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

Karch, François; Török, I.; Tissières, A.

doi:10.1016/0022-2836(81)90536-2

Cited by 163 publications

(77 citation statements)

References 26 publications

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“…The promoters of the genes involved in this study (19,20,31) have at least three sequence elements in common: TATA boxes, GAGA elements, and heat shock elements. Footprinting studies on uninduced hsp70 and hsp26 genes indicate the TATA box (13,32,33) and GAGA elements (ref.…”

Section: Resultsmentioning

confidence: 99%

In vivo transcriptional pausing and cap formation on three Drosophila heat shock genes.

Rasmussen

Lis

1993

Proc. Natl. Acad. Sci. U.S.A.

322

278

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The regulation of many eukaryotic genes occurs at the level of transcriptional elongation. On the uninduced hsp7O gene of Drosophila melanogaster, for example, an RNA polymerase I complex has initiated transcription but has paused early in elongation. In this study, we examine pausing on hsp7O and two of the small heat shock genes (hsp27 and hsp26) at high resolution, using a technique that utilizes paramagnetic particle-mediated selection of terminated run-on transcripts. This technique provides precise information on the distribution ofRNA polymerase within each transcription unit. It also details the progression of 5' cap formation on the elongating transcripts. For each gene, we find polymerases paused over a relatively narrow promoter-proximal region. The regions are generally around 20 nucleotides wide, with two preferred pausing positions spaced roughly 10 nucleotides apart or about one turn of the helix. The bulk ofcapping occurs as transcripts pass between 20 and 30 nucleotides in length. Interestingly, in the three genes examined here, elongational pausing and 5' cap formation appear largely coincident.The transcription of eukaryotic genes is a complex, multistep process. Often, its regulation is assumed to lie mainly in the initiation event, but regulation could potentially be accomplished at any of the numerous steps leading to a full-length transcript: from the binding of RNA polymerase and its accessory factors, through initiation and elongation, to proper termination. For many genes, the control oftranscription occurs after initiation. A blockage or pause during elongation prevents formation of the full-length message; relief of that blockage is required for high-level expression of the gene. This type of transcriptional control has been found in a variety of eukaryotic genes and organisms, including human immunodeficiency virus type 1(1), human and murine c-myc (2-4) and ADA (5, 6), and Drosophila hsp7O (7). (See ref. 8 for a review.)Genes that utilize elongational blockage for transcriptional control have as a result a greater density of RNA polymerase on their 5' ends than on latter regions. Several genes in Drosophila melanogaster have been identified that show such a polymerase distribution. These include heat-inducible (hsp7O, hsp26), metabolic (Gapdh-1, Gapdh-2), and structural genes (f31-Tubulin) (9); thus, the phenomenon appears relatively general. The most thoroughly studied of these is hsp7O. In the uninduced state, it has roughly one RNA polymerase II complex present in the promoter-proximal region of each gene (10,11). This polymerase has initiated transcription but has paused early in the elongational process (7). Upon induction by heat shock, the rate of escape of these paused polymerases into elongation rises dramatically, leading to a >100-fold increase in hsp7O transcription (12). Interestingly, elongational pausing remains the rate-limitingThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "adv...

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Section: Resultsmentioning

confidence: 99%

In vivo transcriptional pausing and cap formation on three Drosophila heat shock genes.

Rasmussen

Lis

1993

Proc. Natl. Acad. Sci. U.S.A.

322

278

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“…DNA sequence data that include 5' flanking regions are available for the hsp70 (14,40), hsp68 (12), and hsp22, hsp23, hsp26, and hsp27 (13,41) genes of D. melanogaster. Because the hsp70 and hsp68 genes are very similar and probably reflect a relatively recent duplication event (2), we have omitted the hsp68 gene in the figures, though it shows striking homology to hsp70 in the region from -42 to +30 (12).…”

Section: Resultsmentioning

confidence: 99%

Localization of the hsp83 transcript within a 3292 nucleotide sequence from the 63B heat shock locus ofD. melanogaster

Hackett¹,

Lis²

1983

Nucl Acids Res

109

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We have determined the complete nucleotide sequence of a 3292 bp cloned segment derived from the 63B heat shock cytogenetic locus of D. melanogaster. Within this segment we have positioned the start of transcription and RNA splice sites of the unique gene that encodes the 83,000 d heat shock polypeptide (hsp83 gene) by SI mapping and synthesis of cDNA from restriction fragment primed mRNA. The sequence begins at a point 879 bp upstream from the transcription start and includes the 149 bp nontranslated first exon, the 1139 bp intron and extends 1125 bp into the protein coding region. These data identify a single open translation reading frame for the first 375 amino acids of the 83,000 d polypeptide, beginning with the first ATG codon located at the 3' intron-exon junction. We discuss and demonstrate the use of E. coli exonuclease III generated single-strand DNA probes as an alternative to strand separation for S1 mapping of mRNA. We also use homology search criteria based upon known protein-DNA binding sites to compare our hsp83 sequence with other sequenced Drosophila heat shock genes. These comparisons indicate that a large region of approximately 80 bp centered around the transcription initiation point of the hsp83 gene shares only a 31% homology with the corresponding region of the hsp70 gene, whereas the hsp22, 23, 26, and 27 genes share a 54% homology with hsp70 in this region. The lower homology of the hsp83 gene is consistent with the deviant nature of this heat shock gene.

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“…In noninduced cells, a very low, but detectable, level of RNA polymerase II cross-links to the 0.97-kb fragment (Fig. 2B, NHS, hybridizing doublet is a minor product of partial XhoI digestion beginning at the +65 PvuII site and extending upstream to a second XhoI site [18].) In contrast, a high level of RNA polymerase II cross-links to the 0.97-kb fragment in heat shock-induced cells (Fig.…”

Section: Methodsmentioning

confidence: 93%

RNA polymerase II interacts with the promoter region of the noninduced hsp70 gene in Drosophila melanogaster cells.

Gilmour¹,

Lis²

1986

Mol. Cell. Biol.

307

222

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By using a protein-DNA cross-linking method (D. S. Gilmour and J. T. Lis, Mol. Cell. Biol. 5:2009Biol. 5: -2018Biol. 5: , 1985, we examined the in vivo distribution of RNA polymerase II on the hsp7O heat shock gene in Drosophila melanogaster Schneider line 2 cells. In heat shock-induced cells, a high level of RNA polymerase II was detected on the entire gene, while in noninduced cells, the RNA polymerase II was confined to the 5' end of the hsp7O gene, predominantly between nucleotides -12 and +65 relative to the start of transcription. This association of RNA polymerase II was apparent whether the cross-linking was performed by a 10-min U irradiation of chilled cells with mercury vapor lamps or by a 40-ps irradiation of cells with a high-energy xenon flash lamp.We hypothesize that RNA polymerase II has access to, and a high affinity for, the promoter region of this gene before induction, and this poised RNA polymerase II may be critical in the mechanism of transcription activation.The Drosophila melanogaster hsp7O promoter region is structurally complex even in noninduced cells. Nucleasehypersensitive regions include an interval upstream of the transcription start, as well as an interval immediately downstream of the transcription start, which has a peak of sensitivity at position +42 (8, 32, 33). These features indicate the absence of standard nucleosomes and suggest the presence of nonhistone proteins (10, 11). Wu (33) discovered that a barrier to exonuclease III digestion exists in the -40 to -12 interval of the hsp7O promoter in the nuclei of noninduced cells. This barrier may be the result of a TATA box-binding protein, which has been partially purified and characterized by in vitro binding assays (25). Inspection of the pattern of exonuclease digestion (33) reveals another barrier 65 base pairs downstream of the transcription initiation site. Thus, the start of the hsp7O transcription unit may also be associated with protein. In the present paper, we discuss evidence obtained with a protein-DNA cross-linking method (13-15) which leads us to conclude that RNA polymerase II is part of this promoter complex in noninduced cells. MATERIALS AND METHODSThe protein-DNA cross-linking method was executed as previously described by Gilmour and Lis (14) for the experiments presented in Fig. 1 and 2. Antiserum against the 215,000-dalton subunit of RNA polymerase 11 (30) was used to recover RNA polymerase II-DNA adducts, and antiserum against Escherichia coli RNA polymerase (13) was used to assess nonspecific background. Experiments in Fig. 3 and 4 had the following modifications. Ten-milliliter portions of Schneider line 2 cells (26) were transferred from a spinner flask to a rectangular, siliconized glass dish (6 by 8 cm) and irradiated for either four flashes separated by 1-s intervals or a single flash from a Xenon lamp (model N734; Xenon Corp.). For the experiment with four flashes, the lamp was positioned 10 cm above the cells, whereas for the single-flash Xenon model 457 micropulser set at 7,000 V. A trigger wi...

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

Cited by 163 publications

References 26 publications

In vivo transcriptional pausing and cap formation on three Drosophila heat shock genes.

In vivo transcriptional pausing and cap formation on three Drosophila heat shock genes.

Localization of the hsp83 transcript within a 3292 nucleotide sequence from the 63B heat shock locus ofD. melanogaster

RNA polymerase II interacts with the promoter region of the noninduced hsp70 gene in Drosophila melanogaster cells.

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