Transcriptional regulation in Drosophila during heat shock: a nuclear run-on analysis.

We used a nuclear run-on assay as a novel approach to study the changes in transcriptional activity that take place in Drosophila melanogaster during heat shock. In response to a rapid temperature upshift, total transcriptional activity in cultured KC161 cells decreased proportionally to the severity of the shock. After extended stress at 37 degrees C (15 min or more), transcription was severely reduced, and at 39 degrees C most transcription was instantaneously arrested. However, strikingly different responses were observed for individual genes. Transcription of histone H1 genes was severely inhibited even under mild heat shock conditions. Transcription of the actin 5C gene decreased progressively with increasing temperature, while transcription of the core histone genes or of the heat shock cognate genes was repressed only under severe heat shock conditions. Transcriptional activation of the D. melanogaster heat shock genes was also investigated. In unshocked cells, hsp84 was moderately transcribed, while transcriptional activity at the other protein-coding heat shock genes was undetectable (less than 0.2 polymerases per gene). Engaged but paused RNA polymerase molecules were found at the hsp70 and hsp26 genes, but not at the other heat shock genes. The rates of transcription increased with increasing temperature with a peak of expression at around 35 degrees C. At 37 degrees C, induction was less efficient, and no induction was achieved after a rapid shift to 39 degrees C. Increased transcription of the heat shock genes was observed within 1-2 min of heat shock, and maximal rates were reached within 2-5 min. Despite very similar profiles of response, different heat shock genes were transcribed at strikingly different rates, which varied over a 20-fold range. The noncoding heat shock locus 93D was transcribed at a very high rate under non-heat shock conditions, and showed a transcriptional response to elevated temperatures different from that of protein-coding heat shock genes. An estimation of the absolute rates of transcription at different temperatures was obtained.

Heat shock response in Drosophila.

Major alterations in genetic activity have been observed in every organism after exposure to abnormally high temperatures. This phenomenon, called the heat shock response, was discovered in the fruit fly Drosophila. Studies with this organism led to the discovery of the heat shock proteins, whose genes were among the first eukaryotic genes to be cloned. Several of the most important aspects of the regulation of the heat shock response and of the functions of the heat shock proteins have been unraveled in Drosophila.

Tissue-specific expression of the heat shock protein HSP27 during Drosophila melanogaster development.

The alpha-crystallin-related heat shock (stress) protein hsp27 is expressed in absence of heat shock during Drosophila melanogaster development. Here, we describe the tissue distribution of this protein using an immunoaffinity-purified antibody. In embryos, hsp27 translated from maternal RNA is uniformly distributed, except in the yolk. During the first, second, and early third larval stages, hsp27 expression is restricted to the brain and the gonads. These tissues are characterized by a high level of proliferating cells. In late third instar larvae and early pupae, in addition to the central nervous system and the gonads, all the imaginal discs synthesize hsp27. The disc expression seems restricted to the beginning of their differentiation since it disappears during the second half of the pupal stage: no more hsp27 is observed in the disc-derived adult organs. In adults, hsp27 is still present in some regions of the central nervous system, and is also expressed in the male and female germ lines where it accumulates in mature sperm and oocytes. The transcript and the protein accumulate in oocytes since the onset of vitellogenesis with a uniform distribution similar to that found in embryos. The adult germ lines transcribe hsp27 gene while no transcript is detected in the late pupal and adult brain. These results suggest multiple roles of hsp27 during Drosophila development which may be related to both the proliferative and differentiated states of the tissues.

Expression of the small heat shock genes during Drosophila development: comparison of the accumulation of hsp23 and hsp27 mRNAs and polypeptides.

Seven heat shock genes are clustered within 15 kilobases of DNA at the Drosophila melanogaster chromosomal site 67B. They show a complex pattern of expression in the absence of external stress during normal development of this organism. In this paper, we quantitatively compare the abundance of the messenger RNAs for these seven genes at all major stages of Drosophila development and then focus on hsp23 and hsp27 for which available antibodies allow the comparison between the accumulation of the mRNAs and that of their corresponding polypeptides. Transcripts for both genes are maximally abundant in white prepupae. We observe that the amount of hsp23 message decreases more rapidly than that of hsp27 mRNA throughout the pupal period. The maximal abundance of the proteins occurs at the middle of the pupal stage, when their corresponding RNAs have almost completely disappeared. The peaks of expression of the proteins are also broader than those of their transcripts, indicating that the half-lives of the polypeptides are longer. These observations suggest that complex mechanisms regulate the expression of the small heat shock genes during Drosophila development.

Several hundred base pairs upstream of Drosophila hsp23 and 26 genes are required for their heat induction in transformed flies.

We have used the P-element-mediated transformation of Drosophila germ line to study the 5' DNA sequences involved in the thermal inducibility of the genes for heat shock proteins hsp23 and 26. The results are strikingly different from those previously obtained in heterologous systems. For hsp23, each successive shortening of the promoter region from 618 to 402, 321 and 263 bp clearly decreased the expression. A construct with only 149 bp was not inducible at all. For hsp26, all the regulatory elements appear to be clustered in the first 350 bp upstream from the cap site. Clones with 171 bp showed a 4- to 10-fold decrease in induction depending on the transformed line, and those with only 52 bp were not expressed. The results suggest that at least three Pelham consensus sequences are required for the full expression of these two genes. The direct involvement of one of these consensus sequences has been assessed: a 6-bp deletion within the proximal element of the hsp26 gene strongly reduced its inducibility. Our results also indicate that X-linked hsp genes exhibit either partial dosage compensation or none at all.

Locus 67B of Drosophila melanogaster contains seven, not four, closely related heat shock genes.

The four small hsp genes of Drosophila melanogaster as well as three genes regulated during development (genes 1, 2 and 3) are localized at the chromosomal locus 67B. The four small hsp genes share strong sequence homologies between themselves which were detected here by cross-hybridization. Under the same stringency conditions, each of the genes 1, 2 and 3 hybridize to some of the small hsp genes. By DNA sequencing of gene 1, the homology was localized within the same two regions already conserved between the small hsp genes: a central region of 83 amino acids, homologous with the mammalian alpha crystallin and the first 15 N-terminal amino acids. The transcriptional inducibility of the genes 1, 2 and 3 was also compared with that of the four small hsp genes during various stages of Drosophila development at either the normal growth temperature or after a heat shock. We confirm previous reports on the developmental patterns of all seven genes and find moreover that genes 1, 2 and 3 are heat-shock inducible at any of the stages tested. We conclude that genes 1, 2 and 3 are also heat shock genes. Therefore, the locus 67B contains seven, not four, small heat shock genes.

Nucleotide sequences responsible for the thermal inducibility of the Drosophila small heat-shock protein genes in monkey COS cells.

The promoter regions of the Drosophila melanogaster small heat-shock protein genes have been analysed in order to localize those sequences responsible for their heat-shock transcriptional inducibility. Different lengths of the 5' DNA sequences of these four genes were each fused individually to the Herpes simplex virus thymidine kinase (HSV-tk) transcription unit. These hybrid genes were constructed in a simian virus 40 recombinant vector for transfection in permissive monkey COS cells and tested for their heat-shock inducibility. The hsp22/HSV-tk and hsp26/HSV-tk fusion genes were found to be heat-inducible at 43 degrees C, giving rise to correctly initiated transcripts, but transcriptionally quiescent at 37 degrees C (control temperature). The hsp23 and hsp27 fusion gene constructs are, however, not heat-shock-inducible; no transcripts being detectable from hsp27/HSV-tk constructs at either temperature and all hsp23/HSV-tk clones being faithfully but constitutively expressed at low levels at both temperatures. By testing a series of 5' deletion mutants in hsp22/HSV-tk, a homologous sequence located adjacent to the TATA box in both the hsp22 and hsp26 genes was identified as being responsible for their heat-shock activation. This control element corresponds to the Pelham "consensus sequence", previously described for the Drosophila hsp70 genes. The possible modes of transcriptional induction of all four genes are discussed.

A DNA segment isolated from chromosomal site 67B in D. melanogaster contains four closely linked heat-shock genes.

cDNA clones coding for two different small heat-shock polypeptides were isolated. Both clones hybridize exclusively to the heat-shock puff site 67B, and restriction mapping of embryonic Drosophila melanogaster DNA showed that the two genes probably occur as single copies and are closely linked. The analysis was extended by isolating genomic clones, which contain these genes and two additional ones. The four different genes code for heat-induced poly(A)+ RNAs. These genes are clustered within an 11 kb segment and are separated by spacers of 1.0-4.7 kb. Three of the genes were found to exhibit alternating polarities. Thus in spite of their close linkage, the four heat-induced genes are most likely organized in individual transcription units.

Physical map of two D. melanogaster DNA segments containing sequences coding for the 70,000 dalton heat shock protein.

The isolation of the two hybrid plasmids 56H8 and 132E3, which contain D. melanogaster (Dm) DNA sequences complementary to the mRNA coding for the 70,000 dalton heat shock protein, has been reported (Schedl et al., 1978). Here we compare the sequence arrangement in the two cloned Dm DNA segments by restriction, cross-hybridization and heteroduplex analysis. The results show that the two cloned DNA segments derive from nonoverlapping regions of the Dm genome; that they contain homologous regions present once in 56H8 and twice in 132E3; and that each homologous region is composed of three distinct contiguous sequence elements, x, y and z, which together define a 3 kb common unit. While the 2.5 kb z elements show a high degree of sequence homology in all three common units, the three x and y elements display an intriguing relationship. The localization of the mRNA coding sequences within each of these common units is presented in the accompanying paper (Artavanis-Tsakonas et al., 1979).

Two hybrid plasmids with D. melanogaster DNA sequences complementary to mRNA coding for the major heat shock protein.

The isolation and partial characterization of two cloned segments of Drosophila melanogaster DNA containing "heat shock" gene sequences is described. We have inserted sheared embryonic D. melanogaster DNA by the poly(dA-dt) connector method (Lobban and Kaiser, 1973) into the R1 restriction site of the ampicillin-resistant plasmid pSF2124 (So, Gill and Falkow, 1975). A collection of independent hybrid plasmids was screened by colony hybridization (Grunstein and Hogness, 1975) for sequences complementary to in vitro labeled polysomal poly(A)+ heat shock RNA. Two clones were identified which contain sequences complementary to a heat shock mRNA species that directs the in vitro synthesis of the 70,000 dalton heat-induced polypeptide. Both cloned segments hybridize in situ to the heat-induced puff sites located at 87A and 87C of the salivary gland polytene chromosomes.

Heat shock of Drosophila melanogaster induces the synthesis of new messenger RNAs and proteins.

The heat shock proteins, labelled in vivo with [35S]methionine, were separated by sodium dodecylsulphate-polyacrylamide gel electrophoresis and fingerprinted after tryptic digestion. Eight distinct heat shock polypeptides are characterized in this way. Heat shock messenger RNAs were isolated and partially purified. Assayed in vitro for protein synthesis, they were found to code for heat shock polypeptides. Some parameters of the kinetics of in vivo synthesis of the heat shock proteins are presented.