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.
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
Shige H
SH
Yoshimura
Joongbaek
J
Kim
Kunio
K
Takeyasu
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