John H. Frenster
Division of Oncology
Stanford University School of Medicine
Stanford, California 94305 

As in the previous study (5), a characteristic electron-dense reaction product, approximately 0.1 um in diameter, is displayed in cells which are first reacted with 10^-3 M acridine orange and are then digested with DNAase (Fig. 1).
Fig. 1. Human lymphocyte exposed to acridine orange probe while in the living state.The discrete 0.1 um electron-dense reaction product is seen preferentially distributed throughout the extended euchromatin of the cell nucleus. Little or no reaction product is seen in the cell cytoplasm or in the condensed heterochromatin portion of the cell nucleus. Lymphocytes isolated in sterile conditions from the peripheral blood of normal fasting sunjects as previously described (6), and incubated at a concentration of 10⁶ cells/ml. at 37^o C for 30 min. in the dark (7) in autologous heparinized plasma diluted 1:5 with medium 199 (Gibco) and containing heparin, 4 U.ml.; penicillin, 0.06 mg/ml., streptomycin 0.06 mg/ml.; and 10^-3 M acridine orange (K & K Labs, re-crystallized twice). After incubation, cells were washed three times in this medium omitting acridine orange, were fixed in 5 % glutaraldehyde in medium 199 for 30 min. at 4^o C, were again washed three times in the wash medium, and were incubated at 37^o C for 30 min. with DNase (Worthington, electrophoretically pure) at a concentration of 1.0 mg. enzyme/ml. in Eagle's minimal essential medium (Gibco). The cells were then post-fixed in 1 % OsO₄, dehydrated, embedded in 'Epon', sectioned 0.1 um thickness, stained with 5 % uranyl acetate, and examined at 80 kV on a 'Siemens 1A' electron microscope (5, 8). Control experiments either omitted acridine orange or substituted 10^-3 M carbodiimide (9) (Aldrich Chem., re-crystallized twice). Other control experiments either omitted DNase or substituted RNase (Worthington, electrophoretically pure) or trypsin (Worthington, re-crystallized three times). None of the control experiments demonstrated the presence of any reaction product (5). (X 7,500).

The reaction product is found only in the nucleus of each cell, and is confined to the extended euchromatin portion active in gene transcription (8). Again, as in the previous study of fixed cells (5), if acridine orange is omitted from the reaction sequence, or if carbodiimide (another ligand to DNA) (9) is substituted for acridine orange, then the reaction product is not observed. Similarly, if DNase is omitted from the reaction sequence, or if RNase or trypsin is substituted for DNase, then the characteristic reaction product is not observed (5). These experimental data indicate that both the introduction of the acridine orange probe and the enzymatic digestion with DNase are necessary conditions for visualization of the reaction product. They also strongly suggest that the reaction product is formed as a result of the interaction of acridine orange with DNA binding sites within the extended euchromatin portion of the living cell nucleus (5).

In cell-free systems, the prior presence of histones on the DNA template effectively decreases the reactivity of such DNA to acridine orange probes (4). The preferential localization (Fig. 1) of of DNA-bound acridine orange probes to the active euchromatin poprtion of the living lymphocyte nucleus is thus consistent with previous from isolated chromatin fractions (2) indicating a partial displacement of histones from underlying DNA templates within active euchromatin. Conversely, the preferential localization of histone-bound ³H-phytohemagglutinin probes to the repressed heterochromatin portion of the living lymphocyte nucleus (10) is also consistent with the previous data from isolated chromatin fractions (2). These data indicated that displaced histones within active euchromatin are tightly bound by diverse species of nuclear polyanions and hydrophobic ligands (2), while the histones within repressed heterochromatin are bound only to the underlying DNA templates (2) and are thus free to react (1) with histone-binding probes such as phytohemagglutinin (10).

These ultrastructural studies of molecular probes utilizing either DNA ligands or histone ligands within living human lymphocytes thus confirm the previous conformational data from studies on isolated chromatin fractions (1, 2), and from studies on fixed cells (3-5). Other nuclear ligands, either binding directly to DNA (11) or acting as counter-ligands by binding to histones (12), may prove useful as sensitive probes of gene derepression in living cells, and as probes of the strand separations which occur within DNA molecules during active transcription (13, 14) and gene dosage compensation (15).

This work was supported by grants from the U.S. Public Health Service and the Leukemia Society.

References:

1. Frenster JH, Nature 206: 1269 (1965).

2. Frenster JH, Nature 206: 680 (1965).

3. Killander D, and Rigler R, Exp. Cell Res. 54: 163 (1969).

4. Rigler R, and Killander D, Exp. Cell Res. 54: 171 (1969).

5. Frenster JH, Cancer Res. 31: 1128 (1971).

6. Frenster JH, and Rogoway WM, in Proc. Fifth Annual Leukocyte Culture Conf. (edit. by Harris JE), 359, (Academic Press, London and New York, 1970).

7. Hill RB, Bensch KG, and King DW, Exp. Cell Res. 21: 106 (1960).

8. Frenster JH, Allfrey VG, and Mirsky AE, Proc. Nat. U.S. Acad. Sci. 50: 1026 (1963).

9. Augusti-Tocco G, and Brown GL, Nature 206: 683 (1965).

10. Stanley DA, Frenster JH, and Rigas DA, in Proc. Fourth Annual Leukocyte Culture Conf. (edit. by McIntyre OR), 1 (Appleton-Century-Crofts, New York, 1971).

11. Frenster JH, Nature 208: 1093 (1965).

12. Frenster JH, in "Handbook of Molecular Cytology" (edit. by Lima-de-Faria A), 251 (North-Holland, Amsterdam and London, 1969).

13. Frenster JH, Nature 208: 894 (1965).

14. Crick F, Nature 234: 25 (1971).

15. Herstein PH, and Frenster JH, in Proc. Second Conf. on Embryonic and Fetal Antigens in Cancer (edit. by Anderson NG, and Coggin JH), 1 (National Technical Information Service, U.S. Dept. of Commerce, Springfield, Virginia, 1972). 

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