Mol Gen Genet (1986) 205:193-200 © Springer-Verlag1986 Characterization of an rbcS gene from Nicotiana plumbaginifolia and expression of an rbcS-CAT chimeric gene in homologous and heterologous nuclear background Carsten Poulsen 1, Robert Fluhr 1, John M. Kauffman 1, Marc Boutry 2, and Nam-Hai Chua 1 1 Laboratory of Plant Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10021-6399, USA 2 Laboratory of Enzymology, Place Croix du Sud, 1, Box 8, B-1348 Louvain-la-Neuve, Belgium Summary. We have cloned a gene (rbcS-SB) encoding the small subunit of ribulose bisphosphate carboxylase from Nicotiana plumbaginifolia and analyzed it with respect to structure and expression. The nucleotide sequence of the rbcS-8B coding region is highly conserved when compared to rbcS sequences from N. tabacum and N. sylvestris. The first two of the three Nicotiana rbcS introns are also well conserved, whereas the third intron of the N. plumbaginifo- lia rbcS gene is different from that of Nicotiana tabacum except for sequences close to the intron borders. Upstream of the transcription start sites there are about 400 bp con- taining 90% homology with respect to the N. tabacum gene. A region that contains an SV40 enhancer core sequence and covering the -120 to -155 region is tandemly re- peated in the - 555 to - 585 region of the N. plumbaginifo- lia gene. The rbcS-8B gene is expressed predominantly in leaves of N. plumbaginifolia and its expression is regulated by light. An 1.07 kbp 5' flanking sequence (- 1,038 to + 32) of the rbcS-8B gene is sufficient to confer regulated expres- sion on the coding sequence of bacterial chloramphenicol acetyl transferase (CAT) in transgenic plants. Expression levels of the rbcS-8B-CAT chimeric gene are uniform among transgenic clones of N. plumbaginifolia and N. taba- cum but some variation is seen among those of Petunia hybrida. Key words: Nucleotide sequence - Transcription - Light induction - Organ specificity - Transgenic plants Introduction The enzyme ribulose-l,5-bisphosphate carboxylase-oxygen- ase (RubisCO) is found in all photosynthetic organisms, eukaryotic as well as prokaryotic. It is composed of two types of subunits in most cases, eight identical large sub- units of 52,00(~56,000 Mr and eight small subunits of 12,000-15,000 Mr. In eukaryotes, the large subunit is en- coded by a single gene (rbcL) on the multicopy chloroplast genome (Poulsen 1984), the gene is transcribed by a chloro- plast RNA polymerase (Hanley-Bowdoin et al. 1985) and translated on 70S ribosomes in the organelle (Mishkind and Offprint requests to : C. Poulsen Schmidt 1983). The small subunit (rbcS) is encoded by small multi-gene families of 4-12 members in the nuclear genomes of pea (Coruzzi et al. 1984), soybean (Berry-Lowe et al. 1982), Petunia (Dunsmuir et al. 1983), wheat (Broglie et al. 1983), Lemna (Stiekema et al. 1983), and possibly in to- bacco (Mazur and Chui 1985). Expression of the rbcS and rbcL genes is regulated by light. The rbcS gene transcription is controlled by the phytochrome system (Silverthorne and Tobin 1984) whereas the rbcL gene might be regulated both at the transcriptional and also at the translational levels (Poulsen 1984). Expression of the two RubisCO genes is confined to photosynthetic tissues (Coruzzi et al. 1984). The separation of rbcS and rbcL genes in two different cellular compartments raises questions about the nature of the factors that coordinate their expression. One approach toward answering these questions is the investigation of sequence elements in the genes of importance for develop- mental expression. To this end, gene expression experiments involving transfer of pea rbcS genes and pea rbcS chimeric gene constructs into Nicotiana species or into P. hybrida, have been carried out (Morelli et al. 1985). Reports on work in Drosophila (Amin et al. 1985; Dudler and Travers 1984; Pauli et al. 1986) have indicated that sequence elements re- quired for proper developmental expression in heterologous gene transfer systems are not as stringent as in homologous systems. To compare the expression pattern of a develop- mentally regulated plant gene in homologous and heterolo- gous nuclear background, we have isolated and character- ized an rbcS gene (rbcS-8B) from N. plumbaginifolia. An upstream fragment of rbcS-8B including the promoter and the transcription start site was used for chimeric gene con- struction. After plant transformation we have studied regu- lated expression of the chimeric construct in the homolo- gous N. plumbaginifolia and also in transgenic N. tabacum and P. hybrida. Materials and methods Isolation of N. plumbaginifolia nucleic acids. Nuclear DNA was isolated and cloned in the EMBL4 vector as previously described (Boutry and Chua 1985). For total RNA isola- tion, plant materials were homogenized in a guanidinium thiocyanate buffer (Chirgwin et al. 1979). The cleared ho- mogenate was loaded onto a CsC1 cushion and RNA was