Comparative analysis of DNA methylation in tobacco heterochromatic sequences

Cytosine methylation levels and susceptibility to drug-induced hypomethylat ion have been studied in several Nicotiana tabacum (tobacco) DNA repetitive sequences. It has been shown using HapII, MspI, BamHI and Sau3AI methylati on-sensitive restriction enzymes that the degree of 5'-(m)C(m)CG-3' methyla tion varied significantly between different repeats. There were almost satu ration levels of 5-methylcytosine at the inner (3') cytosine position and v ariable degrees of methylation at the outer (5') cytosine at the enzyme rec ognition sites. The non-transcribed high copy satellite sequences (HRS60, G RS) displayed significant heterogeneity in methylation of their basic units while middle repetitive sequences (R8.1, GRD5, 5S rDNA) were more uniforml y modified at both cytosine residues. Dihydroxypropyladenine (DHPA) treatme nt, which is thought to reduce DNA methyltransferase activity by increasing S-adenosylhomocysteine levels, resulted in extensive demethylation of the outer cytosine in all repeats, and the partial hypomethylation of cytosines at the inner positions in less densely methylated repeats such as HRS60 an d GRS. The results suggest that hypomethylation of 5'-(m)C(m)CG-3' sites wi th DHPA is a gradual non-random process proceeding in the direction (m)C(m) CG --> C(m)CG --> CCG. The 18S-5.8S-25S rDNA was remarkably hypomethylated relative to the 5S rDNA at all restriction sites studied. Fluorescence in-s itu hybridization showed that DNA decondensation within and between the 18S -5.8S-25S and 5S rDNA loci was variable in different nuclei. All nuclei had condensed and decondensed sequence. The chromatin of 18S-5.8S-25S rDNA was more readily digested with micrococcal nuclease than the 5S rDNA suggestin g that the overall levels of decondensation were higher for 18S-5.8S-25S rD NA. Variable decondensation patterns within and between loci were also obse rved for GRS and HRS60. Cytosine methylation of the tobacco repeats is disc ussed with respect to transcription, overall levels of condensation and ove rall structure.