Molecular and cytogenetic characterization of a transgene locus that induces silencing and methylation of homologous promoters in trans.

One type of homology-dependent gene silencing in transgenic plants involves a silencing locus that is able to transcriptionally inactivate and methylate an unlinked target locus with which it shares sequence identity in promoter regions. In a manner resembling paramutation of endogenous genes, the target locus reactivates and loses methylation progressively over several generations after segregating away from the silencing locus, which autonomously acquires stable methylation. To investigate the origins of trans-silencing ability and susceptibility, we have analyzed the structures, flanking DNA sequences and chromosomal locations of a nopaline synthase promoter silencing locus, H2, and a sensitive target locus, K81. A partially resistant target locus, K alpha has been characterized molecularly. The complex and scrambled H2 locus comprises six copies of the nopaline synthase promoter, two of which are collinear with prokaryotic non-T-DNA sequences, and is integrated close to a region of intercalary heterochromatin. These features probably contribute collectively to the silencing ability because H2 subclones reintroduced into random locations in the K81 genome did not frequently induce silencing. Both the K81 and K alpha loci have simple structures, although the former contains non-T-DNA prokaryotic sequences that are also present at H2, and they are flanked by low copy plant DNA. H2 and K81 might interact effectively because they are present on morphologically similar chromosomes from the T subgenome of allotetraploid tobacco.