Hybrid genes containing the 5' region of Drosophila heat shock protein (hsp) genes, ligated to the herpesvirus thymidine kinase (tk) gene, were transfected into Drosophila line S3 cells. Constructions containing sequences upstream from hsp7O, or from any of the small hsp genes, showed heat-inducible tk expression. Ecdysterone-inducible tk expression was seen only in transfections with small hsp-tk hybrid genes. Plasmids containing a synthetic "heat shock consensus sequence" or a deletion of the hsp23 upstream region were totally inactive in transfection studies.The small heat shock protein (s-hsp) genes of Drosophila, designated hsp22, hsp23, hsp26, and hsp27, are expressed in response to high temperature shock (1) and at two points in development. During pupation, all four of the genes are transcribed and translated in imaginal discs (2, 3). During oogenesis, both hsp26 and hsp27 are expressed in ovarian nurse cells (4). The expression of the four s-hsp genes can also be induced in vitro in both established tissue culture cell lines and in immature imaginal discs (2, 5) by treatment with physiological concentrations of the molting hormone, ecdysterone.Our finding of regulated gene expression by two independently acting stimuli raises several questions at the molecular level. Among these is whether recognition and binding of the ecdysterone-receptor complex (6, 7) and of the endogenous factor(s) implicated in heat shock induction (8, 9) occur at the same upstream regulatory site.We have recently developed a functional assay for analyzing s-hsp gene regulation in a homologous transient expression system (10). Initial studies, using a cloned gene that specifies an electrophoretic hsp22 gene variant, demonstrated that the transfected gene could rapidly acquire the normal pattern of heat shock regulation. We now describe experiments designed to examine the regulated expression of hybrid genes containing upstream promoter regions from each of the s-hsp genes ligated to the herpesvirus thymidine kinase (tk) gene. We find that these hybrid genes acquire both heat shock induction and ecdysterone induction. Preliminary results on the nature and location of these regulatory sites are discussed. METHODS AND MATERIALSCell Culture and Transfection. Drosophila line S3 cells were cultured in Schneider's medium (GIBCO) supplemented with 10% fetal bovine serum (GIBCO) at 25°C. Ecdysterone-insensitive line F6 cells were grown in ZH1 medium (11). For transfection, 37 ,tg of CsCl-purified plasmid DNA (12) was added as a CaCl2 coprecipitate (13) to each 75-cm2 flask of confluent cells in 10 ml of medium. For heat shock, the flasks were floated in a 370C water bath for 1 hr after a 24-hr transfection. In hormone studies, 6 hr after transfection began, ecdysterone (Calbiochem) was added to 1 uM final concentration, and the flasks were cultured for an additional 24 hr at 250C.Slot Blot Analysis. Total RNA was recovered from cells by using the guanidine-HCl/CsCl method (14) with the addition of a DNase digestion step (13). Aliqu...

Both spontaneous and thyroid hormone (TH)-induced metamorphosis of Rana catesbeiana are accompanied by a marked increase in the activity of the urea cycle enzyme carbamyl phosphate synthetase (CPS). The increase induced by exogenous TH is de novo synthesis of enzyme and appears to be secondary to an increase in the CPS mRNA level resulting from the elevated plasma TH. Since endogenous TH levels rise sharply during spontaneous metamorphosis, a similar sequence of events would be anticipated. However, after midclimax, CPS activity continues to increase, while plasma TH levels steadily decline, suggesting that other factors are involved. To obtain insight into this problem, the changes in CPS mRNA level during spontaneous development were determined using a mammalian CPS cDNA probe and correlated with changes in CPS activity and plasma T3 concentration. CPS mRNA level and CPS activity were barely detectable until midprometamorphosis, but both increased rapidly during the latter half of this phase. CPS activity continued to rise, reaching a maximum in the adult frog. The CPS mRNA level, however, was highest during the first half of climax, but declined after midclimax and was relatively low in the adult frog. Studies were also performed in which the rise and fall in the plasma T3 concentration typical of metamorphic climax were induced by exposure of premetamorphic tadpoles to T3, followed by its withdrawal. Both CPS activity and CPS mRNA level were induced by T3, but when plasma T3 levels fell after removal of the exogenous T3, CPS mRNA level, but not CPS activity, also decreased. Additional studies indicated that the TH-induced increase in CPS mRNA was evident within 24 h, could be prevented by simultaneous injection of actinomycin-D, and could not be induced in tadpoles undergoing climax; in this phase the T3 receptors are fully occupied with endogenous TH. When premetamorphic tadpoles were immersed in T3-containing water (0-500 nM) for 6 days, CPS mRNA, CPS activity, and plasma T3 concentration increased in parallel, reaching a maximum at 50 nM. At 50 nM T3, the plasma T3 level was sufficient to saturate the receptors, and no additional increase in CPS mRNA level or CPS activity was obtained at higher concentrations of T3. These studies indicate that the CPS mRNA level during spontaneous development correlates with the plasma T3 concentration and suggest that it is a function of T3 receptor occupancy. The data are also consistent with an effect of TH on transcription of the CPS gene and with a relatively long half-life of its protein product, the CPS enzyme.

A cloned Drosophila heat shock protein 22 gene was transfected into two independently established Drosophila cell lines. Each line carried a different heat shock protein 22 allele, distinguishable by electrophoresis of the protein. The transfected gene was not expressed at 25 degrees C but could be induced at 36 degrees C. In one line, two heat shock protein 22 electromorphs were synthesized.

When Drosophila cell lines are exposed to physiological doses of the steroid molting hormone, ecdysterone, they enter mitotic arrest and differentiate morphologically. These responses are accompanied by specific changes in gene expression. Several enzyme activities (acetylcholinesterase, beta-galactosidase, dopa decarboxylase, and catalase) are induced and the synthesis of a cytoplasmic actin and the four small heat-shock proteins is initiated. Several of these ecdysterone inducible genes have been physically isolated and characterized, in several cases by DNA sequencing. Current studies focus on introducing cloned ecdysterone inducible genes into responsive cells by DNA mediated transfection. Once it is clear that these introduced genes acquire the normal pattern of hormone-regulated gene expression in the cell, in vitro mutagenesis can be used before transfection to modify their structure. Transient expression, then, can become a functional assay to define regions of DNA flanking the coding region of inducible genes that are needed for proper gene expression and regulation in cultured cells.

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