Propidium iodide as a probe for the study of chromatin thermal denaturationin situ
- 1 September 1981
- journal article
- research article
- Published by Springer Nature in Journal of Molecular Histology
- Vol. 13 (5), 781-791
- https://doi.org/10.1007/bf01003289
Abstract
The possibility of using propidium iodide, a phenanthridinic fluorochrome specific for double-stranded nucleic acids, for the study of chromatin thermal denaturationin 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°C. The samples were stained with propidium iodide and mounted in buffer or glycerol. Measurements were performed with a microfluorometer at a wavelength of 546 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.This publication has 12 references indexed in Scilit:
- Methods in Ultrastructural Cytochemistry of the Cell NucleusProgress in Histochemistry and Cytochemistry, 1980
- Studies on the role of histones H1 (f1) and H5 (f2c) in chromatin structureExperimental Cell Research, 1976
- Thermal denaturation of DNA in situ as studied by acridine orange staining and automated cytofluorometryExperimental Cell Research, 1975
- Circular dichroism of histone-bound regions in chromatinBiopolymers, 1975
- DEOXYRIBONUCLEIC ACID CYTOPHOTOMETRY OF STAINED HUMAN LEUKOCYTES III. THERMAL DENATURATION OF CHROMATINJournal of Histochemistry & Cytochemistry, 1974
- Cytochemical characterization of deoxyribonucleoprotein by UV-microspectrophotometry on heat denatured cell nucleiExperimental Cell Research, 1972
- Interaction of ethidium bromide with whole and selectively deproteinized deoxynucleoproteins from calf thymusJournal of Molecular Biology, 1972
- Methyl green staining and highly repetitive DNA in polytene chromosomesChromosoma, 1972
- Thermal denaturation and template properties of DNA complexes with purified histone fractionsJournal of Molecular Biology, 1970
- The use of a vertical illuminator with interchangeable dichroic mirrors for fluorescence microscopy with incidental light.1967