The nontranscribed spacer regions (NTS) that adjoin the coding portion of mouse ribosomal DNA are protected in nucleoli against exhaustive DNase I digestion. Since these sequences are degraded by the enzyme after they are extracted by phenol, the protection is suggested to result from the binding of specific proteins. The nucleolar structure would thus be organized to protect NTS sequences and expose the coding sequences for transcription. We show here that these protected sequences include tracts of poly(dG-dT)-poly(dAdC). We also report that these sequences are localized in regions flanking the rRNA transcription unit. These sequences can potentially form Z-DNA. The organized DNase I-resistant NTS structure in which they participate could be involved in structuring the nucleolus or in regulating transcription because poly(dG-dT)-poly(dA-dC) sequences and portions of spacer rDNA can serve as transcriptional enhancer elements.The mammalian genome is peppered with repetitive sequences that have been judged to be anything from selfish DNA to components of transcriptional enhancers. Furthermore, the evaluation ofthese sequences is complicated by the indeterminate borders of individual genes. Ribosomal DNA (rDNA) provides one case in which alternative possibilities may be resolved. The rDNA is organized in tandem repeat units, so that a gene equivalent can be precisely defined. The rDNA is not only itself a model repetitive DNA, but it also includes several sequences repeated in other parts of the genome (1-3). Here we report on one kind of repetitive element found in the nontranscribed spacer (NTS) region of the rDNA repeat. We show that regions of NTS, unlike the transcribed portion of rDNA, which is uniformly 65% G+C, contain tracts of 50% G+C with poly(dG-dT) on one strand and poly(dA-dC) on the other. These tracts, which can potentially adopt an alternate left-handed Z-DNA conformation (4), may help structure the nucleolus.
MATERIALS AND METHODSCore nucleolar DNA was purified from mouse L-cell nucleoli by exhaustive DNase I treatment as earlier described (5). Polyacrylamide gels (10%) and agarose gels (2%) contained lx TBE (50 mM Tris borate/2.5 mM EDTA, pH 8.3) and were electrophoresed with 1x TBE as running buffer. The gels were stained with ethidium bromide. Agarose gels were prepared for Southern transfer (6) by denaturing for 30 min in 0.5 M NaOH/1.5 M NaCl and neutralizing for 45 min in 0.5 M Tris HCl/1 M NaCI. Transfer to nitrocellulose filters was carried out for 16 hr in 20x SSC (lx SSC = 0.15 M NaCl/0.015 M sodium citrate). The filters were air-dried and the DNA was fixed onto the filter at 80°C for 90 min in a vacuum oven. Nick-translation of synthetic poly(dGdT)-poly(dA-dC) (Pharmacia) and of core nucleolar DNA was by standard procedures (7). Specific activities of poly(dGdT)-poly(dA-dC) and of nucleolar DNA were 4.6 x 107 cpm/,ug and 2.5 x 106 cpm/,4g, respectively. Hybridization was done at a probe concentration of 0.1 ,g/ml. Filters were hybridized for 18 hr and then washed in 2x SSC/0.1%...