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

In this paper, tapping mode atomic force microscopy was applied to visualize the higher order chromatin structure reconstituted from calf thymus DNA and calf thymus whole histone in vitro. All the revealed reconstituted chromatin structures consisted of similar well-defined subunits. In some of the chromatin structures, four to six subunits per turn were wrapped right-handed to form a flattened solenoid with a height distribution of 12 ± 2 nm; and the other Yand V-shaped chromatin structures were formed by aggregation of these solenoids, which presented similar heights as the solenoids. Typical atomic force microscopy (AFM) images revealed relatively regular solenoid structure (containing six nucleosomes per turn) with a ratio of diameter to pitch of 3 : 1, which is in good agreement with that of 30 : 11 in the chromatin solenoid model proposed by Finch and Klug. Bio-gel elution was used to separate reconstituted chromatin from uncombined histone particles, which proved to be helpful for the AFM imaging of the higher order chromatin structure with nearly no disturbance of histone particles and salt in the background. The AFM observations showed that the higher order chromatin structure was preserved well enough during the bio-gel elution process. The cause for the stability of higher order chromatin structure during the bio-gel elution was analysed. Electron microscopy analysis and circular dichroism spectroscopy investigation of the DNA–histone complexes indicated the formation of ordered aggregates. Copyright  2001 John Wiley & Sons, Ltd.