In order to identify specific cis-acting elements which regulate the expression of the divergent Cab22R and Cab22L genes of Petunia, we conducted systematic mutational studies of the 1 kb intergenic promoter region. Sequence analysis revealed three GATA box sequence repeats positioned between the TATA and CAAT box elements. These GATA elements are conserved in corresponding promoter regions of all LHCII Type I Cab genes in Petunia and other dicotyledonous plants we have examined. Site-specific mutations in the CAAT box and the GATA box elements of the Cab22R promoter resulted in 8-fold and 5-fold reductions in Cab22R transcript levels respectively. A deletion of 52 bp, adjacent and upstream from the CAAT box (-92 to -145) in the Cab22R promoter reduced transcript levels 20-fold. This deletion contains a region of 13 bp which is conserved between many Petunia Cab genes. These results indicate that the quantitative expression of the Cab22 promoters is regulated by multiple cis-acting elements including CAAT and GATA box elements as well as sequences located between -92 and -145. The deletion of the region between -92 and -145 is partially compensated by homologous sequences present in the adjacent divergent promoter Cab22L.

We investigated the basis for quantitative differences in leaf expression of the petunia genes (rbcS) encoding the small subunit of ribulose bisphosphate carboxylase. The most abundantly, SSU301, and the most weakly, SSU911, expressed petunia rbcS genes maintained their differential expression when transferred to tobacco, indicating that the determinants of quantitative expression are intrinsic to these rbcS genes. Analysis of chimeric genes in which the sequences of SSU301 and SSU911 had been exchanged at the translation start showed that sequences both 5' and 3' to the start codon contribute to differences in steady-state mRNA levels. The sequences 3' to the translation initiation codon were investigated by preparing chimeric genes in which sequences of the SSU301 and SSU911 were exchanged between each intron and at the translation termination codon. The results showed that sequences downstream of the coding region contribute to quantitative differences in expression of SSU301 and SSU911, and nuclear run-on transcription experiments indicated that the 3' sequences affect transcription rates of the rbcS genes.

We have studied the expression of four sets of tandem gene fusions in transgenic tobacco plants. This was to determine if the problem of between-transformant variability in expression of introduced genes could be overcome by using a linked reference gene as a co-ordinately expressed control. Tandem gene fusions containing identical 5' flanking regions (SSU301-ocs with either SSU301-cat or SSU301-SSU911) were not co-ordinately expressed in the transgenic tobacco plants whereas the tandem gene fusions containing similar but not identical 5' flanking regions (SSU301-ocs with SSU911-cat or SSU911-SSU301) were co-ordinately expressed. The lack of co-ordinate expression of some of the tandem gene fusions appears to be partially explained by absence of the corresponding genomic DNA segments in the transgenic plants.

We investigated the basis for quantitative differences in leaf expression of the petunia genes (rbcS) encoding the small subunit of ribulose bisphosphate carboxylase. The most abundantly, SSU301, and the most weakly, SSU911, expressed petunia rbcS genes maintained their differential expression when transferred to tobacco, indicating that the determinants of quantitative expression are intrinsic to these rbcS genes. Analysis of chimeric genes in which the sequences of SSU301 and SSU911 had been exchanged at the translation start showed that sequences both 5' and 3' to the start codon contribute to differences in steady-state mRNA levels. The sequences 3' to the translation initiation codon were investigated by preparing chimeric genes in which sequences of the SSU301 and SSU911 were exchanged between each intron and at the translation termination codon. The results showed that sequences downstream of the coding region contribute to quantitative differences in expression of SSU301 and SSU911, and nuclear run-on transcription experiments indicated that the 3' sequences affect transcription rates of the rbcS genes.

No abstract