Several hundred base pairs upstream of Drosophila hsp23 and 26 genes are required for their heat induction in transformed flies.

We have used the P-element-mediated transformation of Drosophila germ line to study the 5' DNA sequences involved in the thermal inducibility of the genes for heat shock proteins hsp23 and 26. The results are strikingly different from those previously obtained in heterologous systems. For hsp23, each successive shortening of the promoter region from 618 to 402, 321 and 263 bp clearly decreased the expression. A construct with only 149 bp was not inducible at all. For hsp26, all the regulatory elements appear to be clustered in the first 350 bp upstream from the cap site. Clones with 171 bp showed a 4- to 10-fold decrease in induction depending on the transformed line, and those with only 52 bp were not expressed. The results suggest that at least three Pelham consensus sequences are required for the full expression of these two genes. The direct involvement of one of these consensus sequences has been assessed: a 6-bp deletion within the proximal element of the hsp26 gene strongly reduced its inducibility. Our results also indicate that X-linked hsp genes exhibit either partial dosage compensation or none at all.     

Heat-inducible expression of a reporter gene detected by transient assay in zebrafish

Heat-inducibility of two reporter constructs expressing lacZ gene under the control of mouse and Xenopus hsp70 promoters was tested in zebrafish (Danio rerio) embryos using a transient expression system. Cells expressing beta-galactosidase were stained blue by histochemical staining and their average number per embryo was used as an indicator of the expression level of the reporter gene. Both constructs were heat-inducible in the embryonic tissues and showed similar heat dependence (increasing expression levels from 35-36 degrees C up to 39 degrees C with an apparent decrease at 40 degrees C), resembling that of the zebrafish hsp70 genes. However, their induction kinetics were different, which might be due to differences in their 5' UTRs. Spatial expression patterns of the two hsp/lacZ constructs and an endogenous hsp70 gene were mostly similar on the RNA level. These results indicate that our approach is applicable for in vivo analysis of the heat-shock response and that exogenous heat-shock promoters may be useful for inducible expression of transgenes in fish.     

P-transposable vectors expressing a constitutive and thermoinducible hsp82-neo fusion gene for Drosophila germline transformation and tissue-culture transfection

Three P-transposable vectors (approx. 16, 12, and 9 kb) were constructed containing a hsp82-neo fusion gene encoding a truncated heat-shock protein 82 of Drosophila pseudoobscura and the bacterial neomycin phosphotransferase (NPT). In transgenic Drosophila melanogaster, hsp82-neo exhibits high levels of housekeeping gene promoter and NPT activities in all cells in the absence of heat-shock and is further induced (fivefold) by elevated temperatures (35 degrees-36 degrees C). The hsp82-neo selection of transformants is possible from embryo to adulthood. The hsp82-neo insertion in a P-element plasmid carrying an alcohol-dehydrogenase-encoding gene (Adh) produced plasmids pHS22 (approx. 16 kb) and pHS24 (approx. 12 kb), in which both genes were expressed, as observed in 13 transgenic strains. Cloning of DNA fragments up to at least 16 kb in a third vector, pHS85 (approx. 9 kb), lacking the Adh cointegrate is facilitated by a 104-bp multiple cloning site (MCS) positioned downstream (3') from hsp82-neo. To accept inserts of nonselectable foreign genes, MCS provides 20 restriction sites, eight of them unique. The hsp82-neo-expressing vectors also function in cell-culture transfection assays. The hsp82-neo fusion gene (3.73 kb) may be of wide application as a dominant selection marker in other animal systems and plants.     

Localized heat-shock induction in Drosophila melanogaster

We describe a technique for inducing localized expression of genes fused to heat-shock gene promoters. We demonstrate that a localized heat-shock response can be induced in Drosophila melanogaster at any developmental stage after formation of the cellular blastoderm by contacting a region of the animal with a heated needle. The size of the induced region can be altered by varying parameters such as the temperature and size of the needle tip. The test system utilized here is a D. melanogaster strain transformed with a fusion of the Drosophila hsp26 gene and the E. coli lacZ gene; the activity of this hybrid gene is monitored in whole animals by staining for beta-galactosidase activity. Induced beta-galactosidase activity is confined to the cells in the region of heating; the beta-galactosidase activity can still be detected 48 hr after the heat shock. Given the heat inducibility of Drosophila heat-shock promoters in heterologous systems, we suggest that this technique will be useful for allowing spatially controlled induction of a gene of interest in any organism into which fusion genes can be introduced. Additional uses of the technique for following cell movements during development are discussed.