Gene conversion of ribosomal DNA in Nicotiana tabacum is associated with undermethylated, decondensed and probably active gene units

We examined the structure, intranuclear distribution and activity of riboso mal DNA (rDNA) in Nicotiana sylvestris (2n=2x=24) and N. tomentosiformis (2 n=2x=24) and compared these with patterns in N. tabacum (tobacco, 2n=4x=48) . We also examined a long-established N. tabacum culture, TBY-2. Nicotiana tabacum is an allotetraploid thought to be derived from ancestors of N. syl vestris (S-genome donor) and N. tomentosiformis (T-genome donor). Nicotiana sylvestris has three rDNA loci, one locus each on chromosomes 10, 11, and 12. In root-tip meristematic interphase cells, the site on chromosome 12 re mains condensed and inactive, while the sires on chromosomes 10 and 11 show activity at the proximal end of the locus only. Nicotiana tomentosiformis has one major locus on chromosome 3 showing activity and a minor, inactive locus on chromosome II. In N. tabacum cv. 095-55, there are four rDNA loci on T3, S10, S11/t and S12 (S11/t carries a small T-genome translocation). T he locus on S12 remains condensed and inactive in root-tip meristematic cel ls while the others show activity, including decondensation at interphase a nd secondary constrictions at metaphase. Nicotiana tabacum DNA digested wit h methylcytosine-sensitive enzymes revealed a hybridisation pattern for rDN A that resembled that of N. tomentosiformis and not N. sylvestris. The data indicate that active, undermethylated genes are of the N. tomentosiformis type. Since S-genome chromosomes of N. tabacum show rDNA expression, the re sult indicates rDNA gene conversion of the active rDNA units on these chrom osomes. Gene conversion in N. tabacum is consistent with the results of pre vious work. However, using primers specific for the S-genome rDNA intergeni c sequences (IGS) in the polymerase chain reaction (PCR) show that rDNA gen e conversion has not gone to completion in N. tabacum. Furthermore, using m ethylation-insensitive restriction enzymes we demonstrate that about 8% of the rDNA units remain of the N. sylvestris type (from ca. 75% based on the sum of the rDNA copy numbers in the parents). Since the active genes are li kely to be of an N. tomentosiformis type, the N. sylvestris type units are presumably contained within inactive loci (i.e. on chromosome S12). Nicotia na sylvestris has approximately three times as much rDNA as the other two s pecies, resulting in much condensed rDNA at interphase. This species also h as three classes of IGS, indicating gene conversion has not homogenised rep eat length in this species. The results suggest that methylation and/or DNA condensation has reduced or prevented gene conversion from occurring at in active genes at rDNA loci. Alternatively, active undermethylated units may be vulnerable to gene conversion, perhaps because they are decondensed and located in close proximity within the nucleolus at interphase. In TBY-2, re striction enzymes showed hybridisation patterns that were similar to, but d ifferent from, those of N. tabacum. In addition, TBY-2 has elevated rDNA co py number and variable numbers of rDNA loci, all indicating rDNA evolution in culture.