We have developed a method to identify non-histone proteins involved in organizing the nucleoprotein fiber of metaphase chromosomes isolated from HeLa cells. The approach is to remove histones from the metaphase chromosomes by competition with polyanions such as dextransulfate and heparin. The histone-depleted chromosomes can be isolated by sucrose gradient sedimentation. They contain a highly-folded DNA fiber, organized by repetitive attachment to a central matrix (chromosomal scaffold) consisting of non-histone proteins. The chromosomal scaffold can be isolated as a structurally independent entity following removal of most of the DNA and has the shape of a metaphase chromosome. The main focus of this proposal is to study the histone-depleted chromosome structurally and biochemically. We will identify the proteins involved, study the DNA-protein interactions and deduce the overall folding of the DNA fiber by electron microscopy.