Propidium iodide as a probe for the study of chromatin thermal denaturation in situ

The possibility of using propidium iodide, a phenanthridinic fluorochrome specific for double-stranded nucleic acids, for the study of chromatin thermal denaturation in situ has been examined. Smears of lymphocytes and hepatocyte nuclei from 15-day-old rats were fixed in acetic acid--ethanol (1:3 v/...

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Bibliographic Details
Published in:The Histochemical journal 1981-09, Vol.13 (5), p.781-791
Main Authors: Barni, S, de Piceis Polver, P, Gerzeli, G, Nano, R
Format: Article
Language:English
Subjects:
Animals
Chromatin - ultrastructure
Deoxyribonucleoproteins - analysis
DNA - analysis
Kinetics
Liver - analysis
Lymphocytes - analysis
Nucleic Acid Denaturation
Nucleoproteins - analysis
Phenanthridines
Propidium
Protein Denaturation
Rats
Rats, Inbred Strains
Spectrometry, Fluorescence
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Summary:The possibility of using propidium iodide, a phenanthridinic fluorochrome specific for double-stranded nucleic acids, for the study of chromatin thermal denaturation in situ has been examined. Smears of lymphocytes and hepatocyte nuclei from 15-day-old rats were fixed in acetic acid--ethanol (1:3 v/v), treated with RNAse and submitted to different protein extraction procedures, namely, incubation with pepsin, trypsin and sodium chloride. Denaturation experiments were performed in Sörensen buffer at pH 7.4 containing 10% formamide at temperatures between 27 and 95 degrees C. The samples were stained with propidium iodide and mounted in buffer or glycerol. Measurements were performed with a microfluorometer at a wavelength of 446 nm. The results indicate a higher thermostability of lymphocytes as compared to hepatocytes. The denaturation pattern suggests a certain organization complexity of chromatin, better emphasized by the derivative curves which show the presence of at least three fractions with different melting points. After protein extraction, the denaturation curves exhibit a somewhat simplified pattern, with the disappearance of the most stable peak in the derivative curves. The samples mounted in glycerine exhibit a better stability of staining with time, and an increased quantum efficiency of the fluorochrome with regard to those mounted in buffer. These data confirm the importance of protein--DNA interactions in the organization of chromatin and point to some differences, depending on the cell type and on functional activity.
ISSN:0018-2214
1573-6865
DOI:10.1007/BF01003289