SUSCEPTIBILITY OF TRANSGENE LOCI TO HOMOLOGY-DEPENDENT GENE SILENCING

Previous work has shown that two unlinked, partially homologous transg ene loci can interact in plant nuclei, leading to reversible methylati on and inactivation of one transgene locus in the presence of the seco nd. To study whether the chromosomal location of a transgene influence s its susceptibility to trans-inactivation, we retransformed four tran sgenic lines, which contained the same construct (H) integrated in dif ferent chromosomal locations, with a second, partially homologous cons truct (K). At least 50 double transformants (DTs) were regenerated fro m each single transformant (ST) and screened for inactivation of marke rs [chloramphenicol acetyltransferase (CAT); hygromycin resistance (HY G(R))] at the resident H locus. For two STs, H locus markers were inac tivated in less than 1% of the DTs, suggesting that, at these integrat ion sites, H was relatively resistant to trans-inactivation. In contra st, the other two STs appeared to be more sensitive to trans-inactivat ion: 4-10% of the DTs were CAT(-) and/or Hyg(s). Inactivation of H loc us markers could be attributed to two distinct phenomena: 1. Regenerat ion from cells containing different epigenetic states of H, in which e ither both, one or none of the H alleles was active. This instability in the expression of the H locus, which was independent of K, was more pronounced in the homozygous state, and was associated with cellular mosaicism of expression and methylation. 2. The presence of an unlinke d K locus could weaken the Hyg(R) phenotype by transcriptional inactiv ation and increased methylation of the hph gene at the H locus. These results indicated that a susceptible transgene locus is inherently uns table and partially methylated, and that these characteristics are exa cerbated when the locus is homozygous for the transgene and/or when an unlinked homologous transgene is present.