Evaluation of the activities of the medfly and Drosophila hsp70 promoters in vivo in germ‐line transformed medflies

Abstract

The promoter of the hsp70 gene of Drosophila melanogaster has been widely used for the expression of foreign genes in other insects. It has been generally assumed that because this gene is highly conserved, its promoter will function efficiently in other species. We report the results of a quantitative comparison of the activities of the medfly and D. melanogaster hsp70 promoters in vivo in transformed medflies. We constructed transformed lines containing the lacZ reporter gene under the control of the two promoters by using Minos-mediated germ-line transformation. The activity of each promoter was evaluated in 15 transformed lines by beta-galactosidase quantitative assays. The heat-inducible activity of the medfly promoter was found several times higher than the respective activity of the heterologous D. melanogaster promoter. These results were confirmed by northern blot analysis and indicate that the D. melanogaster promoter does not work efficiently in medfly. The -263/+105 medfly promoter region that was used in this study was found able to drive heat shock expression of the lacZ reporter gene in all stages of medfly, except early embryonic stages, in a similar fashion to the endogenous hsp70 genes. However the heat inducible RNA levels driven from this promoter region were significantly lower than the endogenous hsp70 RNA levels, suggesting that additional upstream and/or downstream sequences to the -263/+105 region may be necessary for optimum function of the medfly hsp70 promoter in vivo.

Structural characterization of the medfly hsp83 gene and functional analysis of its proximal promoter region in vivo by germ‐line transformation

“…In agreement with previous medfly transformation experiments (Christophides et al, 2000;Kalosaka et al, 2006), we observed strong chromosomal position effects and promoter interference phenomena as can be seen from the vari-Archives of Insect Biochemistry and Physiology January 2008 doi: 10.1002/arch. ability of β-galactosidase levels and eye coloration among the P L -lacZ lines (Table 3).…”

“…On the other hand, the -380/+139 region of the gene drove significant lacZ expression under normal conditions, indicating that the responsible sequences for the constitutive expression of the Cchsp83 gene reside in the interval between positions -380 and -86. The activity of this Cchsp83 promoter region is similar to the heat-inducible activity of the medfly hsp70 promoter (Kalosaka et al 2006). However, as was shown in Figure 5, the levels of constitutive expression of the lacZ RNA in two P L -lacZ lines with moderate expression were significantly lower than the respective levels of the Cchs83 RNA.…”

“…Heat shock treatments were performed as previously described (Kalosaka et al, 2006). For RT-PCR experiments, the flies were used immediately after heat shock, while for β-galactosidase assays the heat shocked flies were allowed to recover for 90 min at 25°C before protein extraction.…”

Functionality of the GAL4/UAS system in Tribolium requires the use of endogenous core promoters

“…Similar experience has recently been described for the tephritid fruit fly Ceratitis capitata [80,81]. On the other hand, Drosophila heat shock constructs have been shown to work in the silkworm Bombyx mori [46] and the butterfly Bicyclus anynana [52].…”

The heat shock 70 genes of the olive pest Bactrocera oleae: genomic organization and molecular characterization of a transcription unit and its proximal promoter region

“…In Drosophila and other Diptera, three to seven inducible hsp70 genes have been identified, organized at one or two chromosomal loci (for a review and references therein, see Bettencourt and Feder 2001;Evgen'ev et al 2004). The promoter of the Drosophila melanogaster hsp70 gene has been used in genetic transformation systems for conditional expression of several genes in a variety of insects, exhibiting, however, lower activity in nondrosophilid species including B. oleae (Komitopoulou et al 2004;Kalosaka et al 2006;Koukidou et al 2006). …”