RESUMO
Protein C23, a predominant nucleolar phosphoprotein and a putative nucleolus organizer protein, was analyzed for its general DNA binding characteristics and for its selectivity in binding plasmid DNAs containing cloned fragments of the genes that code for ribosomal RNA (rDNA). By use of nitrocellulose filter disk assays, the protein bound saturably to nuclear DNA with a relatively high affinity. Binding was maximal at low ionic strength (0-0.1 M KCl) with progressively decreasing binding at or above 0.2 M. In competition assays protein C23 showed a marked preference for linear single-stranded vs. double-stranded DNA and little or no affinity for ribosomal RNA. The relative affinities of rDNA sequences for protein C23 were determined with cloned fragments spanning 15.8 kilobases (kb) of DNA starting approximately 3.7 kb upstream from the initiation site for 45S preribosomal RNA to near the 3' end of the sequence coding for 28S RNA. Of the five linearized plasmids tested, only one (pKW1) was an effective competitor for 32P-labeled nuclear DNA. As measured by the concentration of competing DNA required to achieve 50% competition, pKW1 was approximately 20-fold more effective than the second best competitor. The DNA insert in pKW1 is a 3.5-kb sequence which is located in the nontranscribed spacer region less than 0.5 kb upstream from the initiation site for 45S preribosomal RNA. These results suggest that protein C23 has a preference for binding DNA sequences in the nontranscribed spacer of rDNA.