindsten, J., Lomakka, G., Moller, A., Zech, L.: The use of fluorescence techniques for the recognition of mammalian chromosomes and chromosome regions. Int. Rev. Cytol. 11, 1–72 (1972)

    Google Scholar 

  • Chen, R.F.: Practical aspects of the calibration and use of the Aminco-Bowman Spectrofluorometer. Anal. Biochem. 20, 339–357 (1967)

    Google Scholar 

  • Chen, R.F.: Measurements of absolute values in biochemical fluorescence spectroscope. Nat. Bur. Stand. (Special Pub.) 378, 183–196 (1973)

    Google Scholar 

  • Comings, D.E., Drets, M.E.: Mechanisms of chromosome banding IX. Are variations in base composition adequate to account for quinacrine, Hoechst 33258, and Duanomycin banding? Chromosoma (Berl.) 56, 199–211 (1976)

    Google Scholar 

  • Ellison, J.R., Barr, H.J.: Quinacrine fluorescence of specific chromosome regions. Chromosoma (Berl.) 36, 375–390 (1972)

    Google Scholar 

  • Förster, T.: Delocalized excitation and excitation transfer. Modern quantium chemistry (O. Sinanoglu, ed.), 3, 43–75 Academic Press, New York 1965

    Google Scholar 

  • Gill, J.E., Jotz, M.M., Young, S.G., Modest, E.J., Sengupta, S.K.: 7-amino-actinomycin D as a cytochemical probe. J. Histochem. Cytochem. 23, 793–799 (1975)

    Google Scholar 

  • Isenberg, I., Dyson, R.D.: Analysis of fluorescence decay by a method of moments. Biophys. J. 9, 1337–1350 (1969)

    Google Scholar 

  • Isenberg, I., Mullouly, J.P., Dyson, R.D., Hanson, R.: Studies on the analysis of fluorescence decay data by the methods of moments. Biophysical J. 13, 1090–1115 (1973)

    Google Scholar 

  • Jensen, R.H., Langlois, R.G., Mayall, B.H.: Strategies for choosing a deoxyribonucleic acid stain for flow cytometry of metaphase chromosomes. J. Histochem. Cytochem. 25, 954–964 (1977)

    Google Scholar 

  • Jorgenson, K.F., Sande, J.H. Van de, Lin, C.C.: The use of base pair specific DNA binding agents as affinity labels for the study of mammalian chromosomes. Chromosoma (Berl.) 68, 287–302 (1978)

    Google Scholar 

  • Kurnick, N.B., Foster, M.: Methyl green. III. Reaction with deoxyribonucleic acid, stoichiometry, and behavior of the reaction product. J. gen. Physiol. 34, 147–159 (1950)

    Google Scholar 

  • Kurnit, D.M., Shafit, B.R., Maio, J.J.: Multiple satellite deoxyribonucleic acids in the calf and their relation to the sex chromosomes. J. molec. Biol. 81, 274–285 (1973)

    Google Scholar 

  • Langlois, R.G., Jensen, R.H.: Interactions between pairs of DNA specific fluorescent stains bound to mammalian cells. J. Histochem. Cytochem. 27, 72–78 (1979)

    Google Scholar 

  • Latt, S.A.: Fluorometric detection of deoxyribonucleic acid synthesis; possibilities for interfacing bromodeoxyuridine dye techniques with flow fluorometry. J. Histochem. Cytochem. 25, 913–926 (1977a)

    Google Scholar 

  • Latt, S.A.: Fluorescent probes of chromosome structure and replication. Canad. J. Genet. Cytol. 19, 603–623 (1977b)

    Google Scholar 

  • Latt, S.A.: Fluorescent probes of DNA microstructure and synthesis. In: Flow cytometry and sorting, (M. Melamed, P. Mullaney, and M. Mendelsohn, eds.) pp. 263–284. New York: J. Wiley, 1979

    Google Scholar 

  • Latt, S.A., Brodie, S., Munroe, S.J.: Optical studies of complexes of quinacrine with DNA and chromatin: Implications for the fluorescence of cytological chromosome preparations. Chromosoma (Berl.) 49, 17–40 (1974)

    Google Scholar 

  • Latt, S.A., Cheung, A.H.T., Blout, E.R.: Energy transfer, a system with relatively fixed donor-acceptor separation. J. Amer. Chem. Soc. 87, 995–1003 (1965)

    Google Scholar 

  • Latt, S.A., Sahar, E., Eisenhard, M.E.: Pairs of fluorescent dyes as probes of DNA and chromosomes. J. Histochem. Cytochem. 27, 65–71 (1979)

    Google Scholar 

  • Latt, S.A., Wohlleb, J.: Optical studies of the interaction of 33258 Hoechst with DNA, chromatin and metaphase chromosomes. Chromosoma (Berl.) 52, 297–316 (1975)

    Google Scholar 

  • Leemann, U., Ruch, F.: Selective excitation of mithramycin or DAPI fluorescence on double-stained cell nuclei and chromosomes. Histochemistry 58, 329–334 (1978)

    Google Scholar 

  • Lilie, R.D., Conn's, H.J.: Biological Stains, 8th edit. Baltimore: Williams and Wilkins, 1969

    Google Scholar 

  • Lin, M.S., Comings, D.E., Alfi, O.S.: Optical studies of the interaction of 4′,6-diamidino-2-phenylindole with DNA and metaphase chromosomes. Chromosoma (Berl.) 60, 15–25 (1977)

    Google Scholar 

  • Lubit, B.W., Phron, T.D., Miller, O.J., Erlanger, B.F.: Localization of 5-methylcytosine in human metaphase chromosomes by immunoelectron microscopy. Cell 9, 503–509 (1976)

    Google Scholar 

  • Miller, O.J., Schnedl, W., Allen, J., Erlanger, B.F.: 5-methylcytosine localized in mammalian constitutive heterochromatin. Nature (Lond.) 251, 636–637 (1974)

    Google Scholar 

  • Modest, E.J., Sengupta, S.K.: 7-substituted actinomycin D (NSC-3053) analogs as fluorescent DNA-binding and experimental antitumor agents. Cancer Chemother. Rep. 58, 35–48 (1974)

    Google Scholar 

  • Mueller, W., Crothers, D.M.: Studies of the binding of actinomycin and related compounds to DNA. J. molec. Biol. 35, 251–290 (1968)

    Google Scholar 

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

    Google Scholar 

  • Nyak, R., Sirsi, M., Posser, S.K.: Spectrophotometric studies on the interaction of chromomycin A3 with DNA and chromatin of normal and neoplastic tissue. Biochim. biophys. Acta. (Amst.) 378, 195–204 (1975)

    Google Scholar 

  • Pachmann, U., Rigler, R.: Quantum yield of acridines interacting with DNA of defined base sequence. Exp. Cell Res. 72, 602–608 (1972)

    Google Scholar 

  • Prantera, G., Bonaccorsi, S., Pimpinelli, S.: Simultaneous production of Q- and R-bands after staining with chromomycin A3 or olivomycin. Science 204, 79–80 (1979)

    Google Scholar 

  • Sahar, E., Latt, S.A.: Enhancement of banding patterns in human metaphase chromosomes by energy transfer. Proc. nat. Acad. Sci. (Wash.) 75, 5650–5654 (1978)

    Google Scholar 

  • Schnedl, W., Breitenbach, M., Mikelsaar, A.V., Stranziger, G.: Mithramycin and DIPI: A pair of fluorochromes specific for G-C and A-T rich DNA, respectively. Hum. Genet. 36, 299–305 (1977)

    Google Scholar 

  • Schnedl, W., Dev, V.G., Tantravahi, R., Miller, D.A., Erlanger, B.F., Miller, O.J.: 5-methylcytosine in heterochromatic regions of chromosomes. Chimpanzee and gorilla compared to the human. Chromosoma(Berl.) 52, 59–66 (1975)

    Google Scholar 

  • Schreck, R.R., Dev, V.G., Erlanger, B.F., Miller, O.J.: The structural organization of mouse metaphase chromosomes. Chromosoma (Berl.) 62, 337–350 (1977)

    Google Scholar 

  • Schweizer, D.: Reverse fluorescent chromosome banding with chromomycin and DAPI. Chromosoma (Berl.) 58, 307–324 (1976)

    Google Scholar 

  • Schweizer, D., Ambros, P., Andrle, M.: Modification of DAPI banding on human chromosomes by prestaining with a DNA binding oligopeptide antibiotic, distamycin A. Exp. Cell Res. 111, 327–332 (1978)

    Google Scholar 

  • Sengupta, S.K., Schaer, D.: The interaction of 7-substituted actinomycin D analogs with DNA, Biochim. biophys. Acta (Amst.) 521, 89–100 (1978)

    Google Scholar 

  • Stryer, L.: Fluorescence energy transfer as a spectroscopic ruler. Ann. Rev. Biochem. 47, 819–840 (1978)

    Google Scholar 

  • Van de Sande, J.H., Lin, C.C., Jorgenson, K.F.: Reverse banding on chromosomes produced by a guanosine-cytosine specific DNA binding antibiotic olivomycin. Science 195, 400–402 (1977)

    Google Scholar 

  • Ward, D.C., Reich, E., Goldberg, I.H.: Base specificity in the interaction of polynucleotides with antibiotic drugs. Science 149, 1259–1263 (1965)

    Google Scholar 

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

    Google Scholar 

  • Wartell, R.M., Larson, E.J., Wells, R.D.: Netropsin, a specific probe for A-T regions of duplex deoxyribonucleic acid. J. biol. Chem. 249, 6719–6731 (1974)

    Google Scholar 

  • Wartell, R.M., Larson, J.E., Wells, R.D.: The compatibility of netropsin and actinomycin binding to natural deoxyribonucleic acid. J. biol. Chem. 250, 2698–2702 (1975)

    Google Scholar 

  • Weisblum, B.: Why centric regions of quinacrine-treated mouse chromosomes show diminished fluorescence. Nature (Lond.) 246, 150–151 (1973)

    Google Scholar 

  • Weisblum, B., Haseth, P.L. de: Quinacrine, a chromosome stain specific for deoxyadenylate-deoxy-thymidylate-rich regions in DNA. Proc. nat. Acad. Sci. (Wash.) 69, 629–632 (1972)

    Google Scholar 

  • Wells, R.D., Larson, J.G.: Studies on the binding of actinomycin D to DNA and DNA model polymers. J. molec. Biol. 49, 319–346 (1970)

    Google Scholar 

  • Yguerabide, J.: Nanosecond fluorescence spectroscopy of macromolecules. In: Methods in enzymology (C.C.H.W. Hirs and S.N. Timasheff, eds.), vol. 25, pp. 498–578. New York: Academic Press 1973

    Google Scholar 

  • Zelenin, A.V., Kirianova, E.A., Kolesnikov, V.A., Stepanova, N.G.: Use of actinomycin D for the specific quenching of fluorescence of deoxyribonucleic acid in cells stained with acridine aminoderivatives. J. Histochem. Cytochem. 24, 1169–1172 (1976)

    Google Scholar 

  • Zimmer, Ch., Reinert, K.E., Luck, G., Wahnert, U., Lober, G., Thrum, H.: Interaction of the oligopeptide antibiotics. Netropsin and Distamycin A with nucleic acids. J. molec. Biol. 58, 329–348 (1979)

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Genetics Division and Mental Retardation Center, Children's Hospital Medical Center, USA

    Elhanan Sahar & Samuel A. Latt

  2. Department of Pediatrics, Harvard Medical School, 02115, Boston, Massachusetts, USA

    Elhanan Sahar & Samuel A. Latt

Authors
  1. Elhanan Sahar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Samuel A. Latt
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sahar, E., Latt, S.A. Energy transfer and binding competition between dyes used to enhance staining differentiation in metaphase chromosomes. Chromosoma 79, 1–28 (1980). https://doi.org/10.1007/BF00328469

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00328469

Keywords

Search

Navigation