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.     

Continuous wave and simulated GSM exposure at 1.8 W/kg and 1.8 GHz do not induce hsp16-1 heat-shock gene expression in Caenorhabditis elegans

Recent data suggest that there might be a subtle thermal explanation for the apparent induction by radiofrequency (RF) radiation of transgene expression from a small heat-shock protein (hsp16-1) promoter in the nematode, Caenorhabditis elegans. The RF fields used in the C. elegans study were much weaker (SAR 5-40 mW kg(-1)) than those routinely tested in many other published studies (SAR approximately 2 W kg(-1)). To resolve this disparity, we have exposed the same transgenic hsp16-1::lacZ strain of C. elegans (PC72) to higher intensity RF fields (1.8 GHz; SAR approximately 1.8 W kg(-1)). For both continuous wave (CW) and Talk-pulsed RF exposures (2.5 h at 25 degrees C), there was no indication that RF exposure could induce reporter expression above sham control levels. Thus, at much higher induced RF field strength (close to the maximum permitted exposure from a mobile telephone handset), this particular nematode heat-shock gene is not up-regulated. However, under conditions where background reporter expression was moderately elevated in the sham controls (perhaps as a result of some unknown co-stressor), we found some evidence that reporter expression may be reduced by approximately 15% following exposure to either Talk-pulsed or CW RF fields.     

Expression of microinjected hsp 70/CAT and hsp 30/CAT chimeric genes in developing Xenopus laevis embryos

The expression of microinjected chimeric genes containing Drosophila hsp 70 and Xenopus hsp 70 and hsp 30 promoters linked to the reporter gene coding for bacterial chloramphenicol acetyltransferase (CAT) was examined during early development of Xenopus laevis. Heat-inducible expression of fusion genes containing either the Drosophila hsp 70 promoter (1100 bp) or the Xenopus hsp 70 promoter (750 bp) was first detectable after the midblastula stage of development. This coincides with the embryonic stage at which the endogenous hsp 70 gene is first heat-inducible. A Xenopus hsp 30/CAT fusion gene containing 350 bp of promoter sequences was also heat-inducible after the midblastula stage unlike the endogenous hsp 30 genes which were not heat-inducible until the early tailbud stage (stage 23-24). Sequences that are present within either the coding or 3' region of the hsp 30 clone do not cause the microinjected hsp 30 gene to be developmentally regulated in a normal manner. Additionally, microinjected hsp 30 gene sequences have no effect on the developmental regulation of endogenous hsp 30 genes which continue to be activated at the tailbud stage of development. Our data suggest, that an inhibitory system, which may control the expression of the endogenous hsp 30 gene during development, does not regulate the expression of the injected hsp 30 gene.     

Efficient transfection of chicken cells by lipofection, and introduction of transfected blastodermal cells into the embryo.

Chicken blastodermal cells (CBCs) and primary chicken fibroblasts (PCFs) have been lipofected with a variety of lacZ constructs encoding Escherichia coli beta-galactosidase (beta-gal). A reporter construct (phspPTlacZpA) containing a mouse heat-shock protein 68 gene (hsp 68) promoter was used to establish conditions for efficient lipofection. The construct, in circular or linear plasmid form or as reporter sequences alone, was transferred efficiently by incubating the cells for 3.5 h in a mixture of 6.2 micrograms Lipofectin (a cationic liposome preparation from Bethesda Research Laboratories) and 1.55-3.1 micrograms DNA per mL DMEM. These lipofection conditions were used to transfer a reporter construct (pCBcMtlacZ) containing a Zn(2+)-inducible chicken metallothionein (cMt) promoter, and constructs showing constitutive expression due to Rous sarcoma virus plus chicken beta-actin (pmiwZ) or cytomegalovirus (pMaori3) promoters. Endogenous chicken beta-gal and transferred bacterial beta-gal activity could be distinguished clearly by incubating the cells with the substrate, Xgal, at pH 4.3 or 7.4, respectively. Expression of phspPTlacZpA in chicken cells did not appear to require specific induction of the mouse hsp68 promoter, whereas expression of pCBcMtlacZ required treatment of the cells for 6-12 h with 150 microM ZnCl2. Bacterial beta-gal activity was observed following lipofection of CBCs that were cultured in suspension or plated. The efficiency of lipofection was at least 1 in 25 for CBCs, judging by the proportion of cells shown to have beta-gal activity 16-24 h after lipofection treatment began; these events could represent transient or stable incorporation of the construct.(ABSTRACT TRUNCATED AT 250 WORDS)     

A muscle-specific intron enhancer required for rescue of indirect flight muscle and jump muscle function regulates Drosophila tropomyosin I gene expression.

The control of expression of the Drosophila melanogaster tropomyosin I (TmI) gene has been investigated by P-element transformation and rescue of the flightless and jumpless TmI mutant strain, Ifm(3)3. To localize cis-acting DNA sequences that control TmI gene expression, Ifm(3)3 flies were transformed with P-element plasmids containing various deletions and rearrangements of the TmI gene. The effects of these mutations on TmI gene expression were studied by analyzing both the extent of rescue of the Ifm(3)3 mutant phenotypes and determining TmI RNA levels in the transformed flies by primer extension analysis. The results of our analysis indicate that a region located within intron 1 of the gene is necessary and sufficient for directing muscle-specific TmI expression in the adult fly. This intron region has characteristics of a muscle regulatory enhancer element that can function in conjunction with the heterologous nonmuscle hsp70 promoter to promote rescue of the mutant phenotypes and to direct expression of an hsp70-Escherichia coli lacZ reporter gene in adult muscle. The enhancer can be subdivided further into two domains of activity based on primer extension analysis of TmI mRNA levels and on the rescue of mutant phenotypes. One of the intron domains is required for expression in the indirect flight muscle of the adult. The function of the second domain is unknown, but it could regulate the level of expression or be required for expression in other muscle.     

BIOLOGICAL EFFECTS OF ELF-EMF ENHANCED STRESS RESPONSE: NEW INSIGHTS AND NEW QUESTIONS

The effect of extremely low frequency electromagnetic fields (ELF-EMF) on gene expression and early development has been investigated in two different transgenic animals, the nematode Caenorhabditis elegans and Drosophila melanogaster, respectively. ELF-EMF enhanced biological reactions considerably in the presence of a second stressor (mild heat shock). In C. elegans, this effect could be demonstrated at the level of heat shock protein (hsp) gene expression by means of a lacZ reporter gene controlled by an hsp 16 or hsp 70 promoter. In Drosophila, the same experimental strategy led to ELF-EMF induced developmental defects, as well as to a considerable retardation of development. The findings are discussed with respect to possi- ble molecular mechanisms that might explain the observed synergistic ELF-EMF induced enhancement of the stress response. An experimental approach is suggested which may help to unravel the involved signal transduction pathways.     

Involvement of differential gene expression and mRNA stability in the developmental regulation of the hsp 30 gene family in heat-shocked Xenopus laevis embryos

Four complete hsp 30 genes have been isolated from Xenopus laevis: hsp 30A, hsp 30B (a pseudogene), hsp 30C, and hsp 30D. The hsp 30A and hsp 30C genes are first heat inducible at the early tailbud stage, as determined by RNase protection and RT-PCR assays. In this study, we determined by RT-PCR that the hsp 30D gene was first heat inducible (33^oC for 1 h) at the mid-tailbud stage, approximately 1 day later in development than hsp 30A and hsp 30C. Furthermore, using Northern blot analysis, we detected the presence of very low levels of hsp 30 mRNA at the heat-shocked late blastula stage. The relative levels of these pre-tailbud (PTB) hsp 30 mRNAs increased at the gastrula and neurula stage followed by a dramatic enhancement in heat shocked tail-bud and tadpole stage embryos (50- to 100- fold relative to late blastula). Interestingly, treatment of blastula or gastrula embryos at high temperatures (37^oC for 1 h) or with the protein synthesis inhibitor, cycloheximide, followed by heat shock, led to enhanced accumulation of the pre-tailbud (PTB) hsp 30 mRNAs. hsp 70, hsp 87, and actin messages were not stabilized at high temperatures or by cycloheximide treatment. Finally, hsp 30D mRNA was not detected by RT-PCR analysis of cycloheximidetreated, heat-shocked blastula stage embryos, confirming that it is not a member of the PTB hsp 30 mRNAs. This study indicates that differential gene expression and mRNA stability are involved in the regulation of hsp 30 gene expression during early Xenopus laevis development. © 1995 Wiley-Liss, Inc.     

Limitation in use of heterologous reporter genes for gene promoter analysis. Silencer activity associated with the cloramphenicol acetyltransferase reporter gene

Various heterologous reporter genes have been widely used for the functional characterization of gene promoters. Many such studies often found weak to very strong silencer activities to be associated with specific parts of the basal promoter or further upstream regions. In this study, we carried out a systematic study on human blood coagulation factor IX (hFIX) and anti-coagulant protein C (hPC) genes, previously shown to have silencer activities associated with their 5'-flanking regions containing promoter sequences. With newly constructed chloramphenicol acetyltransferase (CAT) reporter vectors carrying hFIX or hPC gene promoter sequences, we confirmed the strong silencer activities associated with the regions nt -1895 through nt -416 of the hFIX gene or with the region nt -802 through nt -82 of the hPC gene. However, no such silencer activities associated with the specific regions were found when autologous hFIX cDNA, hFIX minigenes, or hPC minigenes were used as reporters in the expression vector system. Relative levels of CAT, hFIX, and hPC proteins produced in the transient assays correlated well with their mRNA levels. Human FIX minigene constructs containing a simian virus 40 (SV40) 3'-untranslated region (UTR) taken from the CAT reporter gene showed no silencer activity, indicating that SV40 3'-UTR sequence of the CAT reporter gene does not contribute to the silencer activity. Expression vectors constructed with the beta-galactosidase gene under the control of hFIX gene promoter sequences also showed no silencer activity associated with the region nt -1895 through nt -416. These findings indicate that silencer activities associated with specific regions of promoter sequences as analyzed with CAT reporter genes may represent artifacts specific to the CAT reporter genes. Our findings strongly suggest a need for re-examination of promoter characterizations of many eukaryotic genes, which have been studied to date with CAT reporter genes.     

A small temperature rise may contribute towards the apparent induction by microwaves of heat-shock gene expression in the nematode Caenorhabditis Elegans

We have previously reported that low intensity microwave exposure (0.75-1.0 GHz CW at 0.5 W; SAR 4-40 mW/kg) can induce an apparently non-thermal heat-shock response in Caenorhabditis elegans worms carrying hsp16-1::reporter genes. Using matched copper TEM cells for both sham and exposed groups, we can detect only modest reporter induction in the latter exposed group (15-20% after 2.5 h at 26 degrees C, rising to approximately 50% after 20 h). Traceable calibration of our copper TEM cell by the National Physical Laboratory (NPL) reveals significant power loss within the cell (8.5% at 1.0 GHz), accompanied by slight heating of exposed samples (approximately 0.3 degrees C at 1.0 W). Thus, exposed samples are in fact slightly warmer (by < or =0.2 degrees C at 0.5 W) than sham controls. Following NPL recommendations, our TEM cell design was modified with the aim of reducing both power loss and consequent heating. In the modified silver-plated cell, power loss is only 1.5% at 1.0 GHz, and sample warming is reduced to approximately 0.15 degrees C at 1.0 W (i.e., < or =0.1 degrees C at 0.5 W). Under sham:sham conditions, there is no difference in reporter expression between the modified silver-plated TEM cell and an unmodified copper cell. However, worms exposed to microwaves (1.0 GHz and 0.5 W) in the silver-plated cell also show no detectable induction of reporter expression relative to sham controls in the copper cell. Thus, the 20% "microwave induction" observed using two copper cells may be caused by a small temperature difference between sham and exposed conditions. In worms incubated for 2.5 h at 26.0, 26.2, and 27.0 degrees C with no microwave field, there is a consistent and significant increase in reporter expression between 26.0 and 26.2 degrees C (by approximately 20% in each of the six independent runs), but paradoxically expression levels at 27.0 degrees C are similar to those seen at 26.0 degrees C. This surprising result is in line with other evidence pointing towards complex regulation of hsp16-1 gene expression across the sub-heat-shock range of 25-27.5 degrees C in C. elegans. We conclude that our original interpretation of a non-thermal effect of microwaves cannot be sustained; at least part of the explanation appears to be thermal.     

Heat shock and developmental regulation of the Drosophila melanogaster hsp83 gene.

In contrast to the hsp70 gene, whose expression is normally at a very low level and increases by more than 2 orders of magnitude during heat shock, the hsp83 gene in Drosophila melanogaster is expressed at high levels during normal development and increases only severalfold in response to heat shock. Developmental expression of the hsp83 gene consists of a high level of tissue-general, basal expression and a very high level of expression in ovaries. We identified regions upstream of the hsp83 gene that were required for its developmental and heat shock-induced expression by assaying beta-galactosidase activity and mRNA levels in transgenic animals containing a series of 5' deletion and insertion mutations of an hsp83-lacZ fusion gene. Deletion of sequences upstream of the overlapping array of a previously defined heat shock consensus (HSC) sequence eliminated both forms of developmental expression of the hsp83 gene. As a result, the hsp83 gene with this deletion mutation was regulated like that of the hsp70 gene. Moreover, an in vivo polymer competition assay revealed that the overlapping HSC sequences of the hsp83 gene and the nonoverlapping HSC sequences of the hsp70 gene had similar affinities for the factor required for heat induction of the two heat shock genes. We discuss the functional similarity of hsp70 and hsp83 heat shock regulation in terms of a revised view of the heat shock-regulatory sequence.