Studies of cloned sequences from four Drosophila heat shock loci.

DNA cloned from the D. melanogaster (Oregon R) heat shock loci at 63BC and 95D codes for the 83,000 and the 68,000 dalton heat shock proteins, respectively. Both coding sequences occur once per haploid genome. Sequences complementary to messenger RNA for the 70,000 dalton heat shock protein are represented five times, twice at 87A and three times at 87 C. The copies at 87A differ characteristically from those at 87C in an interval of a few hundred bp near the 5' end of the messenger sequence, and the corresponding two classes of hsp 70 messenger RNA are found on polysomes after heat shock. Within this differential region, there is about 15% divergence between messenger sequences cloned from the two loci, while in the rest of the messenger region examined the homology is much closer although still imperfect. Unexpectedly, considerable homology is found between the sequence for the 68,000 dalton heat shock protein at 95D and the sequences for the 70,000 dalton protein at 87A and 87C, and between these sequences and a site in 87D. Messenger RNA molecules of 2.4, 2.55 and 3.05 kb code for the 68,000, 70,000 and 83,000 dalton heat shock proteins and hybridize to apparently uninterrupted DNA sequences of 2.1, 2.25 and 2.6 kb, respectively.     

Phosphorylation of Drosophila heat shock transcription factor.

The role that phosphorylation plays in regulating heat shock factor (HSF) function and activity has been the subject of several studies. Here, we demonstrate that Drosophila melanogaster HSF (DmHSF) is a phosphoprotein that is multiply phosphorylated at some sites and is dephosphorylated at others upon heat shock. However, the steady-state level of phosphorylation of Drosophila HSF remains unchanged after heat shock. Phosphoamino-acid analysis reveals that predominantly serine residues are phosphorylated for both the non-shocked and heat shocked molecules. Gel mobility shift assays using extracts from SL2 cells treated with a variety of phosphatase and kinase inhibitors show little or no effect on the heat shock induced DNA binding activity of HSF or on its recovery. We conclude that phosphorylation plays no significant role in regulating the heat induced DNA binding activity of Drosophila HSF.     

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.     

Intracellular localization of heat shock proteins in Drosophila

When cells and tissues of Drosophila are subjected to elevated temperatures, the pattern of protein synthesis shifts from the production of a broad spectrum of different proteins to the vigorous production of a small number of new, heat shock proteins. The intracellular distribution of these proteins has been investigated through autoradiographic analysis of cells labeled with ³H-leucine at 25° and 37°C. After examining sections of cultured cells from D. melanogaster and polytene cells of D. virilis by electron and light microscopy, we conclude that little (if any) heat shock protein becomes associated with mitochondria, despite the many lines of evidence linking the response to respiratory stress. Confirming earlier reports on the presence of heat shock proteins in nuclei, we find the proteins are very highly concentrated there and that their transport to the nucleus occurs very rapidly. Interestingly, their free concentration in the nuclear sap is extremely low; they are, in fact, quantitatively associated with chromosomes. This association occurs in a nonrandom manner, their concentration in highly condensed chromatin being very low relative to that of other chromosomal loci.     

Heavy heat shock induced retrotransposon transposition in Drosophila

The phenomenon of transposition induction by heavy heat shock (HHS) was studied. Males of a Drosophila isogenic line with a mutation in the major gene radius incompletus (ri) were treated by HHS (37 degrees C for 1 h followed by 4 degrees C for 1 h, with the cycle repeated three times) and crossed to untreated females of the same line. The males were crossed 5 d after heat shock, and also 9 d after HHS. Many transpositions were seen in the F1 larvae by in situ hybridization. The rate of induced transposition was at least 2 orders of magnitude greater than that of the control sample, and was estimated to be 0.11 events per transposable element copy per sperm. Two 'hot' subdivisions for transpositions, induced probably during the post-meiotic stage of spermiogenesis, were found: 43B and 97DE. Three-quarters of all transpositions were localized in these positions. In other sites the rates of induced transpositions were (1.3-3.2) x 10(-2) events per occupied segment per sperm, 1 order of magnitude greater than those of the control.     

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.     

果蝇热激蛋白的研究进展

热休克蛋白(heat shock proteins,HSPs)是生物体受到应激刺激时诱导产生的一组保守性蛋白,普遍存在于各种生物体中。近年来,果蝇Drosophila作为生命科学与人类疾病研究的重要模式生物,其热激蛋白的研究取得了许多新的进展。文章对果蝇热激蛋白的类别、热激蛋白基因的表达调控机制、热激蛋白的分子伴侣功能、调节细胞存亡和影响发育及寿命等相关生物学功能进行综述,并对热激蛋白在神经退行性疾病治疗中的应用前景作展望。     

Evidence for complex mutations at microsatellite loci in Drosophila.

Fifteen lines each of Drosophila melanogaster, D. simulans, and D. sechellia were scored for 19 microsatellite loci. One to four alleles of each locus in each species were sequenced, and microsatellite variability was compared with sequence structure. Only 7 loci had their size variation among species consistent with the occurrence of strictly stepwise mutations in the repeat array, the others showing extensive variability in the flanking region compared to that within the microsatellite itself. Polymorphisms apparently resulting from complex nonstepwise mutations involving the microsatellite were also observed, both within and between species. Maximum number of perfect repeats and variance of repeat count were found to be strongly correlated in microsatellites showing an apparently stepwise mutation pattern. These data indicate that many microsatellite mutation events are more complex than represented even by generalized stepwise mutation models. Care should therefore be taken in inferring population or phylogenetic relationships from microsatellite size data alone. The analysis also indicates, however, that evaluation of sequence structure may allow selection of microsatellites that more closely match the assumptions of stepwise models.     

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.