Extensive regions of homology in front of the two hsp70 heat shock variant genes in Drosophila melanogaster

The major heat shock protein of 70,000 M_r in Drosophila melanogaster is encoded by two variant gene types located, respectively, at the chromosomal sites 87A7 and 87C1. We present the DNA sequence of a complete hsp70² gene of the 87A7 type and of the adjacent regions from both variants, extending to 1·2 × 10³ bases upstream from the start of the messenger coding region. We find an untranslated region of 250 nucleotides at the 5′ end of the messenger coding sequence in both variants. There is only one open reading frame which allows coding of a 70,000 M_r protein within the 87A7 variant, as found for an 87C1 variant (Ingolia et al., 1980). We observe 4·2% nucleotide divergence between these two variants with complete conservation of the reading frame. There is a conserved sequence of 355 nucleotides in front of each hsp70 gene, which is 85% homologous between the two variants. The presence of the same sequence element in γ, in front of the αβ heat shock genes (R. W. Hackett & J. T. Lis, personal communication) suggests that this element contains the regulatory signals for the coordinate expression of both the hsp70 and the αβ heat shock genes. Finally we find a very A + T-rich sequence of 150 basepairs which is highly conserved (91·8%) 0·6 × 10³ bases upstream from two hps70 gene variants.     

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.     

Multiple phases of nucleosomes in the hsp 70 genes of Drosophila melanogaster.

The arrangement of the nucleosomes with respect to the DNA sequence has been examined in the genes coding for the major heat shock protein (hsp 70) in Drosophila. In the repressed state of the genes, the nucleosomes are precisely phased in at least three frames.     

A human gene family with sequence homology to Drosophila melanogaster Hsp70 heat shock genes

A growing literature describes the structure and regulation of prokaryotic and eukaryotic heat shock genes. We here report the isolation of several members of a human heat shock protein 70 (hsp 70) multigene family which contains at least 10 different genes and/or pseudogenes exhibiting sequence homology to the hsp70 gene of Drosophila melanogaster. Eight nonoverlapping recombinant lambda phages from a lambda-Charon4A human genomic library were studied by restriction mapping. One lambda clone was sequenced and characterized as a hsp70 pseudogene inserted into a rearranged human HindIII 1.9-kilobase repeated DNA sequence. This pseudogene is probably located on the X chromosome. Its predicted amino acid sequence shows extensive homology to those of Drosophila hsp70, trout hsp70, Xenopus hsp70, yeast hsp70, and some homology to the heat-inducible dnaK gene product of Escherichia coli. Amino acid homology is clustered, suggesting evolutionary conservation of domains critical to the function of this protein in both prokaryotic and eukaryotic cells.     

Degradation of hsp70 and other mRNAs in Drosophila via the 5' 3' pathway and its regulation by heat shock

Two general pathways of mRNA decay have been characterized in yeast. Both start with deadenylation. The major pathway then proceeds via cap hydrolysis and 5'-exonucleolytic degradation whereas the minor pathway consists of 3'-exonucleolytic decay followed by hydrolysis of the remaining cap structure. In higher eukaryotes, these pathways of mRNA decay are believed to be conserved but have not been well characterized. We have investigated the decay of the hsp70 mRNA in Drosophila Schneider cells. As shown by the use of reporter constructs, rapid deadenylation of this mRNA is directed by its 3'-untranslated region. The main deadenylase is the CCR4.NOT complex; the PAN nuclease makes a lesser contribution. Heat shock prevents deadenylation not only of the hsp70 but also of bulk mRNA. A completely deadenylated capped hsp70 mRNA decay intermediate accumulates transiently and is degraded via cap hydrolysis and 5'-decay. Thus, decapping is a slow step in the degradation pathway. Cap hydrolysis is also inhibited during heat shock. Degradation of reporter RNAs from the 3'-end became detectable only upon inhibition of 5'-decay and thus represents a minor decay pathway. Because two reporter RNAs and at least two endogenous mRNAs were degraded primarily from the 5'-end with cap hydrolysis as a slow step, this pathway appears to be of general importance for mRNA decay in Drosophila.     

Nucleotide sequences at the boundaries between gene and insertion regions in the rDNA of Drosophila melanogaster

Ribosomal RNA genes interrupted by type 1 insertions of 1 kb and 0.5 kb have been sequenced through the insertion region and compared with an uninterrupted gene. The 0.5 kb insertion is flanked by a duplication of a 14 bp segment that is present once in the uninterrupted gene; the 1 kb insertion is flanked by a duplication of 11 of these 14 bp. Short insertions are identical in their entire length to downstream regions of long insertions. No internal repeats occur in the insertion. The presence of target site duplications suggests that type 1 insertions arose by the introduction of transposable elements into rDNA. Short sequence homologies between the upstream ends of the insertions and the 28S' boundaries of the rRNA coding region suggest that short type 1 insertions may have arisen by recombination from longer insertions.We have sequenced both boundaries of two molecules containing type 2 insertions and the upstream boundary of a third; the points of interruption at the upstream boundary (28S' site) differ from each other in steps of 2 bp. Between the boundary in the 0.5 kb type 1 insertion and the type 2 boundaries there are distances of 74, 76, and 78 bp. At the downstream boundary (28S' site) the two sequenced type 2 insertions are identical. The rRNA coding region of one molecule extends across the insertion without deletion or duplication, but a 2 bp deletion in the RNA coding region is present in the second molecule. Stretches of 13 or 22 adenine residues occur at the downstream (28S') end of the two type 2 insertions.     

Effect of heat shock,pretreatment and hsp70 copy number on wing development in Drosophila melanogaster

Naturally occurring heat shock (HS) during pupation induces abnormal wing development in Drosophila; we examined factors affecting the severity of this induction. The proportion of HS-surviving adults with abnormal wings varied with HS duration and intensity, and with the pupal age or stage at HS administration. Pretreatment (PT), mild hyperthermia delivered before HS, usually protected development against HS. Gradual heating resembling natural thermal regimes also protected wing development against thermal disruption. Because of the roles of the wings in flight and courtship and in view of natural thermal regimes that Drosophila experience, both HS-induction of wing abnormalities and its abatement by PT may have marked effects on Drosophila fitness in nature. Because PT is associated with expression of heat-inducible molecular chaperones such as Hsp70 in Drosophila, we compared thermal disruption of wing development among hsp70 mutants as well as among strains naturally varying in Hsp70 levels. Contrary to expectations, lines or strains with increased Hsp70 levels were no more resistant to HS-disruption of wing development than counterparts with lower Hsp70 levels. In fact, wing development was more resistant to HS in hsp70 deletion strains than control strains. We suggest that, while high Hsp70 levels may aid cells in surviving hyperthermia, high levels may also overly stimulate or inhibit numerous signalling pathways involved in cell proliferation, maturation and programmed death, resulting in developmental failure.     

Heat shock and developmental regulation of the Drosophila melanogaster hsp83 gene.

In contrast to the hsp70 gene, whose expression is normally at a very low level and increases by more than 2 orders of magnitude during heat shock, the hsp83 gene in Drosophila melanogaster is expressed at high levels during normal development and increases only severalfold in response to heat shock. Developmental expression of the hsp83 gene consists of a high level of tissue-general, basal expression and a very high level of expression in ovaries. We identified regions upstream of the hsp83 gene that were required for its developmental and heat shock-induced expression by assaying beta-galactosidase activity and mRNA levels in transgenic animals containing a series of 5' deletion and insertion mutations of an hsp83-lacZ fusion gene. Deletion of sequences upstream of the overlapping array of a previously defined heat shock consensus (HSC) sequence eliminated both forms of developmental expression of the hsp83 gene. As a result, the hsp83 gene with this deletion mutation was regulated like that of the hsp70 gene. Moreover, an in vivo polymer competition assay revealed that the overlapping HSC sequences of the hsp83 gene and the nonoverlapping HSC sequences of the hsp70 gene had similar affinities for the factor required for heat induction of the two heat shock genes. We discuss the functional similarity of hsp70 and hsp83 heat shock regulation in terms of a revised view of the heat shock-regulatory sequence.     

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.     

DNA sequences flanking the starts of the hsp 70 and alpha beta heat shock genes are homologous

To determine whether ribosomes have a role in the postfertilization activation of protein synthesis in sea urchin eggs, we measured the translational activity of ribosomes isolated from unfertilized eggs and embryos of Strongylocentrotus purpuratus. Numerous previous studies have indicated few if any differences in the activity of such ribosomes. However, by using improved physiological isolation and in vitro conditions, we have found important differences in the activities of egg and embryo ribosomes. Ribosomes obtained from blastula polyribosomes were active in translating reticulocyte mRNA in a ribosome-dependent cell-free translation system, whereas ribosomes obtained from unfertilized eggs became fully active only after a characteristic, reproducible delay of up to 15 min at 26°C. The extent of this delay varied with incubation pH, but not with concentrations of K⁺, Mg²⁺, initiation factors, or mRNA. However, at incubation pH between 6.90 and 7.65, the egg ribosomes were always less active than blastula ribosomes.