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Showing papers on "Upstream activating sequence published in 1982"


Journal ArticleDOI
TL;DR: Experiments employing strains inducible (GAL80) or constitutive (gal80) for GAL10 expression indicate that an additional component of glucose repression is inducer exclusion.
Abstract: We have identified the promoter region of the GAL10 gene (whose product is UDP-galactose epimerase) of Saccharomyces cerevisiae; this promoter mediates galactose induction of transcription in conjunction with the product of the GAL4 regulatory gene. This identification was achieved by excising a 365-base-pair fragment of GAL10 leader DNA with a GAL10 proximal endpoint greater than 100 base pairs upstream of the transcriptional start site and substituting it in place of the upstream activation site of the CYC1 (iso-1-cytochrome c) promoter [Guarente, L. & Ptashne, M. (1981) Proc. Natl. Acad. Sci. USA 78, 2199-2203]. The hybrid promoter is composed of DNA encoding CYC1 mRNA start sites and the GAL segment upstream of these sites. This promoter is regulated in a manner analogous to GAL10; i.e., it is induced by galactose and responds to mutations in the GAL4 and GAL80 regulatory loci. The activity of the hybrid promoter requires sequences in the region of the CYC1 mRNA start sites but does not require a precise spacing between these sequences and the GAL segment. The transposed GAL segment appears not to contain sequences that mediate glucose repression. Thus, the picture of the GAL10 promoter that emerges is one of an upstream activation site that responds to the GAL4 product plus galactose, and a region of transcription initiation that may contain sequences that mediate glucose repression. Experiments employing strains inducible (GAL80) or constitutive (gal80) for GAL10 expression indicate that an additional component of glucose repression is inducer exclusion.

506 citations


Journal ArticleDOI
TL;DR: The transcriptional heat‐shock response is mediated by some factor that interacts with this sequence, and sequences similar to the consensus CT‐GAA‐TTC‐AG from synthetic oligonucleotides are constructed upstream of the TATA box of the herpes virus thymidine kinase gene, in place of the normal upstream promoter element.
Abstract: Previous deletion analysis of the Drosophila hsp70 heat-shock promoter has identified a sequence upstream of the TATA box that is required for heat induction. This region contains homology to other heat-shock promoters, and it was proposed that the common sequence is an important element in the regulation of the heat-shock genes. We have constructed sequences similar to the consensus CT-GAA-TTC-AG from synthetic oligonucleotides and placed them upstream of the TATA box of the herpes virus thymidine kinase gene, in place of the normal upstream promoter element. The resultant genes are heat-inducible both in monkey COS cells and in Xenopus oocytes. We conclude that the transcriptional heat-shock response is mediated by some factor that interacts with this sequence.

281 citations


Journal ArticleDOI
TL;DR: It is shown that sequences located upstream from the T-A-T-A box, between positions -97 and -34, are necessary for efficient in vivo transcription from the adenovirus serotype 2 major late promoter.
Abstract: We show that sequences located upstream from the T-A-T-A box, between positions -97 and -34, are necessary for efficient in vivo transcription from the adenovirus serotype 2 major late promoter. The effect of these upstream sequences was also investigated in vitro using a whole cell or an S100 extract and circular or linear templates. With the whole cell extract, the in vivo effect of the upstream sequences was reproduced in vitro. With the S100 extract, some effect of the upstream sequences was observed with circular, but not with linear, templates.

124 citations


Journal ArticleDOI
TL;DR: It is concluded that sequences between -- 34 and -- 12, upstream from the transcribed region, represent an essential control region for the initiation of transcription in vitro and may be functionally analogous to the T-A-T-A box of RNA polymerase II promoters.
Abstract: The nucleotide sequence(s) specifying RNA polymerase I initiation has been investigated by studying the transcription of deleted and nondeleted mouse ribosomal RNA gene (rDNA) templates in vitro. The deletion of 5'-flanking sequences upstream from position -- 39 did not affect transcriptional activity, but removal of sequences between positions -- 39 and -- 34 resulted in a 90% decrease of rDNA transcription. The template activity was completely eliminated by the further deletion of nucleotides -- 33 to -- 13. It is concluded that sequences between -- 34 and -- 12, upstream from the transcribed region, represent an essential control region for the initiation of transcription in vitro. Therefore, this region may be functionally analogous to the T-A-T-A box of RNA polymerase II promoters. In addition to this control region, sequences located further upstream (between positions -- 45 and -- 169) may also exert some function in efficient transcription initiation as revealed by competition experiments between wildtype and mutant rDNA templates.

87 citations


Journal ArticleDOI
TL;DR: Sea urchin (psammechinus miliaris) H2A histone genes shown to be promoter mutants from oocyte injection experiments were tested for their ability to initiate transcription in vitro, finding that the effects of these sequences could be mimicked by free DNA ends, suggesting that the function of this in vitro upstream sequence might be to provide an entry side for RNA polymerase II.
Abstract: Sea urchin (psammechinus miliaris) H2A histone genes shown to be promoter mutants from oocyte injection experiments were tested for their ability to initiate transcription in vitro. Circular templates were transcribed with HeLa cell extracts, and the transcripts were assayed by mung bean or S1 nuclease mapping of the 5' ends. The transcripts of the H2A mutants produced in vitro were qualitatively similar and, in most cases, identical to those seen in oocyte injection experiments, but quite large quantitative differences were observed for some H2A mutant genes. Both the T-A-T-A box and far upstream sequences residing in the modulator segment E [Grosschedl, R. & Birnstiel, M. L. (1980) Proc. Natl. Acad. Sci. USA 77, 7102--7106] were found to be essential for maximal transcription in vitro. Deletion of either of these sequence elements reduced transcription to 20%. A similar reduction in the amount of H2a transcripts was found when a T-A-T-A-to-T-A-G-A point mutant was tested in vitro. Essential far upstream sequences were mapped between nucleotides -139 and -111, 5' to the initiation site of transcription. In the standard run-off transcription test using restriction fragments, the effects of these sequences could be mimicked by free DNA ends, suggesting that the function of this in vitro upstream sequence might be to provide an entry side for RNA polymerase II.

71 citations


Journal ArticleDOI
TL;DR: A 505-nucleotide long DNA sequence of a part of the gene cluster for the proton-translocating ATPase ( pap operon) of E. coli was determined and identified as an active promoter of a gene coding for a small RNA of unknown function adjacent to the operon.

41 citations


Journal ArticleDOI
TL;DR: The results suggest that it is the T-A-T-A box sequence which plays a role in the efficient initiation of RNA transcription in vitro and further supports the implication that this region may serve a promoter-related function in eucaryotic transcription.

39 citations


Journal ArticleDOI
TL;DR: The DNA sequences required for promoter function have been identified by using 5′ deletion mutants of the fusion gene AT which contains the 5′‐flanking sequence and capping site of the human corticotropin‐beta‐lipotropin precursor gene and the structural sequence of the herpes simplex virus thymidine kinase gene.
Abstract: The cloned human corticotropin-beta-lipotropin precursor gene, when joined with an SV40 vector and introduced into COS monkey cells, is transcribed from its own promoter. The DNA sequences required for promoter function have been identified by using 5' deletion mutants of the fusion gene AT which contains the 5'-flanking sequence and capping site of the human corticotropin-beta-lipotropin precursor gene and the structural sequence of the herpes simplex virus thymidine kinase gene. The deletion of the sequence located between 53 and 59 bp upstream of the capping site enhances the transcription approximately 3-fold, while the deletion of the TATA box region abolishes the transcription.

29 citations


Journal ArticleDOI
TL;DR: Comparison of the relative efficiencies of transcription on intact wild-type DNA, the two cleaved DNAs, and DNA from a deletion mutant suggests that all or most of the sequences constituting an early promoter lie within the genomic region 60-70 to 140 nucleotides upstream from the principal 5' termini of the early mRNA's.
Abstract: We have studied initiation and regulation of early transcription of simian virus (SV40) DNA in vitro by eucaryotic RNA polymerase II, using both a crude HeLa cell extract and a partially purified calf thymus polymerase supplemented with a HeLa cell S100 fraction. Analysis of initiation sites by primer-directed cDNA synthesis and sequencing of cDNA's has revealed that early transcription is initiated at a multiplicity of sites corresponding to the 5' termini of early viral mRNA's. The pattern of in vitro initiation closely resembles the pattern of 5' termini of early mRNA's late in the lytic cycle, with principal initiations between residues 5184 to 5194, upstream from the early Hogness-Goldberg (TATA) sequence, and at residue 5123, well downstream from this sequence. In vitro transcription is initiated to a lesser extent at sites between residues 5150 and 5155, the principal 5' termini of early mRNA's in transformed cells and early in lytic infection, located 21 to 26 nucleotides downstream from the TATA sequence. Initiation occurs at identical sites and with similar efficiencies on form I and linearized DNA templates. There are minor differences in the efficiency of initiation at specific sites by the two transcriptional systems. Studies using a DNA template cleaved just downstream from the TATA sequence and a second template cleaved through a pair of 72-base-pair tandem repeats starting 87 nucleotides upstream from the TATA sequence have revealed that neither the TATA sequence nor the repeats are essential for early transcription in vitro. However, removal of the TATA and upstream sequences shifts initiation of transcription principally to the residue 5123 site. Comparison of the relative efficiencies of transcription on intact wild-type DNA, the two cleaved DNAs, and DNA from a deletion mutant suggests that all or most of the sequences constituting an early promoter lie within the genomic region 60-70 to 140 nucleotides upstream from the principal 5' termini of the early mRNA's.

28 citations


Book ChapterDOI
01 Jan 1982
TL;DR: In this article, the authors analyzed in vivo and in vitro the sequences required for specific transcription from the Adenovirus-2 major late promoter (Ad2MLP) and showed that, in addition to the TATA box, sequences located further upstream between positions −97 and −34 (position + 1 refering to the capsite), are necessary for efficient in vivo transcription.
Abstract: We have analysed in vivo and in vitro the sequences required for specific transcription from the Adenovirus-2 major late promoter (Ad2MLP). Our results demonstrate that, in addition to the TATA box, sequences located further upstream between positions −97 and −34 (position +1 refering to the capsite), are necessary for efficient in vivo transcription. Recombinant plasmids containing wild-type or deleted Ad2MLP, linked to the SV40 T-antigen coding sequence, were introduced by microinjection or calcium-phosphate transfection into cells in culture. T-antigen expression was assayed by immunofluorescence and specific transcription was assayed by quantitative S1 nuclease mapping. Deletion of sequences located between positions −97 and −34 resulted in a strong reduction of T-antigen production and in a 30-fold decrease in specific RNA production. The effect of these upstream sequences was also investigated in vitro using a whole cell extract or an S100 transcription system and circular or linear templates. In the whole cell extract, the in vivo effect was faithfully reproduced when circular templates were used, but the effect was also seen with linear templates. With the S100 extract, some effect of the upstream sequences on specific in vitro transcription was seen with circular, but not with linear templates.

2 citations