MOLECULAR AND CYTOGENETIC ANALYSES OF STABLY AND UNSTABLY EXPRESSED TRANSGENE LOCI IN TOBACCO

To study the influence of genomic context on transgene expression, we have determined the T-DNA structure, flanking DNA sequences, and chrom osomal location of four independent transgene loci in tobacco, Two of these loci were stably expressed in the homozygous condition over many generations, whereas the other two loci became unstable after several generations of homozygosity. The stably expressed loci comprised rela tively simple T-DNA arrangements that were flanked on at least one sid e by plant DNA containing AT-rich regions that bind to nuclear matrice s in vitro. Of the unstably expressed loci, one consisted of multiple incomplete T-DNA copies, and the second contained a single intact T-DN A; in both cases, however, binary vector sequences were directly conti guous to a right T-DNA border, Fluorescence in situ hybridization demo nstrated that the two stably expressed inserts were present in the vic inity of telomeres. The two unstably expressed inserts occupied interc alary and paracentromeric locations, respectively. Results on the stab ility of transgene expression in F-1 progeny obtained by intercrossing the four lines and the sensitivity of the four transgene loci to inac tivation in the presence of an unlinked ''trans-silencing'' locus are also presented. The findings are discussed in the context of repetitiv e DNA sequences and the allotetraploid nature of the tobacco genome.