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

The transcriptional regulation of the Drosophila melanogaster hsp27 (also called hsp28) gene was studied by introducing altered genes into the germ line by P element-mediated transformation. DNA sequences upstream of the gene were defined with respect to their effect on steroid hormone-induced and heat-induced transcription. These two types of control were found to be separable; the sequences responsible for 80% of heat-induced expression were located more than 1.1 kilobases upstream of the RNA initiation site, while the sequences responsible for the majority of ecdysterone induction were positioned downstream of the site at -227 base pairs. We have determined the DNA sequence of the intergenic region separating hsp23 and hsp27 and have located putative heat shock and ecdysterone consensus sequences. Our results indicate that the heat shock promoter of the hsp27 gene is organized quite differently from that of hsp7O.The expression of eucaryotic genes is regulated in a temporal, spatial, and quantitative fashion during the development of an organism. Discernment of the mechanisms underlying these different levels of control is essential for the understanding of the molecular basis of differentiation and the maintenance of the differentiated state. An important step in defining the mechanisms of gene regulation is the elucidation of the DNA sequences that are responsible for mediating the transcriptional activity of genes.Heat shock, the ubiquitous response of all organisms to a sublethal temperature elevation, has been studied extensively in both Drosophila melanogaster and Escherichia coli (see references 36 and 47 for a review) and constitutes an ideal model system in which to study the molecular basis of gene regulation in eucaryotes. In D. melanogaster, the response involves the abundant production of seven proteins, the heat shock proteins, within minutes of a temperature shift from the normal ambient growth temperature of 18 to 25°C to the elevated temperature level of 35 to 37°C. This response involves both the transcriptional activation of the genes encoding these proteins and the preferential translation of the corresponding mRNAs, while nearly all transcriptional and translational activity of non-heat shock genes is shut off. The molecular analysis of these genes has shown that they do not contain introns (with the exception of hsp83) and are clustered within five cytogenetic loci on chromosome 3. Although the biochemical function of the heat shock proteins remains obscure, it is known that they are vital for protection against heat and other potentially lethal physiological stresses. The recent advances in the understanding of the biochemistry of the bacterial heat shock genes, however, will undoubtedly shed light on the function of the eucaryotic heat shock genes as well (36).Subsets of the D. melanogaster heat shock genes have been shown to be expressed at high levels during specific stages of development, in the absence of a heat shock (28,55). hsp22, hsp23, hsp26, and hsp27 (also called hsp28) can ...

Section: Resultsmentioning

confidence: 77%

mentioning

confidence: 99%

Sequences involved in temperature and ecdysterone-induced transcription are located in separate regions of a Drosophila melanogaster heat shock gene.

Hoffman¹,

Corcés²

1986

“…Previous experiments that have employed transfection of the small heat shock genes of Drosophila into monkey COS cells revealed that the transfected hsp 28 genes were either uninducible or inducible to only very low levels by heat treatment; hsp 23 was constitutive at low levels (3,39 Fig. 8; Table 1).…”

Section: Reagentsmentioning

confidence: 98%

Nucleosomal instability and induction of new upstream protein-DNA associations accompany activation of four small heat shock protein genes in Drosophila melanogaster.

Cartwright¹,

Elgin²

1986

We investigated in detail the structural changes that occur in nuclear chromatin upon activation of the four small heat shock protein genes in D. melanogaster. Both -the chemical cleavage reagent methidiumpropyl-EDTA-iron(II) [MPE Fe(II)] and the nuclease DNase I revealed a complex pattern of four or five hypersensitive sites upstream of each gene before activation. In addition, MPE-Fe(II) detected a short positioned array of nucleosomes located on each coding region. Upon heat shock activation a number of changes in the patterns occurred. For each gene, at least one of the upstream hypersensitive regions was eliminated or substantially shifted in position. Regions were established which became highly refractile to digestion by either MPE -Fe(II) or DNase I and, as such, appeared as small "footprints" in the pattern. The location of these refractile regions relative to the cap site varied for each gene examined. The coding regions themselves became highly accessible to DNase I. The nucleosomal arrays detected by MPE-Fe(II) were characterized by a considerable loss of detail and significantly enhanced accessibility, the extent of which probably reflected the relative transcription rate of each gene. Careful mapping of the location and extent of each upstream footprint and comparison with the DNA sequence revealed the presence at each location of two (or more) contiguous or overlapping segments that bear high homology to the heat shock consensus sequence C-T-N-G-A-A-N-N-T-T-C-N-A-G. A specific protein factor (or factors) is most likely bound at or near these sequences in heat-shocked Drosophila cells.

“…However, the basic mechanisms of HSP gene induction have been extremely well conserved over long periods of evolution. As a result, HSP genes from a variety of organisms have been faithfully expressed in very distantly related hosts, e.g., Drosophila hsp7O and the small HSP genes in mammalian cells (4,8,13,33,37) and Dictyostelium heat shock-inducible genes in Saccharomyces cerevisiae (9). Therefore, we have been able to use a mouse fibroblast cell line (C127) to express the C. elegans hspl6 genes.…”

mentioning

confidence: 99%

Efficient transcription of a Caenorhabditis elegans heat shock gene pair in mouse fibroblasts is dependent on multiple promoter elements which can function bidirectionally.

Kay¹,

Boissy²,

Russnak³

et al. 1986

A divergently transcribed pair of Caenorhabditis elegans hspl6 genes was introduced into mouse fibroblasts by stable transfection with vectors containing bovine papillomavirus plasmid maintenance sequences and a selectable gene. The hspl6 genes were transcriptionally inactive in the mouse cells under normal growth conditions and were strongly induced by heat shock or arsenite. In a cell line with 12 copies of the gene pair, there were estimated to be more than 10,000 hspl6 transcripts in each cell after 2 h of heat shock treatment. The hspl6 transcript levels were more than 100 times higher than those of a gene with a herpes simplex virus thymidine kinase gene promoter carried on the same vector. A single heat shock promoter element (HSE) could activate bidirectional transcription of the two hspl6 genes when placed between the two TATA elements, but the transcriptional efficiency was reduced 10-fold relative to that of the wild-type gene pair. Four overlapping HSEs positioned between the two TATA elements resulted in inducible bidirectional transcription at greater than wild-type levels. The number of HSEs can therefore be a major determinant of the promoter strength of heat-inducible genes in mammalian cells. Partial disruption of an alternating purine-pyrimidine sequence between the two hspl6 genes had no significant effect on their transcriptional activity.