IDENTIFICATION OF A GENERAL SILENCER FOR 19S AND 35S PROMOTERS IN A TRANSGENIC TOBACCO PLANT - 90 BP OF HOMOLOGY IN THE PROMOTER SEQUENCE ARE SUFFICIENT FOR TRANS-INACTIVATION

The gene silencing effects triggered by foreign DNA inserted at a sing le locus in a particular transgenic tobacco plant have been characteri zed. The insertional locus refered as the ''silencing locus'' carries two chimeric genes. The first of these genes, 35S-RiN, consists of a t obacco nitrite reductase (NiR) cDNA cloned in the antisense orientatio n downstream of the promoter of the 35S mRNA of the cauliflower mosaic virus (CaMV), and the second, 19S-npt II, consists of the bacterial n eomycin phosphotransferase (npt II) gene cloned downstream of the prom oter of the 19S mRNA of CaMV. Previous analysis showed that no express ion of host NiR genes was detected in plants carrying the ''silencing locus'', suggesting a true antisense effect of the 35S-RiN transgene. In addition, no expression of the 19S-npt II gene which allowed initia lly the selection of the kanamycin-resistant callus was detected in th e regenerated plant and in its progeny. Southern blot analysis reveale d that multiple copies of the resistance gene were inserted and that s equences carried by the ''silencing locus'' were highly methylated. Re activation of the 19S-npt II gene occurred at low frequency when leaf protoplasts of plants homozygous for the ''silencing locus'' were indu ce to divide and plated on medium containing the antibiotic. Expressio n of ''target transgenes expressed from the 19S promoter or the 35S pr omoter was silenced when brought into the presence of the ''silencing locus'', independent of the coding sequence being expressed and indepe ndent of the position of insertion in the genome. Transgenes expressed from a 90 bp fragment of the 35S promoter were also silenced in trans . Southern blot analysis revealed that ''target'' loci became methylat ed in the presence of the ''silencing locus''. Segregation of the ''si lencing locus'' and ''target locus'' in the progeny led to a progressi ve rather than an immediate re-expression of silenced genes at the ''t arget locus'' These results suggest that the ''silencing locus'' which inhibits the expression of the host NiR genes and which carries silen t copies of the 19S-npt II gene also acts as a general silencer for 19 S and 35S promoters and that modifications imprint a silent state to t ransgenes expressed from these promoters.