POSTTRANSCRIPTIONAL SILENCING OF A NEOMYCIN PHOSPHOTRANSFERASE-II TRANSGENE CORRELATES WITH THE ACCUMULATION OF UNPRODUCTIVE RNAS AND WITH INCREASED CYTOSINE METHYLATION OF 3'-FLANKING REGIONS

The transgenic tobacco plant GVCHS(320)-1 harbours several T-DNAs with the neomycin phosphotransferase II (nptII)-encoding chimeric gene und er control of the cauliflower mosaic virus 35S promoter (CaMV 35S). Th ese T-DNAs are distributed over two loci, named A and B. The primary t ransformant GVCHS(320)-1 had substantially reduced steady state nptII transcript levels due to the activity of a post transcriptional silenc ing mechanism. Silencing of the nptII-encoding transgenes in the proge ny of GVCHS(320)-1 requires the presence of the A locus that consists of two physically separated T-DNAs. Although the B locus contains thre e T-DNAs in the same orientation, it gives rise to high steady-state n ptII mRNA levels and NPTII protein levels even in homozygous condition . The B locus only becomes silenced in trans in the presence of a sile ncing locus. The data suggest that silencing of the nptII transgenes i s reinforced by ageing. It is also suggested that silencing is correla ted with a reduced NPTII protein/nptII mRNA ratio, which may be interp reted as the accumulation of unproductive nptII RNA molecules in silen ced plants. The data further demonstrate that the silenced state is co rrelated with extensive C-methylation of diagnostic sites in the 3' th ree-quarters of the coding sequence and in the 3' region up to 1400 bp downstream of the polyadenylation signal.