Developmental timing of transgene expression is dosage dependent

Expression of a beta-glucuronidase (GUS) gene (uidA) driven by the cauliflo wer mosaic virus 35S promoter was studied during development in progeny fro m a primary transformant of tobacco carrying 3 independently segregating in serts. In the R1S2-generation, plants could be grouped to 4 GUS expression phenotypes, showing either continuous GUS activity [S(-) plant] or silencin g of GUS after 13, 5 or 2 weeks [S(13), S(5) and S(2) plants, respectively] . In S(13), S(5) and S(2) plants, GUS activity was reduced 7-, 17- and 50-f old, respectively, as compared to the S(-) plant, Southern blot and segrega tion analysis showed that S(2) plants contained all 3 inserts of which at l east 1 was homozygous. All S(5) plants contained 2 inserts, 1 of which was homozygous and the other hemizygous. The S(13) plant contained 2 hemizygous inserts, while the S(-) plant contained 1 hemizygous insert. Analysis of t he R1S3-generation showed that the timing and degree of silencing was direc tly linked to copy number and that each of the 3 inserts seemed to contribu te equally to gene silencing, The physically linked neomycin phosphotransfe rase II gene driven by the weak nopaline synthase promoter was also subject to dosage-dependent gene but this occurred later and was less pronounced.