PARAMETERS AFFECTING THE ACTIVITY OF ANTISENSE RNA SEQUENCES IN TOBACCO PROTOPLASTS

Plasmids containing various fragments of the beta-glucuronidase (GUS) gene were placed in antisense orientation downstream of the cauliflowe r mosaic virus 35S promoter and cotransfected with a 35S-gus construct into tobacco mesophyll protoplasts. None of the partial-length sequen ces were as effective as the full-length sequence in reducing GUS acti vity. The presence of a polyadenylation sequence downstream of the ant isense sequence had an enhancing effect. The activity of the antisense sequence was largely affected by the incubation temperature of the tr ansfected protoplasts. The chloramphenicol acetyltransferase (CAT) gen e was fused to the gus coding sequence. When this construct was cotran sfected with an antisense sequence directed against CAT, GUS activity was reduced. The implications of these results for the design and uses of antisense sequences are discussed.