Effect of heat shock on ribosome synthesis in Drosophila melanogaster.

In Drosophila tissue culture cells, the synthesis of ribosomal proteins was inhibited by a 1-h 37 degrees C heat shock. Ribosomal protein synthesis was repressed to a greater extent than that of most other proteins synthesized by these cells at 25 degrees C. After a 1-h heat shock, when the cells were returned to 25 degrees C, the ribosomal proteins were much slower than most other 25 degrees C proteins to return to pre-heat shock levels of synthesis. Relative to one another, all the ribosomal proteins were inhibited and later recovered to normal levels of synthesis at the same rate and to the same extent. Unlike the ribosomal proteins, the precursor to the large rRNAs was continually synthesized during heat shock, although at a slightly reduced level, but was not processed. It was rapidly degraded, with a half-life of approximately 16 min. Pre-heat shock levels of synthesis, stability, and correct processing were restored only when ribosomal protein synthesis returned to at least 50% of that seen in non-heat-shocked cells.     

Prosomes and heat shock complexes in Drosophila melanogaster cells.

Prosomes and heat shock protein (HSP) complexes isolated from the cytoplasm of Drosophila cells in culture were biochemically and immunologically characterized. The two complexes were found to separate on sucrose gradients, allowing the analysis of their protein constituents by two-dimensional polyacrylamide gel electrophoresis and by reaction with anti-HSP sera and prosome-specific monoclonal antibodies. All of the prosomal proteins were found to be clearly distinct from the HSP; none of the prosomal proteins was synthesized de novo in heat shock. However, an antiprosome (anti-p27K) monoclonal antibody (mouse anti-duck) recognizing the Drosophila p29K prosomal protein allowed immunoprecipitation from a heat-shocked postmitochondrial supernatant of the crude HSP complex, including the low- and the high-molecular-weight components, in particular the 70 x 10(3)-molecular weight HSP. The highly purified small 16S HSP complex still contained this preexistent p29K prosomal protein, which thus also seems to be a metabolically stable constituent of the HSP complex. The significance of this structural and possibly functional relationship between prosomes and HSP, involving the highly ubiquitous and evolutionarily conserved prosomal protein p27/29K, remains to be elucidated.     

Synthesis of heat shock proteins in Drosophila melanogaster embryos

The pattern of polypeptides synthesized in a cell-free protein synthesizing system containing polysomes isolated from heat-shocked (37 C) Drosophila embryos showed significant differences when compared with the pattern obtained when polysomes from normal embryos were used. The synthesis of normal embryonal proteins was reduced and the heat shock proteins were the major products of elongation. After short, 10 min, heat treatment mainly quantitative changes were observed suggesting that normal mRNAs were still present on polysomes, and their products could be completed in vitro in the heterologous cell-free system. The mRNAs coding for normal embryonal proteins were present in almost unchanged amounts in heat-shocked embryos as could be judged from the pattern of proteins synthesized in heterologous cell-free system supplemented with cytoplasmic RNA from normal and heat-shocked embryos. Thus the change in protein synthesis in heat-shocked embryos is not associated with degradation of normal embryonal mRNAs but with their inaccessibility for translation.     

Reproductive costs of heat shock protein in transgenic Drosophila melanogaster

Senescence may evolve when genes have antagonistic effects between early reproduction and later age-specific mortality. Although widely consistent with data of quantitative genetics, this model has yet to be validated with the identification of a specific locus presenting such trade-offs. The molecular chaperone hsp70 may be a candidate for such a gene. Heat induced expression of the Hsp70 protein in adults decreases rates of age-specific mortality during normal aging, while maternally experienced heat shock depresses the production of mature progeny. Here we show that maternal heat shock reduces the proportion of egg hatch but not the viability of successfully hatched offspring. To assess whether heat induced maternal expression of hsp70 causes reduced egg hatch, we measured the proportion of eggs that hatch from females engineered to overexpress hsp70 transgenes. We used the same transgenic strains that extend longevity upon hsp70 expression and found that Hsp70 is sufficient to suppress egg hatch. The proportion of egg hatch as a function of hsp70 expression was not reduced in the first eggs laid after maternal heat shock, but appears in later laid eggs, which were at preoogenic and early vitellogenic stages during the maternal expression of hsp70. The contervailing effects of hsp70 upon fecundity and subsequent age-specific mortality exemplify antagonistic pleiotropy, and this trade-off could contribute to the evolution of Drosophila senescence.     

The heat shock consensus sequence is not sufficient for hsp70 gene expression in Drosophila melanogaster.

A hybrid gene in which the expression of an Escherichia coli beta-galactosidase gene was placed under the control of a Drosophila melanogaster 70,000-dalton heat shock protein (hsp70) gene promoter was constructed. Mutant derivatives of this hybrid gene which contained promoter sequences of different lengths were prepared, and their heat-induced expression was examined in D. melanogaster and COS-1 (African green monkey kidney) cells. Mutants with 5' nontranscribed sequences of at least 90 and up to 1,140 base pairs were expressed strongly in both cell types. Mutants with shorter 5' extensions (of at least 63 base pairs) were transcribed and translated efficiently in COS-1 but not at all in D. melanogaster cells. Thus, in contrast to the situation in COS-1 cells, the previously defined heat shock consensus sequence which is located between nucleotides 62 and 48 of the hsp70 gene 5' nontranscribed DNA segment is not sufficient for the expression of the D. melanogaster gene in homologous cells. A second consensus-like element 69 to 85 nucleotides upstream from the cap site is postulated to be also involved in the heat-induced expression of the hsp70 gene in D. melanogaster cells.     

Large changes in intracellular pH and calcium observed during heat shock are not responsible for the induction of heat shock proteins in Drosophila melanogaster.

Heat shock caused significant changes in intracellular pH (pHi) and intracellular free calcium concentration [( Ca2+]i) which occurred rapidly after temperature elevation. pHi fell from a resting level value at 25 degrees C of 7.38 +/- 0.02 (mean +/- standard error of the mean, n = 15) to 6.91 +/- 0.11 (n = 7) at 35 degrees C. The resting level value of [Ca2+]i in single Drosophila melanogaster larval salivary gland cells was 198 +/- 31 nM (n = 4). It increased approximately 10-fold, to 1,870 +/- 770 nM (n = 4), during a heat shock. When salivary glands were incubated in calcium-free, ethylene glycol-bis(beta-aminoethyl ether)-N,N',N'-tetraacetic acid (EGTA)-buffered medium, the resting level value of [Ca2+]i was reduced to 80 +/- 7 nM (n = 3), and heat shock resulted in a fourfold increase in [Ca2+]i to 353 +/- 90 nM (n = 3). The intracellular free-ion concentrations of Na+, K+, Cl-, and Mg2+ were 9.6 +/- 0.8, 101.9 +/- 1.7, 36 +/- 1.5, and 2.4 +/- 0.2 mM, respectively, and remained essentially unchanged during a heat shock. Procedures were devised to mimic or block the effects of heat shock on pHi and [Ca2+]i and to assess their role in the induction of heat shock proteins. We report here that the changes in [Ca2+]i and pHi which occur during heat shock are not sufficient, nor are they required, for a complete induction of the heat shock response.     

A novel gene that is up-regulated during recovery from cold shock in Drosophila melanogaster

Gene expression during recovery at 25°C (rearing temperature) after cold shock (0°C) was studied in Drosophila melanogaster using a subtraction technique. A novel gene (Frost, abbreviated as Fst) was considerably up-regulated during recovery after cold shock. In addition, a prolongation of cold shock was more effective for induction. In contrast to cold shock, Fst gene did not respond to heat shock. This gene is apparently the same as the unidentified gene, CG9434. Fst has high internal repeats not only in nucleotide but also in amino acid sequences. In addition, FST protein has a proline-rich region. The deduced amino acid sequence revealed a modular structure; i.e., a signal peptide in the N-terminal region followed by a long hydrophilic region. Therefore, this protein is likely to be directed into ER and secreted into extracellular space.