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.     

Molecular cloning and analysis of DNA complementary to three mouse Mr = 68,000 heat shock protein mRNAs

The construction and isolation of three recombinant DNAs complementary to different mouse L-cell Mr = 68,000 heat shock protein (hsp68) mRNAs is described. cDNA libraries derived from heat-shocked mouse L-cell poly(A)+ RNA by the vector-linked primer strategy of cDNA synthesis and cloning of Okayama and Berg (Okayama, H., and Berg, P. (1982) Mol. Cell. Biol. 2, 161-170) were screened first with a Drosophila hsp70 heterologous probe and subsequently with a cDNA probe isolated from the first screening. Positive clones were assigned to one of three sets based on their restriction map, and the largest member of each group was chosen for further analysis. All three cDNAs hybrid-select mRNA for the mouse major heat shock protein (hsp68) as assayed by in vitro translation and hybridize preferentially to two heat shock-induced hsp68 mRNAs on Northern blots. The coding regions of the cDNAs are almost identical and closely resemble other HSP70 genes but the 3' untranslated regions diverge considerably. Differences in the lengths of the untranslated regions are responsible for the two different sized induced hsp68 mRNAs in mouse L-cells. The physical maps of these cDNA clones and the limited number of mouse genomic DNA fragments detected on Southern blots suggest that there are at least three closely related heat shock-inducible members of the mouse HSP70 gene family. None of the cloned cDNAs are derived from the two related cognate genes known to be present in the mouse genome.     

Cloning and characterization of the Brucella ovis heat shock protein DnaK functionally expressed in Escherichia coli.

The Brucella ovis dnaK gene, homolog to the eukaryotic hsp70 genes, was cloned by using a Drosophila melanogaster probe. Comparison of B. ovis and Escherichia coli sequences revealed a similar organization for the dnaK and dnaJ genes and putative regulatory signals. In E. coli transfected with the cloned fragment, B. ovis hsp70 was expressed at 30 and 50 degrees C apparently under the control of its own promoter. The recombinant protein and a B. ovis native protein displaying the same molecular weight were both recognized by anti-E. coli DnaK serum. Native B. ovis protein was also recognized by sera of sheep either infected or vaccinated with an attenuated Brucella strain, suggesting that Brucella hsp70 could be up-regulated during host colonization. A thermosensitive E. coli dnaK mutant transfected with the cloned fragment recovered colony-forming ability at 42 degrees C, showing that the B. ovis DnaK protein could behave as a functional heat shock protein in E. coli.     

Isolation and characterization of a sea urchin hsp 70 gene segment

Three clones containing Paracentrotus lividus sea urchin DNA sequences which cross-hybridize to Drosophila heat shock protein (hsp) 70 gene were isolated. The sequence arrangements in the three cloned DNA inserts were compared by restriction and cross-hybridization analysis. The results showed that they contain four different genes related to one Drosophila hsp 70 gene. One of these genes was subcloned, and two of the isolated fragments were shown to hybridize to genomic DNA and to RNA from heat-treated sea urchin embryo.     

Isolation of major heat shock protein (hsp70) gene-related sequences from rat genome

Rat genome was assayed for the presence of hsp70 gene-related sequences. Southern blots prepared from rat DNA digested with EcoRI or HindIII restriction endonucleases were hybridized with mouse, human and fruit fly hsp 70 gene probes at increasing stringencies. At the stringency which allows sequences divergent up to about 30% to form stable complexes all three probes detected 25–30 restriction fragments. Increased stringency of the hybridization reduced the number of detectable bands to a few and among them the DNA fragments hybridizing specifically either with mouse or human hsp70 gene probes were detected. Most of the genomic fragments containing hsp70 gene-related sequences were subsequently isolated by screening the rat genomic library with mouse hsp70 gene probe. 168 positive clones were plaque purified and on the basis of the restriction and hybridization pattern we deduced that inserts represented 20 different genomic regions. Partial restriction maps of all isolated genomic fragments were constructed and regions containing hsp70 gene related as well as highly repetitive DNA sequences were localized. A putative sequence rearrangement in the proximity of the hsp70 gene-related sequence was detected in one of the isolated genomic segments.     

Heat-shock proteins, Hsp84 and Hsp86, of mice and men: two related genes encode formerly identified tumour-specific transplantation antigens

Mouse cDNA clones have been isolated with the help of Drosophila melanogaster 82-kDa heat-shock protein (Hsp82)-coding sequences as hybridization probe. Sequencing of the overlapping mouse clones reveals a long open reading frame (ORF) that encodes a polypeptide of 83.3 kDa which shows about 80% similarity to the respective Drosophila Hsp82 amino acid sequence. The N-terminal half of this cDNA cross-hybridizes to a different class of mouse cDNA clones indicating a related gene. Northern blot hybridization experiments reveal a 2.6-kb poly(A)+RNA when probed with the hsp84 clone and a 2.85-kb signal with the hsp84-related cDNA. The amino acid sequences deduced from the contiguous ORF of the hsp84 and the hsp84-related cDNA coincide with the N-terminal sequence of formerly identified 84-kDa and 86-kDa tumour-specific transplantation antigens (Ullrich et al., 1986). In addition, the amino acid composition of the putative 84-kDa mouse Hsp described here is very similar to that of the 84-kDa tumour antigen described by Ullrich et al. (1986). Both observations corroborate the assumption that these Hsps are identical to the described 84-kDa and 86-kDa tumour-specific transplantation antigens. Using these mouse hsp gene clones as hybridization probes we also isolated the corresponding pair of human cDNA clones. Comparison of the respective sequences reveals a strong evolutionary constraint on these two genes in mouse and man.     

Ecdysterone selectively stimulates the expression of a 23000-Da heat-shock protein-beta-galactosidase hybrid gene in cultured Drosophila cells

To study the regulated expression of cloned heat-shock genes in homologous cells, hybrid Drosophila heat-shock-Escherichia coli beta-galactosidase genes were constructed. Segments of the ecdysterone-inducible 23,000-Da heat-shock protein (hsp23) gene and of two other hsp genes (hsp84 and 70), which are not hormone regulated, were functionally linked to the bacterial coding sequence, and the resulting hybrid genes were introduced into cultured, hormone-responsive Drosophila cells by transfection. All hybrid genes directed the synthesis of E. coli-specific beta-galactosidase in heat-treated cells. hsp23 hybrid gene expression was stimulated strongly by ecdysterone, while the activities of the other hybrid genes were not affected at all by the hormone. A hybrid gene with only 147 bp of hsp23 promoter sequence could not be activated by either heat or ecdysterone treatment. Thus, far upstream sequences contain signals required for the regulated expression of the hsp23 gene in Drosophila cells.     

Hsp70 duplication in the Drosophila melanogaster species group: how and when did two become five?

To determine how the modern copy number (5) of hsp70 genes in Drosophila melanogaster evolved, we localized the duplication events that created the genes in the phylogeny of the melanogaster group, examined D. melanogaster genomic sequence to investigate the mechanisms of duplication, and analyzed the hsp70 gene sequences of Drosophila orena and Drosophila mauritiana. The initial two-to-four hsp70 duplication occurred 10--15 MYA, according to fixed in situ hybridization to polytene chromosomes, before the origin and divergence of the melanogaster and five other species subgroups of the melanogaster group. Analysis of more than 30 kb of flanking sequence surrounding the hsp70 gene clusters suggested that this duplication was likely a retrotransposition. For the melanogaster subgroup, Southern hybridization and an hsp70 restriction map confirmed the conserved number (4) and arrangement of hsp70 genes in the seven species other than D. melanogaster. Drosophila melanogaster is unique; tandem duplication and gene conversion at the derived cluster yielded a fifth hsp70 gene. The four D. orena hsp70 genes are highly similar and concertedly evolving. In contrast, the D. mauritiana hsp70 genes are divergent, and many alleles are nonfunctional. The proliferation, concerted evolution, and maintenance of functionality in the D. melanogaster hsp70 genes is consistent with the action of natural selection in this species.     

Naturally occurring transposable elements disrupt hsp70 promoter function in Drosophila melanogaster

Naturally occurring transposable element (TE) insertions that disrupt Drosophila promoters are correlated with modified promoter function and are posited to play a significant role in regulatory evolution, but their phenotypes have not been established directly. To establish the functional consequences of these TE insertions, we created constructs with either TE-bearing or TE-lacking hsp70 promoters fused to a luciferase reporter gene and assayed luciferase luminescence in transiently transfected Drosophila cells. Each of the four TEs reduces luciferase signal after heat shock and heat inducibility of the hsp70 promoter. To test if the differences in hsp70 promoter activity are TE-sequence dependent, we replaced each of the TEs with multiple intergenic sequences of equal length. These replacement insertions similarly reduced luciferase signal, suggesting that the TEs affect hsp70 promoter function by altering promoter architecture. These results are consistent with differences in Hsp70 expression levels, inducible thermotolerance, and fecundity previously associated with the TEs. That two different varieties of TEs in two different hsp70 genes have common effects suggests that TE insertion represents a general mechanism through which selection manipulates hsp70 gene expression.     

Differential expression of heat-shock proteins and spontaneous synthesis of HSP70 during the life cycle of Blastocladiella emersonii

The heat-shock response in Blastocladiella emersonii is dependent on the developmental stage. Cells exposed to elevated temperatures at different stages of the life cycle (sporulation, germination or growth) show a differential synthesis of heat-shock proteins (hsps). Of a total of 22 polypeptides induced, particular subsets of hsps appear in each phase, demonstrating a non-coordinate heat-shock gene expression. In contrast, heat-shock-related proteins (hsp76, hsp70, hsp39a) are spontaneously expressed at a high level during sporulation. By the criteria of two-dimensional gel electrophoresis and partial proteolysis mapping, the 70,000-Da protein, whose synthesis is induced spontaneously during sporulation, is indistinguishable from the heat-inducible hsp70. The techniques of in vitro translation, and Northern analysis using a Drosophila hsp70 probe, demonstrated that enhanced synthesis of hsp70, which occurs during heat-shock treatment and spontaneously during sporulation, is associated with an accumulation of hsp70 mRNA. These observations suggest that hsp70 gene expression is induced during sporulation.     

A consensus sequence polymer inhibits in vivo expression of heat shock genes.

Promoter function for hsp70 gene expression in Drosophila melanogaster was studied with an in vivo competition assay. A polymer of 40 tandem copies of the pair of regulatory elements of the hsp70 gene was constructed and cloned into a plasmid vector. Various marked genes were cotransfected with the polymer plasmid into Schneider line 2 cells, and their expression was determined by enzyme activity measurements. The polymer dramatically inhibited expression of cotransfected hsp70, hsp26, and hsp83 genes, but not cotransfected copia and histone genes. Our results indicate that in vivo, a trans-acting, positive regulatory factor, which can be titrated by heat shock consensus sequences, is required for activation of heat shock genes and is specific for these genes; the coordinate induction of different heat shock genes appears to be mediated by similar, but not identical, interactions of the trans-acting induction factor and the cis-acting heat shock consensus sequences; and the uninduced or basal level expression of the transfected hsp70 gene is also due to interaction of the consensus sequence with a positively acting factor.