RNA polymerase II is transcriptionally engaged but paused -25 nucleotides from the start site of the hsp70 gene of Drosophila melanogaster in uninduced (non-heat-shocked) flies. Here, we identify regions of the bsp70 promoter that are required for formation of this paused polymerase. Various hsplO promoter sequences are substituted for promoter sequences of a yolk protein gene, ypl, which, in males, is normally not expressed and has no paused polymerase. Run-on assays with nuclei of male transgenic flies are used to measure the level of paused polymerase on the hybrid genes. Sequences that reside upstream of the hsp70 TATA element, when fused upstream of the ypl TATA element, specify the formation of a paused polymerase on the 5' end of this hybrid gene. Within this region are multiple copies of the GAGA element, which is known to bind a constitutively expressed factor. This element appears to play a role in generating the pause. Also, in the absence of much of this upstream region, hspJO sequences in the vicinity of the transcriptional start and pause site participate in specifying the pause. Deletions of the pause site reduce the level of paused polymerase but do not lead to constitutive transcription. However, a connection between transcription and pausing is seen. The level of paused polymerase on the various hybrid hsp70-ypl promoters correlates with the promoter's potential to direct heat-induced transcription.[Key Words: RNA polymerase pausing; transcriptional regulation; heat shock genes; GAGA factor; promoter; Drosophila]Received November 1, 1991; revised version accepted December 23, 1991.Heat shock treatments of Drosophila cells trigger a rapid and dramatic increase in the transcription of the hsplO gene. This upsurge in transcription is detectable within several tens of seconds and the shift to the fully induced level of transcription is complete within 3 min (Belyaeva and Zhimulev 1975; T. O'Brien, E. Wong, and J.T. Lis, unpubl.). During this induction, the density of transcribing RNA polymerase II on the hspVO gene increases ~200-fold, as estimated from measurements of pulse-labeled transcripts (Lis et al. 1981b), amounts of cross-linking of RNA polymerase to the hsplO gene in vivo (Gilmour and Lis 1985), and quantification of transcripts produced by nuclear run-on assays (O'Brien and Lis 1991). The fully induced hsp70 gene is packed with approximately one to two polymerases per 100 bp (Corces et al. 1981).The hspJO promoter in uninduced cells appears poised for a rapid and intense change in transcription rate. The promoter is punctuated by nuclease hypersensitive sites located at known regulatory elements and also at the transcription start and leader regions (Wu 1980;Costlow and Lis 1984), RNA polymerase II is already present on the 5' end of the uninduced hspJO gene as detected both in intact cells by UV cross-linking (Gilmour and Lis 1986) and in nuclei by nuclear run-on assays (Rougvie and Lis 1988). Nuclear run-on assays demonstrate further that in uninduced Drosophila cells, this RNA polyme...

The accessory gland of male insects produces components of the seminal fluid that alter the behavior, physiology and life span of the mated female, and contribute to her efficient storage and utilization of sperm. As a step towards understanding how this occurs, we have isolated genes encoding 12 previously unreported accessory gland-specific mRNAs from the fruit fly Drosophila melanogaster. We report here the restriction maps of the new genes, the chromosome positions--which are all autosomal--of the 11 non-repetitive genes, their expression patterns, and the sequences of the accessory gland proteins (Acps) encoded by nine of the genes. Eight of the proteins predicted from these sequences begin with putative secretion signals. Following their signal sequences, three of the predicted molecules are peptides and the other five are larger polypeptides with characteristics of cleavable prohormones. The ninth molecule, which has an N-terminal hydrophobic region but no consensus signal peptide cleavage site, is predicted to be a 716 amino acid glycoprotein. Of the nine proteins, two have intriguing similarities to sequences in protein databases. Acp76A is a 388 amino acid pro-protein which contains a signature sequence for the serpin class of protease inhibitors. The 115 amino acid Acp62F has a 28 amino acid region of high sequence similarity to a neurotoxin of the Brazilian armed spider Phoneutria nigriventer. Models are discussed in which Acp76A plays a role in the observed regulation of Acp proteolysis and/or in the coagulation of seminal fluid to form a mating plug, and in which Acp62F contributes to the reported toxicity of Drosophila seminal fluid.

Evidence that one or more of the proteins synthesized in response to a sublethal heat shock (18,22,24,26) or other stressful conditions (21, 29) play some mechanistic role in allowing cells to subsequently survive at temperatures which would otherwise be lethal is compelling but circumstantial. Reports indicate that not all of the heat shock proteins (HSPs) need to be involved in this protection (4,7,23), and evidence has also been presented indicating that no HSP synthesis is necessary for the induction of this acquired thermotolerance (13). Whichever the case, the changes brought about in cells which permit survival at these otherwise lethal temperatures are unknown.One cellular function shown to be altered by heat shock in a variety of different cell types is translation (2,3,5,17,19,28,32,33). We therefore sought to determine whether a heat shock-induced change in the translational properties of the cell plays any role in permitting cells to subsequently survive at normally lethal temperatures. We examined the induction and utilization of HSP mRNAs in Tetrahymena thermophila cells transferred to a nonlethal, heat shock-inducing temperature (40°C) (10) and compared the results to those found in a similar experiment in which cells were transferred to a temperature (43°C) which was lethal but to which cells could be adapted by a prior heat shock. The results of these experiments strongly suggested that translational regulation was at least in part involved in the ability of cells to survive at 43°C. Earlier work has indicated that ribosomes are modified during heat shock (9, 31) and altered in their translational properties (32). We therefore treated cells in ways we knew from previous studies (14, 16) elicited changes in ribosome structure and function to determine whether such treatments had any effect on the ability of cells to survive a direct transfer from 30 to 43°C. We found conditions which induced acquired thermotolerance to 43°C * Corresponding author.as efficiently as a prior heat shock, but, surprisingly, unlike previous findings for other organisms (21, 29), HSP synthesis was not detectable during the induction of the acquiredthermotolerance state. The conditions which induced this acquired thermotolerance to 43°C, however, did not induce acquired thermotolerance to a 46°C treatment, a condition which can be induced by a prior heat shock. The results of our studies indicate that there are normally lethal temperatures to which T. thermophila can be pre-adapted with treatments that allow cells to efficiently translate heat shock mRNAs at these temperatures and thus survive. These treatments need not elicit the synthesis of any of the major HSPs prior to the shift to the lethal temperature. There are other (higher) lethal temperatures which can be withstood if and only if the accumulation of HSPs has occurred prior to the shift to these temperatures. MATERIALS AND METHODSCulture conditions. For all experiments, except where noted, we used a single strain of T. thermophila, CU355 (IV). We routinely gre...

Tetrahymena thermophila cells that had been shifted from log growth to a non-nutrient medium (60 mM Tris) were unable, during the first few hours of starvation, to mount a successful heat shock response and were killed by what should normally have been a nonlethal heat shock. An examination of the protein synthetic response of these short-starved cells during heat shock revealed that whereas they were able to initiate the synthesis of heat shock proteins, it was at a much reduced rate relative to controls and they quickly lost all capacity to synthesize any proteins. Certain pretreatments of cells, including a prior heat shock, abolished the heat shock inviability of these starved cells. Also, if cells were transferred to 10 mM Tris rather than 60 mM Tris, they were not killed by the same heat treatment. We found no abnormalities in either heat shock or non-heat shock mRNA metabolism in starved cells unable to survive a sublethal heat shock when compared with the response of those cells which can survive such a treatment. However, selective rRNA degradation occurred in the nonsurviving ceUs during the heat shock and this presumably accounted for their inviability. A prior heat shock administered to growing cells not only immunized them against the lethality of a heat shock while starved, but also prevented rRNA degradation from occurring.

No abstract

Tetrahymena thermophila cells that had been shifted from log growth to a non-nutrient medium (60 mM Tris) were unable, during the first few hours of starvation, to mount a successful heat shock response and were killed by what should normally have been a nonlethal heat shock. An examination of the protein synthetic response of these short-starved cells during heat shock revealed that whereas they were able to initiate the synthesis of heat shock proteins, it was at a much reduced rate relative to controls and they quickly lost all capacity to synthesize any proteins. Certain pretreatments of cells, including a prior heat shock, abolished the heat shock inviability of these starved cells. Also, if cells were transferred to 10 mM Tris rather than 60 mM Tris, they were not killed by the same heat treatment. We found no abnormalities in either heat shock or non-heat shock mRNA metabolism in starved cells unable to survive a sublethal heat shock when compared with the response of those cells which can survive such a treatment. However, selective rRNA degradation occurred in the nonsurviving cells during the heat shock and this presumably accounted for their inviability. A prior heat shock administered to growing cells not only immunized them against the lethality of a heat shock while starved, but also prevented rRNA degradation from occurring.

The sex of Drosophila melanogaster is determined by a hierarchy of genes. The ultimate targets of this regulatory hierarchy are the genes encoding terminal differentiation products of one sex. For one of the best-characterized target genes, that encoding female-specific yolk protein 1 (YP1), sex-specific transcriptional controls have been clearly demonstrated. In addition, sex-specific posttranscriptional controls were suggested from experiments in which YP1 RNA was induced in males with hormones. To determine whether males can efficiently process and translate a transcript which is normaily found only in females, we used a non-sex-specific promoter, the hsp7O gene promoter, to drive YP1 gene transcription in germ line transformed males. The efficiency of expression of the YP1 gene at levels of RNA splicing, translation, and protein secretion in these males was compared with that in wild-type females. These experiments show that there are no sex-specific posttranscriptional controls operating to limit the production of secreted YP1 in males. Promoters containing different numbers of heat shock elements were tested for their ability to drive YP1 gene transcription in males. These results show that incompatibility between the hsp7O gene heat shock elements and the YP1 gene promoter can be overcome by increasing the amount of hsp7O gene sequence up or downstream of the TATA box. In the course of this study, two vectors useful for placing genes under heat shock regulation were constructed. One of these vectors is designed so that the heat-induced transcript produced is the "authentic" primary transcript; it should be useful for studies of posttranscriptional regulation.In Drosophila melanogaster, yolk proteins are synthesized only in the adult female fat body and ovarian follicle cells (10, 38). The DNA sequences responsible for the sex-specific, tissue-specific, and stage-specific transcription of the yolk protein 1 (YP1) gene have been localized to the region between nucleotides -325 and -196 of the YP1 gene (16,17,50). Although transcriptional controls normally restrict YP1 gene expression to females, YP1 RNA synthesis can be forced in males by the administration of high levels of the steroid hormone ecdysone (9, 43). A study of the translational efficiency of this YP1 RNA in males suggested that sex-specific posttranscriptional controls exist that limit YP1 synthesis in males when YP1 RNA has been induced (9). Recently, other genes of D. melanogaster expressed sex specifically have been shown to be posttranscriptionally controlled (1,6,30). In the study presented here the efficiency of splicing the primary YP1 gene transcript, its stability, translation, and the secretion of YP1 are examined in males in which levels of YP1 RNA comparable to those in females have been induced by a means that does not require the administration of high levels of ecdysone.To release the YP1 gene from sex-specific transcriptional controls, it was fused to the heat-inducible promoter of the hsp7O gene of D. melanogaster. The hsp7O ge...

When Tetrahymena thermophila cells growing at 30 degrees C are shifted to either 40 or 43 degrees C, the kinetics and extent of induction of heat shock mRNAs in both cases are virtually indistinguishable. However, the cells shifted to 40 degrees C show a typical induction of heat shock protein (HSP) synthesis and survive indefinitely (100% after 24 h), whereas those at 43 degrees C show an abortive synthesis of HSPs and die (less than 0.01% survivors) within 1 h. Cells treated at 30 degrees C with the drugs cycloheximide or emetine, at concentrations which are initially inhibitory to protein synthesis and cell growth but from which cells can eventually recover and resume growth, are after this recovery able to survive a direct shift from 30 to 43 degrees C (ca. 70% survival after 1 h). This induction of thermotolerance by these drugs is as efficient in providing thermoprotection to cells as is a prior sublethal heat treatment which elicits the synthesis of HSPs. However, during the period when drug-treated cells recover their protein synthesis ability and simultaneously acquire the ability to subsequently survive a shift to 43 degrees C, none of the major HSPs are synthesized. The ability to survive a 1-h, 43 degrees C heat treatment, therefore, does not absolutely require the prior synthesis of HSPs. But, as extended survival at 43 degrees Celsius depends absolutely on the ability of cells to continually synthesize HSPs, it appears that a prior heat shock as well as the recovery from protein synthesis inhibition elicits a change in the protein synthetic machinery which allows the translation of HSP mRNAs at what would otherwise be a nonpermissive temperature for protein synthesis.