Presented at the Ninth Annual Meeting of the American Society for Cell Biology, Detroit, Michigan, November 6-8, 1969, and Published in: J. Cell Biol. vol. 43, p. 39A (1969):

Electron Microscope Localization of Acridine Orange Binding within Nuclei of Human Leukemic Bone Marrow Cells".

John H. Frenster

Department of Medicine, Stanford University School of Medicine, Palo Alto, California, 


Abstract:

During selective gene de-repression witin mammalian cells, histone repressors are partially displaced from DNA templates within the extended euchromatin part of cell nuclei (1965. Nature. 206: 680). Such partial displacement of histones has been confirmed by microspectroflourometry of cells reacted with acridine orange after initial fixation (1969. Exptl. Cell Res. 54: 171; 55: 215). Electron microscopy has now markedly improved the resolution of such acridine orange binding studies within intact cells. Normal and leukemic human bone marrow cells were aspirated and fixed with glutaraldehyde. The fixed cells were then reacted with acridine orange, with carbodiimide, or with neither agent. Loosely bound reagent was removed by repeated washing, and the washed cells were incubated with either DNase, RNase, trypsin, or no enzyme. The incubated cells were then postfixed in osmium tetoxide, embedded, sectioned, stained, and examined by microspectrofluorometry and by electron microscopy. Cells which had been reacted with acridine orange and then incubated with DNase displayed a characteristic electron-opaque reaction product, 0.1 m in diameter, which was confined to the extended euchromatin part of the cell nuclei.

Fig. 1. Myeloblast from human leukemia bone marrow, after acridine orange-DNase I probe, with electron-dense reaction product confined to the euchromatin portion of the cell nucleus. X 15,000.


Condensed heterochromatin of cell nuclei was devoid of reaction product, as was the cytoplasm of reactive cells. Omission of acridine orange resulted in an absence of visible product, as did omission of DNase incubation or substitution of RNase or trypsin during incubation. These data suggest that when acridine orange is bound to cell nuclei, it is confined to the extended euchromatin part of the nuclei, a site to which RNA synthesis is also confined (1963. Proc. Natl. Acad. Sci. U.S. 50: 1026). Partial displacement of histones from underlying DNA templates has been shown to be present within euchromatin and to be causal for both of these processes.

Supported by NIH grant CA-10174.


Additional References:

1. Frenster JH, "Ultrastructural Effects of Mercuric Chloride on Nuclear Heterochromatin within Human Lymphocytes", J. Cell Biol. vol. 43: pp. 39A-40A (1969).

2. Frenster JH, "Ultrastructural Continuity Between Active and Repressed Chromatin", Nature vol. 205: no. 4978. pp. 1341-1342 (March 27, 1965).

3. Frenster JH, "Electron Microscopic Localization of Acridine Orange Binding to DNA Within Human Leukemic Bone Marrow Cells", Cancer Res. vol. 31, pp. 1128-1133 (August, 1971).


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euchromatin:  "the most active portion of the genome within the cell nucleus".