Visualization of reconstituted solenoid chromatin structure by tapping mode atomic force microscopy

In this paper, tapping mode atomic force microscopy was applied to visualiz e the higher order chromatin structure reconstituted from calf thymus DNA a nd calf thymus whole histone in vitro. All the revealed reconstituted chrom atin structures consisted of similar well-defined subunits. In some of the chromatin structures, four to six subunits per turn were wrapped right-hand ed to form a flattened solenoid with a height distribution of 12 +/- 2 nm; and the other Y- and V-shaped chromatin structures were formed by aggregati on of these solenoids, which presented similar heights as the solenoids. Ty pical atomic force microscopy (AFM) images revealed relatively regular sole noid structure (containing six nucleosomes per turn) with a ratio of diamet er to pitch of 3:1, which is in good agreement with that of 30:11 in the ch romatin solenoid model proposed by Finch and Klug. Bio-gel elution was used to separate reconstituted chromatin from uncombined histone particles, whi ch proved to be helpful for the AFM imaging of the higher order chromatin s tructure with nearly no disturbance of histone particles and salt in the ba ckground. The AFM observations showed that the higher order chromatin struc ture was preserved well enough during the bio-get elution process. The caus e for the stability of higher order chromatin structure during the bio-gel elution was analysed. Electron microscopy analysis and circular dichroism s pectroscopy investigation of the DNA-histone complexes indicated the format ion of ordered aggregates. Copyright (C) 2001 John Wiley & Sons, Ltd.