Functional Analysis of Drosophila HSP70 Promoter with Different HSE Numbers in Human Cells

The activation of genetic constructs including the Drosophila hsp70 promoter with four and eight HSE sequences in the regulatory region has been described in human cells. The promoter was shown to be induced at lower temperatures compared to the human hsp70 promoter. The promoter activity increased after a 60-min heat shock already at 38°C in human cells. The promoter activation was observed 24 h after heat shock for the constructs with eight HSEs, while those with four HSEs required 48 h. After transplantation of in vitro heat-shocked transfected cells, the promoter activity could be maintained for 3 days with a gradual decline. The promoter activation was confirmed in vivo without preliminary heat shock in mouse ischemic brain foci. Controlled expression of the Gdnf gene under a Drosophila hsp70 promoter was demonstrated. This promoter with four and eight HSE sequences in the regulatory region can be proposed as a regulated promoter in genetic therapeutic systems.     

Expression of a heat-inducible gene in transfected mosquito cells

The evolutionary conservation of the heat shock response suggests that plasmids containing promoters from Drosophila heat shock protein (hsp) genes will be useful in the development of gene transfer procedures for cell lines representing a variety of insect species. Conditions for induction of endogenous hsp genes and for expression of the chloramphenicol acetyltransferase (CAT) gene regulated by the Drosophila hsp 70 promoter were examined in Aedes albopictus (mosquito) cells. Five hsps, ranging in size from 27,000 to 90,000 D, were induced in A. albopictus cells during incubation at 41°C in medium containing [³⁵S]methionine. Relative synthesis of these proteins at 37 and 41°C indicated that Aedes hsp 66 is homologous to Drosophila hsp 70. Detection of CAT activity in transfected mosquito cells was enhanced 10-fold under heat shock conditions (6 h, 41°C) based on maximal expression of hsp 66, relative to conditions defined for expression of hsp 70 in Drosophila cells. Analysis of the endogenous heat shock response may be essential to the optimal use of plasmids containing the Drosophila hsp 70 promoter with other insect cell types.     

Transient expression of a Drosophila melanogaster hsp 70 promoter/lacZ construct injected into larvae of two species of predatory mites (Acari: Phytoseiidae)

The Drosophila melanogaster heat shock 70 promoter (hsp70) was used to regulate expression of the Escherichia coli -galactosidase gene (lacZ) in transiently-transformed predatory mite larvae. A construct containing the hsp70 promoter upstream of the D. melanogaster alcohol dehydrogenase (adh) translational start site and Escherichia coli lacZ gene fusion (adh/lacZ) was injected into larvae of Metaseiulus occidentalis and Amblyseius finlandicus. LacZ expression was compared to expression of a similar construct lacking any upstream regulatory sequence. Expression from the hsp70 promoter was strong and heat shock-dependent in both species. The Drosophila hsp70 promoter therefore appears useful for regulating expression of exogenous DNA in both phytoseiid species and may be broadly applicable in the Phytoseiidae. Furthermore, the lacZ gene is a useful gene for analysis of expression in both species. Larval microinjection provides a method of assessing transient expression and of examining native regulatory sequences in these two phytoseiids and will likely be useful in other phytoseiid mites with only minor modifications.     

Evolution of the Response to Heat Shock in Genus Drosophila

Thermotolerance was studied in a wide spectrum of Drosophilaspecies and strains originating from different climatic zones and considerably differing from one another in the ambient temperature of their habitats. The species that lived in hot climate have a higher thermotolerance. Most species of the virilisgroup exhibited positive correlation between the HSP70 accumulation after heat exposure and thermotolerance; however, this correlation was absent in some species and strains. For example, the D. melanogasterOregon R strain, which had the highest sensitivity to heat shock (HS) among all strains and species studied, displayed the maximum level of HSP70 proteins after HS. The patterns of induction of various heat shock protein (HSP) families after heat exposure in a wide spectrum of Drosophila species were compared. The results obtained suggest that the HSP40 and low-molecular-weight HSPs (lmwHSPs) play a significant role in thermotolerance and adaptation to hot climate. Polymorphism in hsp70 gene clusters ofDrosophila and variation in the numbers of gene copies andhsp70 isoforms in group viriliswere found. The evolutionary role of the variation in the number of hsp70 gene copies observed in the strains and species of genusDrosophilais discussed.     

Naturally occurring transposable elements disrupt hsp70 promoter function in Drosophila melanogaster

Naturally occurring transposable element (TE) insertions that disrupt Drosophila promoters are correlated with modified promoter function and are posited to play a significant role in regulatory evolution, but their phenotypes have not been established directly. To establish the functional consequences of these TE insertions, we created constructs with either TE-bearing or TE-lacking hsp70 promoters fused to a luciferase reporter gene and assayed luciferase luminescence in transiently transfected Drosophila cells. Each of the four TEs reduces luciferase signal after heat shock and heat inducibility of the hsp70 promoter. To test if the differences in hsp70 promoter activity are TE-sequence dependent, we replaced each of the TEs with multiple intergenic sequences of equal length. These replacement insertions similarly reduced luciferase signal, suggesting that the TEs affect hsp70 promoter function by altering promoter architecture. These results are consistent with differences in Hsp70 expression levels, inducible thermotolerance, and fecundity previously associated with the TEs. That two different varieties of TEs in two different hsp70 genes have common effects suggests that TE insertion represents a general mechanism through which selection manipulates hsp70 gene expression.     

The hsp70 locus of Drosophila auraria (montium subgroup) is single and contains copies in a conserved arrangement

The restriction endonuclease pattern of a number of hsp70-homologous clones isolated from a library of heat shock cDNA from Drosophila auraria, a species belonging to the montium subgroup of the melanogaster species group, reveals two types of clones, A and B, differing in a single restriction site. Both types, as well as hsp70-specific probes derived from both hsp70 loci of Drosophila melanogaster, hybridize in situ with a single band at region 32 A of the 2L polytene arm, indicating a clustered organization of the hsp70 gene copies in D. auraria. The longest type B clone was sequenced and it was found that one strand contains an open reading frame (ORF) exhibiting great identity with a previously described hsp70 gene of D. auraria (now denoted as type A) and with its counterparts of D. melanogaster, while its second strand, unlike the type A clone, does not contain a long antiparallel coupled ORF (LAC ORF) because of a base substitution resulting in a premature stop codon. After additional data had been derived from isolation and characterization of hsp70-homologous genomic clones, together with Southern analysis of genomic DNA, we found that two hsp70 gene copies are present at the above locus of D. auraria with an inverted tandem repeat organization, while the presence of a third hsp70 gene is not clearly evident. The above results are compared with those observed at the homologous loci of some melanogaster subgroup species (D. melanogaster and its sibling species), in which, however, the hsp70 locus is duplicated, and with the more distantly related Dipteran Anopheles albimanus. Received: 22 May 1998; in revised form: 18 September 1998 / Accepted: 21 September 1998