A PLANT BASAL IN-VITRO SYSTEM SUPPORTING ACCURATE TRANSCRIPTION OF BOTH RNA-POLYMERASE II-DEPENDENT AND III-DEPENDENT GENES - SUPPLEMENT OFGREEN LEAF COMPONENT(S) DRIVES ACCURATE TRANSCRIPTION OF A LIGHT-RESPONSIVE RBCS GENE

An in vitro transcription initiation system has been developed from nu clei of rapidly growing, non-green tobacco (Nicotiana tabacum) culture d (BY-2) cells. Conditions for nuclear extraction and in vitro transcr iption reaction have been optimized with a tobacco beta-1,3-glucanase gene, a constitutively expressed gene in BY-2 cells. The in vitro syst em supports accurate transcription of RNA polymerase II-dependent prom oters from not only plant genes (tobacco beta-1,3-glucanase gene, caul iflower mosaic virus 35S promoter) but also animal genes (adenovirus 2 major late promoter, simian virus 40 early major promoter). In additi on, this system drives accurate transcription of an RNA polymerase III -dependent Arabidopsis thaliana U6 snRNA gene. As BY-2 cells do not di fferentiate in response to light or any other stimuli, they would prov ide a basal transcription system which lacks tissue-specific and light -responsive nuclear signals as well as chloroplast-derived signals. Co nsequently, the BY-2 cell-free system is unable to transcribe the toma to gene encoding the small subunit of ribulose-1,5-bisphosphate carbox ylase/oxygenase (rbcS3C) whose expression is tissue-specific and light -inducible. However, the transcription of rbcS3C was obtained by suppl ementing the BY-2 system with a nuclear extract of light-grown tomato seedlings. The promoter regions necessary for rbcS transcription was m apped in vitro using a series of 5' deletion mutants. The 351 bp upstr eam sequence is essential and the further upstream region from -351 to -441 enhances its transcription, The in vitro basal system will be us eful to identify specific signals from both the nucleus and chloroplas t in green leaves and other organs/tissues.