Demystifying chromosome preparation and the implications for the concept of chromosome condensation during mitosis

Abstract

The processes taking place during routine chromosome preparation are not well understood. In this study, the morphological changes in amniotic fluid cells, blood lymphocytes, and bone marrow cells in the metaphase stage were examined under an inverted microscope during chromosome preparation. The putative processes that occur during chromosome preparation were simulated in suspension, and the cells were treated with different mixtures of hypotonic solution, fixative, methanol, acetic acid, and water. Evaporation of the fixative was performed under normal atmospheric conditions and under vacuum at different levels of humidity. Freeze fracture electron microscopy was used to analyze the effects of fixative on the cell membrane. Confocal microscopic analysis was used to investigate three-dimensionally the effects of hypotonic treatment on the positions of chromosomes in fixed mitotic lymphocytes. Chromosome preparation-induced changes in the lengths of single chromosomes were also investigated. The results show that chromosome spreading involves significant water-induced swelling of mitotic cells during evaporation of the fixative from the slide, which is a prerequisite for chromosomal elongation, the production of metaphase spreads for chromosome analysis, and the appearance of Giemsa banding patterns. Hypotonic treatment is essential for well-spread metaphase chromosomes because it moves the chromosomes from a central to a more peripheral position in the cell, where they can be stretched more effectively during mitotic swelling. Like mitotic cells, isolated single chromosomes also have their own potential to swell and lengthen during chromosome preparation. We hypothesize that chromosome preparation leads to a genome-wide chromosomal region–specific opening of chromatin structures as GTG-light bands and sub-bands. Living cells may possess a similar mechanism, which is used only to open single chromatin structures to facilitate transcription. We propose the concept of chromosomal region–specific protein swelling.

New aspects on chromosomal instability: Chromosomal break-points in Fanconi anemia patients co-localize on the molecular level with fragile sites

“…ii) The concordance of FS and FA break-points can be explained by mechanistic reasons: the chromosomal band level, i.e., the decondensation of the chromosome (25) influences the break rate observed in chromosomes (Fig. 5).…”

Microtechnologies Enable Cytogenetics

“…The most common method is a simple air drying for 2 days, which helps the visualization of chromosome structures. Another technique involves dry-heat and chemical ageing, the latter being beneficial for FISH analysis, as it results in better signals and preserves chromosome architecture (Claussen et al, 2002, Henegariu et al, 2001Rønne, 1989). Another factor suspected of affecting chromosome spreads quality is the dropping height, but Claussen et al claim it is not essential.…”

Fluorescence intensity profiles of in situ hybridization signals depict genome architecture within human interphase nuclei

“…Current concepts suggest these proce dures to influence rather chromosome length and spreading of G positive bands than chromatin behaviour as the positioning of chromosomes appears to be relatively conserved during the deliv ery of nuclei/metaphase suspension on slides [19]. Therefore, one can modulate chromatin behavior in living cells skipping sophisticated volumetric preparations.…”