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

固定剂 中期 染色体早凝 有丝分裂 生物 染色体 前期 分子生物学 核型 细胞生物学 遗传学 细胞质 减数分裂 基因
作者
U. Claussen,S. Michel,P. Mühlig,Martin Westermann,U.‐W. Grummt,Katrin Kromeyer-Hauschild,Thomas Liehr
出处
期刊:Cytogenetic and Genome Research [Karger Publishers]
卷期号:98 (2-3): 136-146 被引量:105
标识
DOI:10.1159/000069817
摘要

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.
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