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DNA Binding Fluorochromes as Probes for Histone HI-Chromatin Interactions In Situ
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.
1997 (English)In: Cytometry, ISSN 0196-4763, E-ISSN 1097-0320, Vol. 28, no 3, 212-219 p.Article in journal (Refereed) Published
Abstract [en]

We have investigated using the DNA binding fluorochromes 7-aminoactinomycin (7-AAMD) and 4′,6-diamidino-2-phenylindole (DAPI) as cytochemical probes for linker histone (H1)–chromatin interactions in situ. Human lymphocytes, permeabilized with digitonin, were exposed to increasing concentrations of sodium chloride to remove ionically bound H1 from the nuclei. The cells were stained to equilibrium with 1 μM 7-AAMD or 50 nM DAPI. Lymphocytes stained with 7-AAMD showed a gradual increase from 11% to 36% of HCl treated cell fluorescence intensity when the salt concentration was increased from 0.15 to 0.7 M. The corresponding increase for DAPI was 53–68%. The 7-AAMD obviously showed higher sensitivity for H1–chromatin interactions that DAPI but had disadvantages such as high background fluorescence and an affinity that was dependent on the preparation procedure. DAPI had negligible background fluorescence, and its fluorescence intensity resembles the number of available high-affinity dye-binding sites when used at 50 nM. We conclude that both fluorochromes can be used as probes for H1–chromatin interactions in situ and that our method has a potential to provide new information on such interactions.

Place, publisher, year, edition, pages
1997. Vol. 28, no 3, 212-219 p.
Keyword [en]
DNA, chromatin structure, linker histones, dye binding
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-79456DOI: 10.1002/(SICI)1097-0320(19970701)28:3<212::AID-CYTO5>3.0.CO;2-FOAI: oai:DiVA.org:liu-79456DiVA: diva2:542459
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, HelenaRundquist, Ingemar

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