The organization and substructure of chromatin fibres in the interphase nucleus as studied by scanning electron microscopy.

Abstract

The high packaging ratio of DNA in both interphase nuclei and metaphase chromosomes presents great difficulties to our understanding of the three dimensional organisation of processes such as DNA replication and transcription in the nucleus. Although the higher order structure of DNA, in terms of the way it is organised into the unit fibre of chromatin has received much attention over the last decade, the highest levels of packaging of chromatin in both nuclei and chromosomes have hardly begun to be elucidated. Much of the difficulty in investigating fibre organisation with conventional methods is the inherent two dimensional nature of sectioned or spread material in the transmission microscope. Three dimensional imaging from the SEM has, until recently, been limited by the available resolution. Our own previous studies of chromosome structure have shown that a combination of 'in lens' imaging combined with the high signal generation imparted by osmium impregnation have been adequate to routinely visualise chromatin fibre organisation in metaphase chromosomes, and the changes that occur as a result of a variety of banding techniques. More recent experiments using the same techniques on interphase nuclei extracted from a variety of tissue culture cells have indicated that Scanning electron microscopy of nuclei is a potentially useful technique for studying chromatin organisation, which may be made more accessible by a variety of biochemical extraction methods.