N-band proteins of nucleolar organizers: chromosomal mapping, subnucleolar localization and rDNA binding

The ribosomal DNA(rDNA)-containing chromatin in eukaryotes forms a unique architecture called the "secondary constriction" or "nucleolus organiser region (NOR)" on mitotic chromosomes. To gain more insight into non-histone chromosomal proteins (NHCP), termed "N-band proteins", that are specifically associated with the NOR in a wide variety of eukaryotes, we attempted to: identify the NHCP responsible for N-band staining; determine their stoichiometry; map them on metaphase chromosomes; determine their subnucleolar localization and examine their possible ability to bind rDNA. Based on several criteria, including chromosomal localization, solubility, association with chromatin, and intra-nuclear localization, two of the nucleolus-rich NHCP, termed component B of mol.wt 55,000 and component C of mol.wt. 41,000, were tentatively identified as N-band proteins. Immunological studies using a polyclonal, monospecific antibody raised against component C show that this protein is in fact associated with the chromosomal telomeres where NORs are located. In nucleoli, N-band proteins appear to be compartmentalized into a structure that probably corresponds to fibrillar components. Both components B and C are among several NHCPs that showed, under in vitro conditions, a strong affinity for rDNA cloned in lambda phage but not for calf thymus genomic DNA or phage vector DNA. The antibody against component C effectively suppressed in vitro transcription by RNA polymerase I in nucleoli and nucleolar chromatin. Component C appears to exist in the nucleus at 3.75-5.13 X 10(3) copies per rDNA unit or 0.09-0.13 copy per nucleotide. These findings support the hypothesis that the NOR is a chromosomal site, architecturally not only unique but also different from other chromatin regions in that constituent DNA, i.e., rDNA, is organized in a specific manner by interacting with specific NHCP, i.e., N-band proteins.