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High Affinity Binding of 7-Aminoactinomycin D and 4' ,6-Diamidino-2-Phenylindole to Human Neutrophilic Granulocytes and Lymphocytes
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
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1995 (English)In: Cytometry, ISSN 0196-4763, E-ISSN 1097-0320, Vol. 20, no 4, 296-306 p.Article in journal (Refereed) Published
Abstract [en]

The binding behavior of the DNA binding dyes 7-aminoactinomycin D (7-AAMD) and 4′,6-diamidino-2-phenylindole (DAPI) to human neutrophilic granulocytes and lymphocytes was studied by image cytofluorometry. Peripheral blood leukocytes were prefixed in paraformaldehyde (PFA) and attached to cover glasses. Different fixation, permeabilization, and acid extraction methods were applied before the cells were stained to equilibrium using varying concentrations of 7-AAMD or DAPI. The apparent association constant and number of high affinity dye binding sites were estimated for the different cell types, dyes, and treatments. Acid extracted cells, supposedly containing nucleosome-free DNA, were chosen to represent maximal dye binding. Only about 10% of the 7-AAMD binding sites remained in the unextracted PFA-fixed cells, and the apparent dye affinity was also reduced. We found no major difference in high affinity binding between the cell types, but granulocytes showed more fluorescence from less specifically bound 7-AAMD compared to lymphocytes. DAPI had a much higher affinity than 7-AAMD, independent of the preparation method. It showed a cooperative binding behavior with an apparent saturation of the high affinity binding sites at a dye concentration of about 50 nM. We conclude that both dyes may be useful as probes for chromatin structure in intact cells and that our new technique may contribute to such studies since it allows determination of dye affinities and numbers of high affinity binding sites in situ.

Place, publisher, year, edition, pages
1995. Vol. 20, no 4, 296-306 p.
Keyword [en]
DNA, chromatin structure, dye binding, human leukocytes
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-79455DOI: 10.1002/cyto.990200405OAI: oai:DiVA.org:liu-79455DiVA: diva2:542458
Available from: 2012-08-01 Created: 2012-08-01 Last updated: 2017-12-07Bibliographically approved
In thesis
1. A novel method using DNA binding fluorochromes as indirect probes to study linker histone-chromatin interactions in situ
Open this publication in new window or tab >>A novel method using DNA binding fluorochromes as indirect probes to study linker histone-chromatin interactions in situ
2000 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Over the years, a variety of methods have been used to study chromatin structure. These techniques have provided valuable information about nuclear framework, chromatin organisation, and the proteins that are involved in the formation and maintenance of chromatin. However, neither ultrastructural nor biochemical methods are suitable for investigating the dynamics of chromatin structure in structurally preserved nuclei. DNA binding fluorochromes can be used as indirect probes of chromatin structure, because their accessibility to DNA is detennined by the chromatin structure, which enables cytochemical studies of relatively undamaged cells. The aim of the work presented in this thesis was to develop a cytochemical method using the fluorocbromes 7-aminoactinomycin D (7-AAMD) and 4', 6-diamidino-2- phenylindole (DAPI) to study protein-DNA interactions in essentially intact cell nuclei.

Both fluorochromes proved to be useful as probes of chromatin structure, particularly to study Iinker histone-chromatin interactions. Suitable protocols for permeabilisation, salt extraction of linker histones, fixation, and staining were established. Permeabilisation with digitonin followed by linker histone extraction at various NaCl concentrations and subsequent fixation in paraformaldehyde was found to enable analysis of linker histone affinity for chromatin in situ. Although the cells became fragile during the salt extraction, this protocol provided individual cells that could be measured by image cytofluorometry.

In such analyses, DAPI had several advantages over 7-AAMD. At a concentration of 50 nM DAPI, all of the high-affinity binding sites were occupied, with only minor contributions from binding sites with lower affinity. This approach was used to study linker histone affinity in different types of cells, in cells in different stages of the cell cycle, and during cell differentiation in vivo. Our results indicate differences between the investigated cell types in regard to average Iinker histone affinity. It is generally assumed that increased linker histone affinity is associated with chromatin condensation, but that is not supported by our findings. Human fibroblasts showed substantially lower linker histone affinity for highly condensed metaphase chromatin than for interphase chromatin. Moreover, we found a tendency toward decreasing linker histone affinities in amphibian erytbroblasts that were developing into tenninally differentiated erythrocytes. However, as expected, mature avian erythrocytes showed strongly bound linker histones, probably due to their high arginine content. In conclusion, our results suggest that linker histone affinity is not directly involved in chromatin condensation.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2000. 42 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 625
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25682 (URN)10058 (Local ID)91-7219-582-7 (ISBN)10058 (Archive number)10058 (OAI)
Public defence
2000-05-11, Berzeliussalen, Hälsouniversitetet, Linköping, 09:00 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-11-09Bibliographically approved

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Loborg, HelenaLönn, AnitaRundquist, Ingemar

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