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The involvement of nucleosomes in Giemsa staining of chromosomes

A new hypothesis on the banding mechanism

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Summary

A new hypothesis is proposed on the involvement of nucleosomes in Giemsa banding of chromosomes. Giemsa staining as well as the concomitant swelling can be explained as an insertion of the triple charged hydrophobic dye complex between the negatively-charged supercoiled helical DNA and the denatured histone cores of the nucleosomes still present in the fixed chromosomes. New cytochemical data and recent results from biochemical literature on nucleosomes are presented in support of this hypothesis.

Chromosomes are stained by the Giemsa procedure in a purple (magenta) colour. Giemsa staining of DNA and histone (isolated or in a simple mixture) in model experiments results in different colours, indicating that a higher order configuration of these chromosomal components lies at the basis of the Giemsa method. Cytophotometry of Giemsa dye absorbance of chromosomes shows that the banding in the case of saline pretreatment is due to a relative absence of the complex in the faintly coloured bands (interbands). Pretreatment with trypsin results in an increase in Giemsa dye uptake in the stained bands. Cytophotometric measurements of free phosphate groups before and after pretreatment with saline, reveal a blocking of about half of the free phosphate groups indicating that a substantial number of free amino groups is still present in the fixed chromosomes. Glutaraldehyde treatment inhibited Giemsa-banding irreversibly while the formaldehyde-induced disappearance of the bands could be restored by a washing procedure. These results correlate with those of biochemical nucleosome studies using the same aldehydes.

Based on these findings and on the known properties of nucleosomes, a mechanism is proposed that explains the collapse of the chromosome structure when fixed chromosomes are transferred to aqueous buffer solutions. During homogeneous Giemsa staining reswelling of the unpretreated chromosome is explained by insertion of the hydrophobic Giemsa complex between the hydrophobic nucleosome cores and the superhelix DNA. Selective Giemsa staining of the AT-enriched bands after saline pretreatment is thought to be due to the, biochemically well-documented, higher affinity of arginine-rich proteins present in the core histones for GC-enriched DNA, which prevents the insertion of the Giemsa complex in the interbands. Production of Giemsa bands by trypsin pretreatment can be related to the action of this enzyme on the H1 histones and subsequent charge rearrangements.

This and other cytochemical and biochemical evidence leads to a coherent hypothesis that relates the swelling properties, the purple homogeneous Giemsa staining, as well as the selective band staining after pretreatment of the chromosomes primarily to differences in base composition of the supercoiled DNA helices present in bands and interbands.

Such base sequence differences are known to lead to differences in internal tension in supercoiled DNA helices as present in the nucleosomes as well as to differences in binding strength to the polar parts of histones. The proposed hypothesis eliminates the necessity to postulate a role for, up to now elusive, band-specific non-histone proteins in Giemsa banding.

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  1. A. C. van Prooijen-Knegt

    Present address: Department of Human Genetics, Meibergdreef 15, Amsterdam, The Netherlands

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  1. Department of Histochemistry and Cytochemistry, Medical Faculty, Sylvius Laboratories University of Leiden, Wassenaarseweg 72, NL-2333 AL, Leiden, The Netherlands

    P. van Duijn, A. C. van Prooijen-Knegt & M. van der Ploeg

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van Duijn, P., van Prooijen-Knegt, A.C. & van der Ploeg, M. The involvement of nucleosomes in Giemsa staining of chromosomes. Histochemistry 82, 363–376 (1985). https://doi.org/10.1007/BF00494066

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