Changes in chromosomal surface structure by different isolation conditions.
The human cell cycle was synchronized and the chromosomes were isolated by a centrifugation method using two representative solutions for chromosome isolation (a polyamine buffer, PAB and citric acid solution, CAS) and fixatives. The centrifugation method yielded sufficient amounts of human metaphase chromosomes. Observation of the isolated chromosomes by scanning electron microscopy (SEM) revealed two types of surface structure which have been repeatedly reported to date: the human chromosomes in the PAB were relatively smooth but covered irregularly with scaly structures, while the surface of the chromosomes in the CAS exhibited a dense fibrous structure with a uniform diameter of 50-70 nm. Comparison of proteins extracted from chromosomes isolated with the PAB and CAS clearly indicated the removal of linker histones, H1, from chromosomes isolated with the CAS. These findings imply that the two different images of human chromosomes frequently observed by SEM are due to the removal of peripheral chromosomal materials including linker histones and/or the depletion of linker histones which prevent the surface chromatin fibers from scattering.
Sone
Takefumi
T
Department of Biotechnology, Osaka University, Graduate School of Engineering, Suita, Osaka, Japan.
Iwano
Megumi
M
Kobayashi
Shouhei
S
Ishihara
Takeshi
T
Hori
Naoto
N
Takata
Hideaki
H
Ushiki
Tatsuo
T
Uchiyama
Susumu
S
Fukui
Kiichi
K
D002453:Cell Cycle; D002458:Cell Fractionation / Q000379:methods; D002498:Centrifugation / Q000379:methods*; D002875:Chromosomes / Q000737:chemistry / Q000648:ultrastructure*; D005456:Fluorescent Dyes; D006657:Histones / Q000302:isolation & purification; D006801:Humans; D007211:Indoles; D020014:K562 Cells; D008855:Microscopy, Electron, Scanning / Q000379:methods*; D008856:Microscopy, Fluorescence; D009687:Nuclear Proteins / Q000302:isolation & purification
D005456:Fluorescent Dyes; D006657:Histones; D007211:Indoles; D009687:Nuclear Proteins; C007293:DAPI