DNaseI-sensitive and undermethylated rDNA is preferentially expressed in a maize hybrid

An Eco RI polymorphism, present in the 26S ribosomal RNA gene (rDNA) of the maize hybrid Sx19 (B73 x Mo17), was utilized to correlate DNaseI sensitivity, undermethylation and expression in rDNA. We have previously shown that in double digest experiments with methylation-sensitive restriction enzymes and Eco RI, Sx19 rDNA fragments originating from repeat units with two Eco RI sites (8.0 kb) are undermethylated, whereas the fragments originating from repeat units with a single Eco RI site (9.1 kb) are completely methylated. In the present study, Sx19 rDNA chromatin structure was examined by purifying intact nuclei and digesting them briefly with increasing amounts of DNaseI. Analysis of this DNA with Eco RI showed that the 8.0 kb rDNA fragments are extremely sensitive to DNaseI digestion, while the 9.1 kb rDNA fragments are relatively resistant to digestion even at high levels of DNaseI. Specific sites hypersensitive to DNaseI cleavage were mapped to a region in the intergenic spacer (IGS) near the major undermethylated site. Analysis of polymerase chain reaction (PCR) products synthesized using Sx19, B73, and Mo17 DNAs as templates indicated that the Eco RI polymorphism is due to a base change in the recognition site. Direct rRNA sequencing identified a single-base change in Mo17 rRNA relative to B73 rRNA. Allele-specific oligonucleotide probes containing the region surrounding and including the Eco RI polymorphic site were utilized to detect a nucleolar dominance effect by quantitating levels of rRNA transcripts in Sx19 and the reciprocal cross. Results from these single-base-pair mismatch hybridization experiments indicate that the majority of the rRNA transcripts in Sx19 originate from the DNaseI-sensitive, undermethylated, Eco RI-polymorphic rDNA repeat units.