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The transgenic tobacco plant GVCHS(320)-1 harbours several T-DNAs with the neomycin phosphotransferase II (nptll)-encoding chimeric gene under control of the cauliflower mosaic virus 35S promoter (CaMV 35S). These T-DNAs are distributed over two loci, named A and B. The primary transformant GVCHS(320)-1 had substantially reduced steady-state nptll transcript levels due to the activity of a post-transcriptional silencing mechanism. Silencing of the nptll-encoding transgenes in the progeny of GVCHS(320)-1 requires the presence of the A locus that consists of two physically separated T-DNAs. Although the B locus contains three T-DNAs in the same orientation, it gives rise to high steady-state nptll mRNA levels and NPTII protein levels even in homozygous condition. The B locus only becomes silenced in trans in the presence of a silencing locus. The data suggest that silencing of the nptll transgenes is reinforced by ageing. It is also suggested that silencing is correlated with a reduced NPTII protein/nptll mRNA ratio, which may be interpreted as the accumulation of unproductive nptll RNA molecules in silenced plants. The data further demonstrate that the silenced state is correlated with extensive C-methylation of diagnostic sites in the 3′ three-quarters of the coding sequence and in the 3′ region up to 1400 bp downstream of the polyadenylation signal.