Nuclear scaffolds and scaffold-attachment regions in higher plants

DNA in the nuclei of eukaryotic organisms undergoes a hierarchy of folding to be packaged into interphase and metaphase chromosomes. The first level of packaging is the 11-nm nucleosome fiber, which is further coiled into a 30-nm fiber. Evidence from fungal and animal systems reveals the existence of higher order packaging consisting of loops of the 30-nm fibers attached to a proteinaceous nuclear scaffold by an interaction between the scaffold and specific DNA sequences called scaffold-attachment regions (SARs). Support for the ubiquitous nature of such higher order packaging of DNA is presented here by our work with plants. We have isolated scaffolds from tobacco nuclei using buffers containing lithium diiodosalicylate to remove histones and then using restriction enzymes to remove the DNA not closely associated with the scaffold. We have used Southern hybridization to show that the DNA remaining bound to the scaffolds after nuclease digestion includes SARs flanking three root-specific tobacco genes. This assay for SARs is termed the endogenous assay because it identifies genomic sequences as SARs by their endogenous association with the scaffold. Another assay, the exogenous assay, depends upon the ability of scaffolds to specifically bind exogenously added DNA fragments containing SARs. The tobacco scaffolds specifically bind a well-characterized yeast SAR, and cloned DNA fragments derived from the 3'-flanking regions of the root-specific genes are confirmed to contain SARs by this exogenous assay.