No abstract

The SlPPC2 phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) gene from tomato (Solanum lycopersicum) is differentially and specifically expressed in expanding tissues of developing tomato fruit. We recently showed that a 1966 bp DNA fragment located upstream of the ATG codon of the SlPPC2 gene (GenBank AJ313434) confers appropriate fruit-specificity in transgenic tomato. In this study, we further investigated the regulation of the SlPPC2 promoter gene by analysing the SlPPC2 cis-regulating region fused to either the firefly luciferase (LUC) or the β-glucuronidase (GUS) reporter gene, using stable genetic transformation and biolistic transient expression assays in the fruit. Biolistic analyses of 5′ SlPPC2 promoter deletions fused to LUC in fruits at the 8th day after anthesis revealed that positive regulatory regions are mostly located in the distal region of the promoter. In addition, a 5′ UTR leader intron present in the 1966 bp fragment contributes to the proper temporal regulation of LUC activity during fruit development. Interestingly, the SlPPC2 promoter responds to hormones (ethylene) and metabolites (sugars) regulating fruit growth and metabolism. When tested by transient expression assays, the chimeric promoter:LUC fusion constructs allowed gene expression in both fruit and leaf, suggesting that integration into the chromatin is required for fruit-specificity. These results clearly demonstrate that SlPPC2 gene is under tight transcriptional regulation in the developing fruit and that its promoter can be employed to drive transgene expression specifically during the cell expansion stage of tomato fruit. Taken together, the SlPPC2 promoter offers great potential as a candidate for driving transgene expression specifically in developing tomato fruit from various tomato cultivars.

Vestigial (vg) mutants of Drosophila melanogaster are characterized by atrophied wings. In this paper we show that: (1) aminopterin an inhibitor of dihydrofolate reductase (DHFR) and fluorodeoxyuridine (FUdR), an inhibitor of thymidylate synthetase induce nicks in the wings of wild-type flies and phenocopies of the vg mutant phenotype when vg/+ and vgB/+ flies are reared on these substances (vgB is a deficiency of the vg locus). Only thymidine and thymidylate can rescue the flies from the effect of aminopterin. We propose that the vg phenotype is due to a decrease in the dTMP pool in the wings. (2) Mutant vg strains yield more offspring on medium containing aminopterin than on normal medium. The resistance of vg larvae to the inhibitor seems specific to the gene. This is the first case of aminopterin resistance in living eucaryotes. In contrast sensitivity of the vg larvae to FUdR is observed. (3) An increase in the activity and amount of DHFR is observed in mutant strains as compared with the wild-type flies. Our data suggest that the vg+ gene is a regulatory gene acting on the DHFR gene or a structural gene involved in the same metabolic pathway.

The aim of our work was to compare the mechanisms of resistance to aminopterin, inhibitor of the dihydrofolate reductase enzyme, between different Drosophila species and those described for cultured cells. Moreover we compared the systematic species divisions based on morphological traits and those based on a molecular approach. For this purpose, the effect of aminopterin on viability and wing phenotype was studied in different Drosophila species. Dihydrofolate reductase was measured in adult flies. We found an important dihydrofolate reductase activity in the melanogaster sub-group compared to the other species studies. Wing effect was observed only in this sub-group. The effects of aminopterin on the wing phenotype were very similar to the phenotype of rudimentary mutants. Both deplete the pyrimidine pool and it has been shown by the studies of the structural genes of the nucleotide pyrimidine pathway that the wing tissue is very sensitive to every pertubation of this metabolism. The D. ananassae species was found to be fully resistant at the concentrations of the inhibitor tested. No or very little dihydrofolate reductase activity was detected. The binding of the enzyme to the inhibitor was comparable to that found in the Oregon strain of D. melanogaster. The purine and pyrimidine salvage pathways were investigated and the D. ananassae species displayed an important thymidine kinase activity. The D. ananassae flies were sensitive on Sang medium compared to the Oregon flies but were able to use exogenous bases or nucleosides more efficiently.(ABSTRACT TRUNCATED AT 250 WORDS)