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Cytochemical studies of metaphase chromosomes by flow cytometry

Abstract

The cytochemical properties of metaphase chromosomes from Chinese hamster and human cells were studied by flow cytometry. This technique allows precise quantitation of the fluorescence properties of individual stained chromosome types. Chromosomes were stained with the following fluorescent DNA stains: Hoechst 33258, DAPI, chromomycin A3, ethidium bromide, and propidium iodide. The relative fluorescence of individual chromosome types varied depending on the stain used, demonstrating that individual chromosome types differ in chemical properties. Flow measurements were performed as a function of stain and chromosome concentration to characterize the number and distribution of stain binding sites. Flow analysis of double stained chromosomes show that bound stains interact by energy transfer with little or no binding competition. For most hamster chromosomes, there is a strong correlation between relative fluorescence and stain base preference suggesting that staining differences may be determined primarily by differences in average base composition. A few hamster chromosome types exhibit anomalous staining which suggests that some other property, such as repetitive DNA sequences, also may be an important determinant of chromosomal staining.

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References

  1. Arrighi, F.E., Saunders, G.F.: The relationship between repetitious DNA and constitutive heterochromatin with special reference to man. In: Modern Aspects of Cytogenetics: Constitutive Heterochromatin in Man (R.A. Pfeiffer, ed.), p. 113–133. Stuttgart-New York: Schattauer 1973

  2. Arrighi, F.E., Hsu, T.C., Pathak, S., Sawada, H.: The sex chromosomes of the Chinese hamster: constitutive heterochromatin deficient in repetitive DNA sequences. Cytogenet. Cell Genet. 13, 268–274 (1974)

  3. Atkin, N.B., Mattinson, G., Becak, W., Ohno, S.: The comparative DNA content of 19 species of placental mammals, reptiles, and birds. Chromosoma (Berl.) 17, 1–10 (1965)

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

  5. Brodie, S., Giron, J., Latt, S.A.: Estimation of accessibility of DNA in chromatin from fluorescence measurements of electronic excitation energy transfer. Nature (Lond.) 253, 470–471 (1975)

  6. Brody, T.: Histones in cytological preparations. Exp. Cell Res. 85, 255–263 (1974)

  7. Carrano, A.V., Gray, J.W., Langlois, R.G., Burkhart-Schultz, K.J., Van Dilla, M.A.: Measurement and pruification of human chromosomes by flow cytometry and sorting. Proc. nat. Acad. Sci. (Wash.) 76, 1382–1384 (1979)

  8. Carrano, A.V., Gray, J.W., Moore II, D.H., Minkler, J.L., Mayall, B.H., Van Dilla, M.A., Mendelsohn, M.L.: Purification of the chromosomes of the Indian Muntjac by flow sorting. J. Histochem. Cytochem. 24, 348–354 (1976)

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

  10. Dean, P.N., Pinkel, D.: High resolution dual laser flow cytometry. J. Histochem. Cytochem. 26, 622–627 (1978)

  11. Feller, W.: An Introduction to Probability Theory and its Applications. Vol 1, p. 279–300. New York: John Wiley, 1957

  12. Gosden, J.R., Mitchell, A.R., Buckland, R.A., Clayton, R.P., Evans, H.J.: The location of four human satellite DNA's on human chromosomes. Exp. Cell Res. 92, 148–158 (1975)

  13. Gray, J.W., Carrano, A.V., Steinmetz, L.L., Van Dilla, M.A., Moore II, D.H., Mayall, B.H., Mendelsohn, M.L.: Chromosome measurement and sorting by flow systems. Proc. nat. Acad. Sci. (Wash.) 72, 1231–1234 (1975)

  14. Gray, J.W., Langlois, R.G., Carrano, A.V., Dilla, M.A. Van: High resolution chromosome analysis: one and two parameter flow cytometry. Chromosoma (Berl.) 73, 9–27 (1979)

  15. Hanania, N., Caneva, R., Tapiero, H., Havel, J.: Distribution of repetitious DNA in randomly growing and synchronized Chinese hamster cells. Exp. Cell Res. 90, 79–86 (1975)

  16. Horan, P.K., Wheeless, L.L.: Quantitative single cell analysis and sorting. Science 198, 149–157 (1977)

  17. 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)

  18. 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)

  19. Korenberg, J.R., Engels, W.R.: Base ratio, DNA content, and quinacrine-brightness of human chromosomes. Proc. nat. Acad. Sci. (Wash.) 75, 3382–3386 (1978)

  20. Langlois, R.G., Jensen, R.H.: Interactions between pairs of DNA specific fluorescent stains bound to mammalian cells. J. Histochem. Cytochem. 27, 72–79 (1979)

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

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

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

  24. Lawrence, J.J., Duane, M.: Ethidium bromide as a probe of conformational heterogeneity of DNA in chromatin. The role of histone H1. Biochemistry 15, 3301–3307 (1976)

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

  26. LePecq, J.B.: Use of ethidium bromide for separation and determination of nucleic acids of various conformational forms and measurement of their associated enzymes. Methods of Biochemical Analysis, (D. Glick, ed.), Vol. 20, p. 41–86. New York: Interscience 1971

  27. Le Pecq, J.B., Paoletti, C.: A fluorescent complex between ethidium bromide and nucleic acids. Physical-chemical characterization. J. molec Biol. 27, 87–106 (1967)

  28. 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)

  29. Manuelidis, L.: Chromosomal localization of complex and simple repeated human DNAs. Chromosoma (Berl.) 66, 23–32 (1978)

  30. McGhee, J.D., Hippel, O.H.von: Theoretical aspects of DNA-protein interactions: Co-operative and non-co-operative binding of large ligands to a one-dimensional homgeneous lattice. J. molec. Biol. 86, 469–489 (1974)

  31. Miller, O.J., Miller, D.A., Warburton, D.P.: Application of new staining techniques to the study of human chromosomes. Progr. med. Genet. 9, 1–46 (1973)

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

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

  34. Rigler, R.: Microfluorometric characterization of intracellular nucleic acids and nucleoproteins by Acridine Orange. Acta. Physiol. Scand. 67, Suppl. 267 (1966)

  35. 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)

  36. Scatchard, G.: The attraction of proteins for small molecules and ions. Ann. N.Y. Acad. Sci. 51, 660–672 (1949)

  37. Schnedl, W.: Structure and variability of human chromosomes analyzed by recent techniques. Hum. Genet. 41, 1–9 (1978)

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

  39. Stubblefield, E.: Analysis of the replication pattern of Chinese hamster chromosomes using 5-Bromodeoxyuridine suppression of 33258 Hoechst fluorescence. Chromosoma (Berl.) 53, 209–211 (1975)

  40. Stubblefield, E., Wray, W.: Isolation of specific human metaphase chromosomes. Biochem. biophys. Res. Commn. 83, 1404–1414 (1978)

  41. Sumner, A.T., Evans, H.J., Buckland, R.A.: Mechanisms involved in the banding of chromosomes with Quinacrine and Giemsa: The effects of fixation in methanol-acetic acid. Exp. Cell Res. 81, 214–222 (1973)

  42. Van Dilla, M., Steinmetz, L., Davis, D., Gray, J.W.: High speed cell analysis and sorting with flow systems: biological applications and new approaches. IEEE Trans. Nucl. Sci. NS-21, 714–720 (1974)

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

  44. Yunis, J.J., Yasmineh, W.G.: Heterochromatin, satellite DNA, and cell function. Science 174, 1200–1209 (1971)

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Langlois, R.G., Carrano, A.V., Gray, J.W. et al. Cytochemical studies of metaphase chromosomes by flow cytometry. Chromosoma 77, 229–251 (1980). https://doi.org/10.1007/BF00286050

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Keywords

  • Propidium Iodide
  • Metaphase Chromosome
  • Base Preference
  • Chromosome Type
  • Binding Competition