Nucleolar matrix structures were obtained under different extraction conditions from highly purified isolated nucleoli. Their ultrastructural appearance, protein composition and capacity to bind rDNA preferentially were studied in a model binding system. A region spanning approximately 25 kb in the rat ribosomal gene locus was screened for DNA sites capable of specifically interacting with the proteins of the nucleolar matrix (MARs). Two such sites were identified: one is located on an EcoRV-KpnI fragment in the 5′-nontranscribed spacer region, between two repetitive elements and close to the transcription initiation site; the other MAR is on a PvuII-BamHI fragment located in the 3′-nontranscribed region, encompassing an element 85% homologous to a B2-sequence. The two MARs are located in regions rich in polypyrimidine/polypurine tracks and contain a few elements homologous to the consensus sequence for topoisomerase II. This indicates that the "attachment sites" for the ribosomal genes belong to the same class of sequences as the MARs attaching the chromosomal DNA to the nuclear matrix.
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