On-substrate lysis treatment combined with scanning probe microscopy revealed chromosome structures in eukaryotes and prokaryotes.
The proper function of the genome largely depends on the higher-order architecture of the chromosome. To understand the detailed chromosome structure in a native state, we developed an on-substrate procedure of subcellular fractionation suitable for the observation by atomic force microscopy (AFM). HeLa cells on a coverslip were successively treated with a detergent and a high-salt solution to remove the cytoplasmic and nucleoplasmic materials. A closer observation of the nucleus by AFM revealed that the interphase chromosome is composed of a granular unit of approximately 80 nm in diameter. Subsequent mild treatment with deoxyribonuclease I (10 U ml(-1)) exposed these units more clearly, which enabled us to uncover the 80-nm granules forming a fibre of approximately 80 nm width. In the cytoplasmic regions, cytoskeletal fibres with varying widths (10-70 nm) were observed. These observations suggest that the 80 nm granular fibre is a fundamental structural unit of the interphase chromosome. This on-substrate procedure was also applied to Escherichia coli. Cells attached on a coverslip were successively treated with lysozyme and detergent to partially release the nucleoid onto the substrate. The AFM observation revealed that the approximately 80 nm fundamental structural unit forms a granular fibre similar to that of HeLa cells. These results suggest that the fundamental mechanism of chromosome packing is common in both prokaryotes and eukaryotes.
Yoshimura
Shige H
SH
Laboratory of Plasma Membrane and Nuclear Signaling, Graduate School of Biostudies, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan. yoshimura@lif.kyoto-u.ac.jp
Kim
Joongbaek
J
Takeyasu
Kunio
K
D022003:Cell Nucleus Structures / Q000648:ultrastructure; D002875:Chromosomes / Q000648:ultrastructure*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004926:Escherichia coli / Q000648:ultrastructure*; D005057:Eukaryotic Cells; D006367:HeLa Cells; D006801:Humans; D007399:Interphase; D018625:Microscopy, Atomic Force; D009113:Muramidase / Q000378:metabolism; D013347:Subcellular Fractions
D003850:Deoxyribonuclease I; D009113:Muramidase