Comparison of Surface Structures between Extended and Condensed Stages of Barley Chromosomes Revealed with Atomic Force Microscopy

Abstract

SummaryChromosomes are super-molecules consisting of DNA, histone and chromatin proteins, which specifically appear within a cell at the cell division. We analyzed barley chromosomes by atomic force microscopy (AFM) to elucidate its structural basis. Mitotic chromosomes were taken from root tips of barley (Hordeum vulgare L., cv. Minorimugi) using the EMA (enzymatic maceration and air-drying) method after synchronization of cell cycle. Both the air-dried or critical point dried specimens were observed in air by a dynamic force mode. This observation technique enables to obtain three-dimensional image data on the surface structure of barley chromosomes at high resolution without any metal coating. The details of the higher order chromosomal structure such as chromatin fibers were clarified with the biological significance. Acidic treatment (e.g., acetic acid treatment) for removing proteins was useful to obtain clear images of basic chromosomal structure. Thus, it is concluded that the AFM has a great potential for investigation of molecular structures of chromosomes.Key words Atomic force microscopy (AFM), Chromosomal structure, Barley, Chromatin.The genetic information of the eukaryotic cell is stored in chromosomes. These structures are generated by condensation of chromatin fibers, which consist mainly of DNA and histone proteins. Numbers of papers have reported that modes of chemical modifications in DNA and histone proteins are closely related to the regulation of gene expression (reviewed by Rice and Allis 2001). They have revealed that regulation of the chromatin superstructure is closely related to the basic biological function such as transcription, replication, repair and DNA packaging through the cell cycle (Demeret et al. 2001). Primary control occurs through interactions between specific regulatory DNA sequences and large variety of transcription factors. Transcriptions are also regulated by which post-translational modification of histone proteins produces the structural change of chromatin (Struhl 1998); the phosphorylation of histones, for example, plays a role in inducing chromatin condensation (Dmitry et al. 2001). Acetylation of histone H4 is widely discussed as a factor, which causes the structural change of chromatin and serves for transcriptional regulation (Turner 2000, Wako et al. 2002, 2003.Viewed from the ultra-structural aspect, the basic structure of chromatin is the nucleosomes, which consist of histone octamer (H2A, H2B, H3, H4) and double-stranded DNA; the DNA is wound twice around the histone octamer to produce the "beads-on-a-string" form (reviewed by

Surface structures consisting of chromatin fibers in isolated barley (Hordeum vulgare) chromosomes revealed by helium ion microscopy

“…Light microscopy cannot be used to visualize the chromosome structure due to the resolution of light vision wavelengths at 0.22 μm (Brock 1984). To overcome the limiting resolution of light microscopies, researchers attempted to elucidate the inner and outer structure of chromosome using advanced electron microscopy (EM), including atomic force microscopy (Ohmido et al 2005), super-resolution microscopies (Morse and Simpson 1988), scanning electron microscopy (SEM) (Iwano et al 1997), transmission microscopy (TEM) (Manzanero et al 2002), and focused ion beam-SEM (FIB/SEM) (Uchiyama et al 2005;Poonperm et al 2015;Wako et al 2020). As a result, chromosome fibers were visualized and chromosomal proteins, such as TopoIIα and KIF4, were located.…”