Aging affects expression of 70-kDa heat shock proteins in Drosophila

We examined the effect of cellular aging on adult mortality and hsp70 gene expression in Drosophila melanogaster under thermal stress. The results showed that flies exposed to 37 degrees C for various time intervals had reduced survival rate with age. The level of hsp70 mRNA increases in flies up to 23-28 days of age, but then declines as they get older. When flies are shifted to 25 degrees C after 30 min of heat stress, the time-dependent decrease in hsp70 mRNA levels occurs more rapidly in young flies than in old ones. The hsp70 mRNA present during this recovery period is translated into protein, and senescent flies continue to synthesize this protein for up to 5 h after heat shock. The prolonged expression of hsp70 RNA during recovery from heat shock was also observed in young flies fed canavanine, an arginine analogue. These data suggest that in old insects, the accumulation of conformationally altered proteins plays a role in the regulation of hsp70 RNA expression. These results are discussed in relation to the finding that old flies are more sensitive to thermal stress than young ones.     

Regulated expression of a Drosophila melanogaster heat shock locus after stable integration in a Drosophila hydei cell line.

DNA-mediated cotransformation has been used to transfer a Drosophila melanogaster heat shock locus into cultured Drosophila hydei cells by use of the copia-based selectable vector pCV2gpt and of pMH10A, a cloned 87A7 heat shock locus encoding a mutant heat shock protein (hsp). Transformed lines contain between 50 and 200 copies of both plasmids, each separately organized as a head-to-tail concatemer which is stably maintained in the transformed lines. Exposure of the cotransformants to heat shock temperatures induces the regulated expression of the hsp RNA and the mutant hsp in all the lines analyzed.     

cAMP-dependent protein kinase and the disruption of learning in transgenic flies.

A molecular genetic approach was used to test for a role of cAMP-dependent protein kinase (PKA) in learning and memory in Drosophila. We used genes encoding a peptide inhibitor of PKA, an N-terminal regulatory subunit fragment containing a pseudosubstrate inhibitory domain, and a wild-type catalytic subunit. These dominantly acting genes were placed under control of the hsp70 promoter and transformed into wild-type flies. Induction of the transgenes by 1 hr heat shock results in overproduction of their RNA in adult flies. The same heat shock treatment disrupts the ability of the flies to learn in an odor discrimination task reinforced with electric shock. The results demonstrate the involvement of PKA in the associative learning of Drosophila.     

Two closely linked transcription units within the 63B heat shock puff locus of D. melanogaster display strikingly different regulation

We report the isolation and characterization of a cloned DNA of D. melanogaster, Dm4L, that is derived from the major heat shock puff site at 63B. This segment contains two closely linked genes that are each present once per Drosophila haploid genome. One of these, the hsp 83 gene, encodes an abundant heat shock mRNA that, unlike other major heat shock mRNAs, is also abundant in uninduced (23 degrees) kco cells. Although only a slight increase in the level of total hsp 83 RNA can be detected after heat shock in Kco cells, the level of hsp 83 poly(A)+ mRNA increases more than 6-fold and the level of pulse-labeled hsp 83 RNA in total cellular RNA increases 11-fold relative to uninduced cells. In contrast, the levels of total, poly(A)+, and pulse-labeled RNA homologous to the second gene, 63B-T2, are approximately the same in both induced and uninduced cells. Hence, even though these genes are separated by only one thousand base pairs, and, from in situ hybridization to polytene chromosomes, both lie within the heat shock puff, they display strikingly different regulatory properties, These results demonstrate that close linkage of a gene to a heat shock puff is not sufficient to render its expression heat inducible.     

Expression and localization of Drosophila melanogaster hsp70 cognate proteins.

Monoclonal antibodies have been used to identify three proteins in Drosophila melanogaster that share antigenic determinants with the major heat shock proteins hsp70 and hsp68. While two of the proteins are major proteins at all developmental stages, one heat shock cognate protein, hsc70, is especially enriched in embryos. hsc70 is shown to be the product of a previously identified gene, Hsc4. We have examined the levels of hsp70-related proteins in adult flies and larvae during heat shock and recovery. At maximal induction in vivo, hsp70 and hsp68 never reach the basal levels of the major heat shock cognate proteins. Monoclonal antibodies to hsc70 have been used to localize it to a meshwork of cytoplasmic fibers that are heavily concentrated around the nucleus.     

The use of promoter fusions in Drosophila genetics: isolation of mutations affecting the heat shock response

We have constructed a gene fusion using the promoter of Drosophila hsp70 and the structural gene for Drosophila alcohol dehydrogenase (Adh) and used this construct to transform Adh-deficient flies. In these transformants, Adh is expressed only after heat shock. Like hsp70 itself, this heat-shock-inducible Adh (Adhhs) is induced in a wide variety of tissues. It fails to be induced in primary spermatocytes. Although the tissue distribution of Adh activity is very different from wild type, this does not appear to be deleterious. Indeed, the induction of Adhhs allows flies to survive exposure to ethanol. We have used this latter characteristic to select dominant, trans-acting mutations that alter the response of flies to heat shock.     

Structure, organization, and expression of the 16-kDa heat shock gene family of Caenorhabditis elegans

Exposure of the nematode Caenorhabditis elegans to a heat shock results in the induction of a number of genes not normally expressed in the animals under normal growth conditions. Among these are a family of genes encoding 16 kDa heat shock proteins (hsp16s). The major hsp16 genes have been cloned and characterized, and found to reside at two clusters in the C. elegans genome. One cluster contains two distinct genes, hsp16-1 and hsp16-48, arranged in divergent orientations separated by only 348 base pairs (bp). An identical pair, duplicated and inverted with respect to the first pair, is located 415 bp away. This cluster, located on chromosome V, therefore contains four genes as two identical pairs within less than 4 kilobases of DNA, and the pairs form the arms of a large inverted repeat. A second pair of genes, hsp16-2 and hsp16-41, constitutes a second hsp16 locus with an organization very similar to that of the hsp16-1/48 locus, except that it is not duplicated. Comparisons of the derived amino acid sequences show that hsp16-1 and hsp16-2 form a closely related pair, as do hsp16-41 and hsp16-48. These hsps show extensive sequence identity with the small hsps of Drosophila, as well as with mammalian alpha-crystallins. The coding region of each gene is interrupted by a single intron of approximately 50 bp, in a position homologous to that of the first intron in mouse alpha-crystallin gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mouse and Drosophila genes encoding the major heat shock protein (hsp70) are highly conserved.

We used a cloned Drosophila melanogaster hsp70 gene to hybrid-select heat shock-induced mouse mRNA and showed that this mRNA encodes the major mouse heat shock protein. This result suggests that the sequence of the hsp70 gene(s) is highly conserved.     

Selective translation of heat shock mRNA in Drosophila melanogaster depends on sequence information in the leader.

One of the effects of a temperature increase above 35 degrees C on Drosophila melanogaster is a rapid switch in selectivity of the translational apparatus. Protein synthesis from normal, but not from heat shock, mRNA is much reduced. Efficient translation at high temperature might be a result of the primary sequence of heat shock genes. Alternatively a mRNA modification mechanism, altered as a consequence of heat shock, might allow for efficient high temperature translation of any mRNA synthesized during a heat shock. The gene for alcohol dehydrogenase (Adh) was fused to the controlling elements of a heat shock protein 70 (hsp70) gene. Authentic Adh mRNA, synthesized from this fusion gene at elevated temperatures was not translated during heat shock. A second Adh fusion gene in which the mRNA synthesized contained the first 95 nucleotides of the Hsp70 non-translated leader sequence gave rise, at high temperature, to mRNA which was translated during the heat shock. Thus, the signal(s) in the mRNAs controlling translation efficiency at heat shock temperatures is encoded within the heat shock genes.     

Heat shock proteins affect RNA processing during the heat shock response of Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the splicing of mRNA precursors is disrupted by a severe heat shock. Mild heat treatments prior to severe heat shock protect splicing from disruption, as was previously reported for Drosophila melanogaster. In contrast to D. melanogaster, protein synthesis during the pretreatment is not required to protect splicing in yeast cells. However, protein synthesis is required for the rapid recovery of splicing once it has been disrupted by a sudden severe heat shock. Mutations in two classes of yeast hsp genes affect the pattern of RNA splicing during the heat shock response. First, certain hsp70 mutants, which overproduce other heat shock proteins at normal temperatures, show constitutive protection of splicing at high temperatures and do not require pretreatment. Second, in hsp104 mutants, the recovery of RNA splicing after a severe heat shock is delayed compared with wild-type cells. These results indicate a greater degree of specialization in the protective functions of hsps than has previously been suspected. Some of the proteins (e.g., members of the hsp70 and hsp82 gene families) help to maintain normal cellular processes at higher temperatures. The particular function of hsp104, at least in splicing, is to facilitate recovery of the process once it has been disrupted.