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Mild heat treatments applied to whole animals or cell cultures of Drosophila prior to lethal heat shocks result in increased survival and protection against phenocopy induction. The optimal condition for the preliminary mild heat treatment is that which induces the synthesis of heat-shock proteins but does not turn off the protein synthesis that is in progress. Recovery of protein synthesis but not RNA synthesis following a drastic heat shock is much enhanced by the pretreatments. The results suggest that the protection for survival and against phenocopy induction is due to storage of messenger RNA.Key words: drosophila, gene regulation, heat shock, protection phenocopies, survival I NTROD UCTl ON We recently described [ I ] a series of phenocopies that are induced in Drosophila melanogaster by subjecting pupa to heat shock at specific stages of development. Three of the phenocopies produced by shocks at successive intervals closely resemble the mutants hook and javelin which are involved in determining the structure of the scutellar bristles of the adult fly. We have also shown that the conditions used for phenocopy induction, turn off, for a time, both transciptional and translational activities in Drosophila tissues [ 1 , 2 ] . Subsequently, translation resumes long before reactivation of transcription [ l ] . These findings show that heat shock can result in storage of mRNAs in keeping with the observations of McKenzie et a1 [ 131 and Mirault et a1 [14] . Furthermore these observations provide a reasonable basis for interpretation of the heat-shock protection phenomenon described by Milkman several years ago [3,4] . Milkman and collaborators demonstrated that when Drosophila pupa (24 hours) were subjected to a mild heat shock prior to one that would be lethal alone, some animals would survive. They also reported that a pretreatment of this kind would prevent the induction of a phenocopy of the mutant crossveinless.

Heat shock protein synthesis can be induced during recovery from cold treatment of Drosophila melanogaster larvae. Survival of larvae after a cold treatment is dramatically improved by a mild heat shock just before the cold shock. The conditions which induce tolerance to cold are similar to those which confer tolerance to heat.Heat shock proteins are a group of proteins which are universally expressed in response to heat, heavy metals, and a variety of chemical treatments (1, 5, 10, 11). The major heat shock proteins synthesized in bacteria, plants, and mammals show a high degree of sequence homology (1, 5). These proteins are also synthesized during some periods in normal development.Transient resistance to killing by heat stress has been observed to be induced in many organisms by an initial exposure to a sublethal heat treatment (2, 7, 11). We have shown that conditions which induce thermotolerance in Drosophila melanogaster also induce the synthesis of heat shock proteins (7), and this correlation has been repeated many times in a variety of systems (1,5). Because of this and the universality of the response, it has been suggested that heat shock proteins play a role in protecting cells from damage caused by these stresses. The precise function of the heat shock proteins as well their role in stress protection are still uncertain.We show here that heat shock proteins are synthesized during recovery from prolonged exposure to cold in the absence of heat shock and that a mild heat treatment of the same kind which protects against death from heat shock also prevents death from exposure to cold.Induction of heat shock proteins by cold treatment. Drosophila larvae recovering from cold treatments of more than 8 h at 0°C synthesized the same set of heat shock or stress proteins that are made in response to heat shock (7, 9, 12). Figure 1 shows the proteins synthesized in larval salivary glands during recovery from 14 h at 0°C. Salivary glands were dissected from cold-shocked larvae that had been allowed to recover for 0, 10, 20, 30, or 40 min at 25°C. The salivary glands were labeled for 30 min with ['5S] methionine, and the proteins were separated on sodium dodecyl sulfate-polyacrylamide gels as described previously (8). An untreated 25°C control is shown in Fig. 1, lane C, as is a typical heat shock pattern from animals heated to 37°C (lane H). Clearly, all the major heat shock proteins appeared in the 20-min sample. The strongest heat shock protein synthesis patterns were seen not during the 30 min immediately following the cold treatment, but rather when larvae were allowed to recover for 30 or 40 min before being labeled. This suggests that the induction of the heat shock proteins may be a response to the shift from 0 to 25°C rather than a response to the 0°C treatment itself. We are currently doing * Corresponding author.RNA blots to determine directly the levels of hsp70 mRNA present at 0°C and during the recovery at 25°C.Experiments designed to evaluate the effects of different durations of cold treatments ...

A mild heat shock at 350C, which induces heat shock gene expression, greatly enhances survival and the recovery of protein synthesis in Drosophila cells after a higher temperature heat shock. The 350C treatment is also effective in preventing heat-induced developmental defects in pupae. We show here that the major larval mRNAs are present in approximately normal (25C) concentrations after a 40. 1C heat shock whether or not the animals receive a pretreatment. This indicates that the pretreatment affects translation directly rather than messenger concentration. We also observe selective translation of heat shock messages and some 25WC messages during, recovery from heat shock.Pretreatment at a nonlethal temperature (30 min at 350C) dramatically improves the ability of Drosophila melanogaster larvae, pupae, adults, and cell lines to withstand a normally lethal heat shock. This type of pretreatment also prevents developmental defects (phenocopies) induced in Drosophila pupae by heat shock (1). The acquisition of resistance to heat has been described in a wide variety of plant (2, 3) and animal systems, including mammalian cell lines (4-7). Understanding the molecular basis for this phenomenon is therefore of general interest. We have shown previously that the dramatic effects on survival and phenocopy prevention are paralleled by a much more rapid recovery of protein synthesis in the animals that received the 350C pretreatment (1). We are interested in how the pretreatment improves recovery from heat shock and, in particular, in the possibility that one or more of the heat shock gene products mediates the effects on recovery through effects on regulation of RNA and protein synthesis. In this paper we show that the 350C pretreatment does not affect concentrations of the major larval messages as has been previously suggested (1,8), but rather it affects the ability of the cells to translate mRNA. MATERIALS AND METHODSFly Culture and Labeling in Vivo. Embryos. collected over a 2-hr period (9) were kept at 250C for 31/2 days. Third-instar larvae were washed and heat treated in moist vials submersed in a water bath of the temperatureindicated. Larvae recovering from heat treatment were kept in vials containing a small amount of moist yeast. Larvae were injected with [35S]methionine (3.0 kCi per larva; 1 Ci = 3.7 X 1010 becquerels) as described (10). Thirty minutes after the injection, larvae were ground in cold '10% trichloroacetic acid. Six larvae were used for each time point. The acid precipitate was washed twice with ethanol, dried, and dissolved in sample buffer. One-fifth of each sample was run on the gel.RNA Isolation and Protein Synthesis in Vitro. Larvae, wings, or salivary glands were frozen in liquid nitrogen and ground in a glass grinder in extraction buffer (0. 22 M Tris-HCl, pH 7.5/0.04 M EDTA/0.02 M NaCl/1% sodium dodecyl sulfate) plus an equal volume of phenol. One volume of chloroform was added immediately after grinding and samples were extracted (11,12). Samples were centrifuged and reextracte...

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