A plant basal in vitro system supporting accurate transcription of both RNA polymerase II- and III-dependent genes: supplement of green leaf component(s) drives accurate transcription of a light-responsive rbcS gene.
An in vitro transcription initiation system has been developed from nuclei of rapidly growing, non-green tobacco (Nicotiana tabacum) cultured (BY-2) cells. Conditions for nuclear extraction and in vitro transcription reaction have been optimized with a tobacco beta-1,3-glucanase gene, a constitutively expressed gene in BY-2 cells. The in vitro system supports accurate transcription of RNA polymerase II-dependent promoters from not only plant genes (tobacco beta-1,3-glucanase gene, cauliflower mosaic virus 35S promoter) but also animal genes (adenovirus 2 major late promoter, simian virus 40 early major promoter). In addition, this system drives accurate transcription of an RNA polymerase III-dependent Arabidopsis thaliana U6 snRNA gene. As BY-2 cells do not differentiate in response to light or any other stimuli, they would provide a basal transcription system which lacks tissue-specific and light-responsive nuclear signals as well as chloroplast-derived signals. Consequently, the BY-2 cell-free system is unable to transcribe the tomato gene encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcS3C) whose expression is tissue-specific and light-inducible. However, the transcription of rbcS3C was obtained by supplementing the BY-2 system with a nuclear extract of light-grown tomato seedlings. The promoter regions necessary for rbcS transcription was mapped in vitro using a series of 5' deletion mutants. The 351 bp upstream sequence is essential and the further upstream region from -351 to -441 enhances its transcription. The in vitro basal system will be useful to identify specific signals from both the nucleus and chloroplast in green leaves and other organs/tissues.
Fan
H
H
Center for Gene Research, Nagoya University, Japan.
Sugiura
M
M
D017360:Arabidopsis / Q000235:genetics; D001483:Base Sequence; D002467:Cell Nucleus / Q000235:genetics; D002474:Cell-Free System; D018506:Gene Expression Regulation, Plant / Q000528:radiation effects; D017343:Genes, Plant / Q000235:genetics*; D043326:Glucan 1,3-beta-Glucosidase; D008027:Light; D018551:Solanum lycopersicum / Q000201:enzymology; D008969:Molecular Sequence Data; D018515:Plant Leaves / Q000737:chemistry; D010947:Plants, Toxic; D011401:Promoter Regions, Genetic / Q000235:genetics; D012319:RNA Polymerase II / Q000378:metabolism; D012320:RNA Polymerase III / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000235:genetics; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics*; D013698:Templates, Genetic; D014026:Nicotiana / Q000235:genetics; D014158:Transcription, Genetic; D001617:beta-Glucosidase / Q000235:genetics
D012342:RNA, Small Nuclear; D012319:RNA Polymerase II; D012320:RNA Polymerase III; D001617:beta-Glucosidase; D043326:Glucan 1,3-beta-Glucosidase; D012273:Ribulose-Bisphosphate Carboxylase