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At least 379 records · Page 21Linked to original sources
PubMed1988

Statistical positioning of nucleosomes by specific protein-binding to an upstream activating sequence in yeast.

Arrays of nucleosomes were positioned with respect to the GAL1-GAL10 intergenic region inserted into Saccharomyces cerevisiae minichromosomes. Deletions of DNA flanking the upstream activation sequence left the array unaltered, showing that nucleosome positioning was not a consequence of sequence-specific histone-DNA interactions but depended on proximity to the galactose-responsive upstream activation sequence (UASG). Replacement of the upstream activation sequence by synthetic oligonucleotides with different protein-binding properties identified a short sequence within this region that is responsible for the ordered array. This sequence overlaps a binding site for GAL4 protein, a positive regulator of transcription, but exerts its effect on chromatin structure independently of GAL4, probably through binding a novel factor that is not GAL-specific.

Base Sequence↗
PubMed1991

Factor-independent activation of Escherichia coli rRNA transcription. II. characterization of complexes of rrnB P1 promoters containing or lacking the upstream activator region with Escherichia coli RNA polymerase.

A region upstream from the Escherichia coli rrnB P1 promoter, the upstream activator region (UAR), increases the activity of the promoter in vivo and the rate of association with RNA polymerase (E sigma 70) in vitro in the presence of the two initiating nucleotides. We have used four types of chemical and enzymatic footprinting probes to determine whether rrnB P1-E sigma 70 complexes formed in the presence of the initiating nucleotides (RPinit) differ from typical open complexes (RPo) formed in the absence of the initiating nucleotides and to examine the structural differences between rrnB P1 complexes containing the UAR and those lacking the UAR. We find that the rrnB P1-RPinit complex closely resembles open complexes formed at other E sigma 70 promoters, indicating that the formation of the first phosphodiester bond does not result in a major rearrangement of the promoter-RNA polymerase complex. An unusual potassium permanganate modification at position -18 in both RPo and RPinit indicates the possible presence of a subtle difference in the -10, -35 spacer structure compared to some other E. coli promoters. We show that the E sigma 70-rrnB P1 complex formed with the promoter containing the UAR has DNase I and hydroxyl radical cleavage patterns in the -50 region different from those observed with the same promoter lacking the UAR. These results are interpreted to indicate that E sigma 70 may interact with a region further upstream from that contacted by RNA polymerase bound at most other promoters and/or that unusual structural properties of this region are induced by bound E sigma 70.

Base Sequence↗
PubMed1982

An expandable gene that encodes a Drosophila glue protein is not expressed in variants lacking remote upstream sequences.

The Drosophila melanogaster gene Sgs4 encodes one of the glue polypeptides, sgs-4, synthesized in the larval salivary gland. We have examined the structure and expression of Sgs4 in five strains that produce abundant amounts of sgs-4 and its mRNA and in four that do not. The nonproducers include three Japanese strains that accumulate trace amounts of mRNA and one strain, BER-1, that contains no detectable Sgs4 RNA. Sgs4 carries a tandem array of repeated 21 bp elements within its coding sequence. The number of elements per array varies, causing considerable differences in the lengths of Sgs4 and its mRNA among the strains. These differences in length are not correlated with differences in mRNA abundance; rather, the low or zero abundance in nonproducers correlates with the loss of DNA upstream from the gene. The Japanese nonproducers carry a 52 bp deletion 305 bp upstream from the 5' end of Sgs4, and BER-1 carries a 95 bp deletion 392 bp upstream. Curiously, each deletion encompasses one or more of the salivary-gland-specific DNAase I-hypersensitive sites which are known to flank the Sgs4 gene.

Animals↗
PubMed1983

Multiple origins of transcription in the 4.5 Kb upstream of the epsilon-globin gene.

By a combination of primer extension and S1 mapping, we have identified and characterized at least nine epsilon-globin initiation sites in the 4.5 kb upstream of the canonical mRNA cap site. These occur in four regions, -65 to -250, -900, -1480, and -4500 bp from the translation initiation codon ATG. Transcripts from eight sites yield sufficient material for direct RNA analysis and in all of these instances the molecules are capped. Approximately 10%-15% of epsilon-globin transcripts originate from these upstream sites in both an erythroleukemic cell line and purified erythroblasts from first-trimester human embryos. In three established cell lines derived from adult non-erythroid tissue, low levels of transcription of the epsilon-globin gene occur, but the RNA molecules originate exclusively from one of the upstream sites identified in erythroid cells. These findings suggest modification of the current models of transcription of globin genes.

Cell Line↗
PubMed1985

An RNA polymerase II transcription factor binds to an upstream element in the adenovirus major late promoter.

A gel electrophoresis DNA binding assay has been used to identify proteins in HeLa cell extracts that specifically bind to the major late promoter of adenovirus. A major late promoter transcription factor MLTF has been detected as a discrete protein-DNA complex. MLTF binds specifically and with high affinity to sequences upstream of the TATA box of the major late promoter. This factor protects a 17 bp (-50 to -66) region in a DNAase I footprinting assay. The same region has been shown to be important for efficient transcription from the major late promoter both in vivo and in vitro. MLTF stimulates in vitro transcription only from a template containing this upstream region. The binding, footprinting, and transcription-stimulatory activities of MLTF cofractionate through two chromatographic steps. These results suggest that direct binding of MLTF to an upstream element activates transcription from the major late promoter.

Adenoviruses, Human↗
PubMed1988

Use of a versatile lacZ vector to analyze the upstream region of the Bacillus subtilis spoOF gene.

We have constructed a versatile vector, pIS112, in which lacZ translational fusions can be made in Escherichia coli and then analyzed in Bacillus subtilis in three contexts, without recloning: in multicopy during propagation of the plasmid, in single copy integrated via a Campbell-type mechanism into the wild-type locus of the cloned fragment, or in single copy integrated into a heterologous locus. Upstream regions are reconstituted in the integration into the wild-type locus, but not into the heterologous locus, allowing the identification of upstream regulatory sequences. We have used this vector to analyze the expression of the early sporulation gene, spoOF, which, during early stationary phase, is induced 10-fold from a basal vegetative level. When a region, -50 to -150 bp relative to the transcriptional start site, is removed in the spoOF-lacZ gene, stationary phase induction of beta-galactosidase is lost. The same deletion in the upstream region of the functional spoOF gene results in cells which sporulate very poorly, although they are not blocked at the onset of sporulation, as in an spoOF null mutant. This suggests induction of spoOF expression during the beginning of stationary phase is necessary for wild-type sporulation.

Bacillus subtilis↗
PubMed1996

Structure and polymorphism of the upstream region of the pfg27/25 gene, transcriptionally regulated in gametocytogenesis of Plasmodium falciparum.

Transcription of the early gametocyte-specific gene pfg27/25, and genomic structure and polymorphism of its upstream region were studied in the human malaria parasite Plasmodium falciparum. The upstream genomic sequence of the pfg27/25 gene is characterised by a repetitive region that contains five direct and one inverted repeats of a unit constituted by a perfectly conserved sequence flanked by a poly-dT and a poly-d(AT) tract. Sequences further upstream from the repetitive region are polymorphic in distantly related parasite lines. Nuclear 'run off' experiments indicated that transcription of pfg27/25 is developmentally regulated. Transcription of the gene, undetectable in asexual parasites, is activated at the onset of gametocytogenesis. Te gene is transcribed in a 2.5 kb mature mRNA for the first 2-3 days of sexual differentiation, while transcription is down-regulated in more mature gametocytes.

Animals↗
PubMed1992

Gene for 17 alpha-hydroxylase/C (17-20) lyase P-450: complete nucleotide sequence of the porcine gene and 5' upstream sequence of the rat gene.

We describe the isolation and characterization of a full-length clone for the porcine 17 alpha-hydroxylase/C(17-20) lyase (CYP17) gene. The complete exon and partial intron sequences are presented including approx. 1000 bp of the 5' upstream sequence. In addition we describe the isolation and characterization of the 5' upstream region of the rat CYP17 gene. The sequences of the first exon, part of the first intron, and approx. 3.5 kb of the 5' upstream region are presented.

Amino Acid Sequence↗
PubMed1995

An archaeal gene upstream of grpE different from eubacterial counterparts.

In some eubacteria with a dnaK locus in which grpE is close upstream of dnaK, grpE is preceded by an open reading frame (orf) believed to be a heat-shock gene. We also found an orf, orf16, upstream of grpE in the archaeon Methanosarcina mazei S-6, but this gene differs from the eubacterial counterpart: it is shorter, does not respond to a temperature upshift as heat-shock genes do, and the deduced protein Orf16, does not resemble the proteins coded by the eubacterial equivalents. orf16 is expressed monocistronically, with a transcription initiation site 24 bases upstream of the translation start codon, 22 bases downstream of a putative promoter identical to the consensus promoter for genes in methanogens. This initiation site is used by heat-shocked and non-heat-shocked cells in the two morphologic stages of M. mazei S-6 tested, i.e., packets and single cells. Three transcription termination sites were identified, one of which is detectable only in non-heat-shocked cells. Data from comparative analyses of the Orf16 deduced amino acid sequence and those of other known proteins, as well as the apparent biochemical characteristics of Orf16, suggest that the latter is a membrane molecule.

Amino Acid Sequence↗
PubMed1991

Transcription of sequences upstream of the rat prolactin gene suggests the existence of a second promoter.

The transcription of the rat prolactin gene domain has been examined using a modified Southern blot procedure. Cloned genomic DNAs were resolved by electrophoresis in agarose, transferred to nitrocellulose, and probed with radiolabeled RNA that had been synthesized in vitro by nuclei isolated from pituitary tumor cells. Data presented in this paper illustrate that single copy genomic sequences located within 7.3 kb upstream of exon 1 are transcribed. Single copy or low copy number DNA sequences that reside greater than 7.3 kb upstream of exon 1, or downstream of exon 5 were not transcribed at detectable levels. These data suggest that a second promoter may exist upstream of the rat prolactin gene and that this second promoter may be active in pituitary cells.

Animals↗
PubMed1990

"Upstream" regulation of the release probability in sympathetic nerve varicosities.

The results appear to support the following tentative working hypothesis. (1) Nerve impulse-induced transmitter release from sympathetic nerve varicosities is monoquantal and highly intermittent (probability range: 0-0.03). (2) Nerve impulses invade varicosities as all-or-none, Na+ channel-dependent action potentials; invasion failure may be rare. (3) The release probability is not controlled by properties (amplitude or duration) of the invading action potential or the resulting Ca2+ current, but by the availability of an as yet unidentified permissive factor. (4) The permissive factor is actively transported intra-axonally, probably in association with organelles (LDVs?). (5) The activation and/or transport of the permissive factor are controlled "upstream" of the varicosity; they depend on Ca2+ influx through channels insensitive to nifedipine (hence, not of L-type) but blocked by Cd2+ and apparently opened by slight depolarization of the resting membrane, in this respect behaving more as T- than N-type channels. (6) A high resting K+ efflux "upstream" of the varicosity restricts the availability of the permissive factor; it is the main mechanism maintaining the (economically necessary) low release probability. (7) Prejunctional agonists do not inhibit transmitter secretion by causing a conduction block or by reducing the action potential-induced Ca2+ influx into the varicosity itself, but by depressing the Ca2(+)-dependent activation and/or transport of the permissive factor; they act at least in part via receptors "upstream" of the varicosity. (8) This hypothesis for regulation of the release probability in sympathetic nerves may apply, at least in part, to other neurons as well.

Animals↗
PubMed1984

Sequence polymorphisms in the 5'-upstream region of the fibroin H-chain gene in the silkworm, Bombyx mori.

DNA fragments containing the fibroin H-chain gene from two different strains of Bombyx mori, J-139 and Nd (2) were cloned into phage lambda Charon 4A. Comparison of the restriction sites in these cloned DNAs revealed that in addition to the known polymorphism in the region coding for the repetitive amino acid sequence of the fibroin H-chain [Manning and Gage, J. Biol. Chem. 255 (1980) 9451-9457], at least two other types of polymorphism were present, one around the 5' end of the structural gene, and the other in the far upstream region of the gene. Restriction sites around the 5' end of the gene were well conserved between these strains, but some heterogeneity, suggesting the presence of small insertions, deletions or base changes, was noted. In contrast, DNA sequences of the region 2-4 kb upstream from the 5' end of the gene were markedly different between these two strains, indicating that either a deletion or an insertion of a DNA sequence longer than 2 kb had occurred in this region. Comparison with several other strains suggested that the observed changes in the far-upstream region were unique to the Nd(2) strain.

Animals↗
PubMed1989

The role of bases upstream of the Shine-Dalgarno region and in the coding sequence in the control of gene expression in Escherichia coli: translation and stability of mRNAs in vivo.

A range of translational initiation regions (TIR) was created by combining synthetic DNA fragments derived from the atpB-atpE intercistronic sequence of Escherichia coli with the cDNA sequence encoding mature human interleukin 2 (IL-2), the E. coli fnr gene, or an fnr::lacZ gene fusion. Both the overall rates of gene expression and the relative concentrations and stabilities of the corresponding mRNA species were estimated in strains bearing the constructs on plasmids. These measurements served as the basis for analyses of the relationship between the structure of the TIR and the true rates of translation that it promotes. The constructs involving the IL-2 cDNA were predicted to allow much less stable secondary structure within the TIR than those involving the N-terminal region of the fnr gene. Thus by combining one set of upstream sequences with two different types of N-terminal coding sequence, it was possible to distinguish between the respective influences of primary and secondary structure upon initiation. The data indicate that in the presence of a given Shine-Dalgarno (SD)/start codon combination, the decisive factor for translational initiation efficiency is the stability of base pairing involving, or in the vicinity of, this region. The sequences contributing to this secondary structure can be many bases upstream of the SD region and/or downstream of the start codon. There was no indication that the specific base sequence upstream of the SD region could, other than to the extent that it contributed to the local secondary structure, significantly influence the efficiency of translational initiation.

Amino Acid Sequence↗
PubMed1989

The activity of the Pseudomonas aeruginosa pilin promoter is enhanced by an upstream regulatory site.

Recently, a comparison of the nucleotide sequences upstream of the structural pilin genes (pil) from five different isolates of Pseudomonas aeruginosa identified a sequence homologous to a NifA-binding site, a positive regulatory sequence in Klebsiella pneumoniae which controls the expression of the genes which fix nitrogen [Pasloske et al., J. Bacteriol. 170 (1988) 3738-3741]. This sequence was located between -98 and -114 bp relative to the transcription start point. To establish whether this putative recognition sequence exerted any regulatory effect on pil gene expression, we assayed for the expression of pilin and pili, and assessed the strength of the pilin promoter with and without this upstream sequence. A 48-bp deletion, which removed the putative recognition sequence, decreased the levels of pilin and pilus expression. Also, the equivalent deletion reduced pilin promoter activity by 90% when it was measured using a promoter probe vector. These results provide evidence that a nucleotide sequence upstream of the pilin promoter positively regulates the transcription of the P. aeruginosa pil gene.

Bacterial Outer Membrane Proteins↗
PubMed1992

An upstream activating sequence from the Aspergillus nidulans gpdA gene.

Introduction of a previously identified promoter element of the Aspergillus nidulans gpdA gene (encoding glyceraldehyde-3-phosphate dehydrogenase), the so-called gpd box, into the upstream region of the highly regulated A. nidulans amdS gene (encoding acetamidase), significantly increased (up to 30-fold) the expression of the lacZ reporter gene fused to these expression signals. This increase was dependent on the orientation of the gpd box and on the site of introduction into the amdS upstream region. The presence of additional gpdA sequences which flank the gpd box reduced or even extinguished positive effects of the gpd box. omega-Amino acid and carbon catabolite regulation of the amdS promoter were retained after introduction of the gpd box, indicating that the gpd box does not abolish interactions of the regulatory proteins, AmdR and CreA, with the amdS transcription control sequences. Based on the results, it is suggested that the gpd box comprises at least two separate activities: one being orientation dependent, but relatively independent of position of the gpd box in the upstream region, and the other is only functional near other sites of transcriptional control. Most likely, both activities are not involved in regulation of the amdS promoter.

Aspergillus nidulans↗
PubMed1994

DNase I hypersensitive sites far upstream of the rat tryptophan oxygenase gene direct developmentally regulated transcription in livers of transgenic mice.

Expression of the gene coding for tryptophan oxygenase (TO) is switched on in rat liver about two weeks after birth. We identified two clusters of DNaseI hypersensitive (HS) sites in the TO gene upstream region; one near the promoter, the other at a distant upstream location (-8.5 kb). Hypersensitivity of upstream sites was present in adult and in 7 day old rat liver, but absent in kidney. To investigate their role in transcriptional regulation, a reporter gene controlled by both HS site regions was used to generate transgenic mice. In these animals the transgene followed the cell specific and developmental regulation of the endogenous gene: inactive after birth and active in adult liver. Transgenes containing only the promoter proximal HS site were non-functional.

Aging↗
PubMed2004

Cloning and characterization of 5' upstream promoter region of rat WAP gene.

Regulatory regions of genes encoding milk proteins are frequently used to produce in the mammary gland of transgenic animals a variety of pharmaceutically and medically important human proteins. One such example is the whey acidic protein (WAP) promoter region, identified so far in the genome of mouse, rat, rabbit, camel, pig, brushtail possum and Tammar wallaby. The aim of the present study was cloning and characterization of the 5' upstream promoter region of rat WAP gene. Using Genome Walking procedure, we cloned the region extending from -849 to -3671 bp. We have shown that there are two conserved regions highly similar to hypersensitive sites present in mouse and rabbit upstream region of WAP gene with binding sites for STAT5 transcription factor, essential for expression of WAP gene in mammary glands during lactation. We characterized dispersed and tandem repeats in the upstream region of rat WAP gen localized not far away from the translation initiation site.

5' Flanking Region↗
PubMed2005

Cloning of the hamster p16 gene 5' upstream region and its aberrant methylation patterns in pancreatic cancer.

The hamster model of pancreatic carcinogenesis is useful for understanding the development of human pancreatic cancer. However, there is only a small amount of hamster genetic information available for analyzing the gene alterations in hamster pancreatic cancers. Here, we determined the nucleotide sequence of the 5' upstream region of the hamster p16 gene using a suppression polymerase chain reaction method combined with gene-specific primers. Based on this sequence, we analyzed the methylation status of the 5' region by bisulfite sequencing in three normal pancreatic tissues and five pancreatic duct adenocarcinomas (PDAs). All five PDAs were highly methylated in the 5' upstream region and showed reduced expressions of the p16 gene, while the three normal samples were demethylated. The method described in this study is a highly effective and rapid technique for determining the 5' upstream region, and is applicable to epigenetic studies of the methylation status of this region.

5' Flanking Region↗