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PubMed1992

Protein-binding to reiterated motifs within the wheat rRNA gene promoter and upstream repeats.

The binding of specific proteins in wheat nuclear extracts to the -34 to +10 region of the wheat ribosomal RNA gene promoter has been detected. DNase I footprinting revealed two binding regions, one spanning the transcription initiation site from -7 to +2, and a larger one further upstream from around -29 to -15. An oligonucleotide made to the -30 to -14 promoter region binds a protein in a sequence-specific manner. This protein also binds specifically to sites with related sequences further upstream in the promoter and in the associated intergenic spacer repeats. A consensus sequence, CATGG--GC-AAAAC, was defined from the different binding sites of the protein. These results, taken together with in vivo results on the enhanced activity of nucleoli containing ribosomal RNA genes with more intergenic spacer repeats, endorse the hypothesis that ribosomal RNA gene transcription is regulated by the extent of binding of a protein to many sites upstream of the transcriptional start site.

Base Sequence↗
PubMed1986

Immunocytochemical demonstration of growth hormone, prolactin and somatostatin-like immunoreactivities in the brain of larval, young adult and upstream migrant adult sea lamprey, Petromyzon marinus.

Growth hormone, prolactin and somatostatin-like immunoreactivities were demonstrated in the brains of larval, young adult (parasitic) and upstream migrant adult sea lampreys, Petromyzon marinus, by means of immunoperoxidase techniques. Growth hormone (GH) and prolactin (PRL) were observed within separate perikarya in the nucleus praeopticus, within fibers in the commissura praeinfundibularis, and in nerve endings within the neurohypophysis of larval and adult-stage lampreys. Cell bodies demonstrating immunoreactive growth hormone were more numerous than those reactive for prolactin. Unlike in the upstream migrant adult lamprey, no GH or PRL was demonstrated in the adenohypophysis of larval or parasitic lamprey. Somatostatin (SRIF)-like immunoreactive neurons were demonstrated in the nucleus commissurae praeinfundibularis, anterior and posterior pars ventralis hypothalami, pars dorsalis thalami, and the tegmentum motorium rhombencephali of larval, parasitic and upstream migrant adult lampreys. Many of the SRIF containing neurons within the hypothalamus were cerebrospinal fluid (CSF)-contacting cells. SRIF fibers were found throughout most of the brain predominating within the nucleus praeopticus, pars ventralis hypothalami, and the nucleus interpeduncularis. No SRIF immunoreactivity was found within the neurophyophysis. The possible functions of these peptides within the brain of the lamprey are discussed.

Aging↗
PubMed1992

Synergistic effect of upstream sequences, CCAAT box elements, and HSE sequences for enhanced expression of chimaeric heat shock genes in transgenic tobacco.

The thermoregulated expression of the soybean heat shock (hs) gene Gmhsp17.3-B is regulated via the heat shock promoter elements (HSEs), but full promoter activity requires additional sequences located upstream of the HSE-containing region. Structural features within this putative enhancer region include a run of simple sequences which are also present upstream of HSE-like sequences of other soybean hs genes, and three perfect CCAAT box sequences located immediately upstream from the most distal HSE of the promoter. A series of heterologous and homologous promoter fusions linked to the chloramphenicol acetyl transferase (CAT) gene was constructed and examined in transgenic tobacco plants. The region containing the AT-rich domain of the 5' flanking region was unable to direct transcription from the TATA box of a truncated delta CaMV35S promoter. Heat-inducible CAT activity was detectable when additional sequences from the native promoter containing three CCAAT boxes and a single HSE were present in the constructions. Complete reconstitution of the native hs promoter/enhancer region increased hs specific CAT activities only very little, but deletion of CCAAT box sequences reduced CAT expression five-fold. Our results suggest that AT-rich sequences have a moderate effect on thermoinducible expression levels of the soybean heat shock gene and that CCAAT box sequences act cooperatively with HSEs to increase the hs promoter activity.

Base Sequence↗
PubMed1991

Upstream sequences regulating legumin gene expression in heterologous transgenic plants.

We have previously isolated a legumin gene LeB4 from Vicia faba and shown that a 4.7 kb DNA fragment containing the gene leads to seed-specific expression in transgenic tobacco plants. Here we report that the 2.4 kb upstream sequence alone, when fused to either the neomycin phosphotransferase II (nptII) gene or the beta-glucuronidase (uidA) gene, leads to high enzyme levels in transgenic seeds of both tobacco and Arabidopsis. beta-Glucuronidase (GUS) activity is especially intense in the cotyledons fading out towards the embryonal root tip, a result confirmed by in situ hybridization. Staining of endosperm cells is consistent in both species. Analysis of a series of promoter deletion mutants fused to the nptII gene and introduced into tobacco plants revealed that about 1 kb of 5'-flanking sequence is sufficient for high-level expression but indirect evidence suggests the presence of weak positive regulatory elements further upstream. Deletions leaving only 0.2 kb of upstream sequence reduce enzyme levels to less than 10%. A deletion which destroys the legumin box with its seed protein gene-specific CATGCATG motif has no obvious effects on expression levels.

Blotting, Southern↗
PubMed1991

A multi-component upstream activation sequence of the Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase gene promoter.

The majority of the activation potential of the Saccharomyces cerevisiae TDH3 gene promoter is contained within nucleotides -676 to -381 (relative to the translation initiation codon). An upstream activation sequence (UAS) in this region has been characterized by in vitro and in vivo assays and demonstrated to be composed of two small, adjacent DNA sequence elements. The essential determinant of this upstream UAS is a general regulatory factor 1 (GRF1) binding site at nucleotides -513 to -501. A synthetic DNA element comprising this sequence, or an analogue in which two of the degenerate nucleotides of the GRF1 site consensus sequence were altered, activated 5' deleted TDH3 and CYC1 promoters. The second DNA element of the UAS is a 7 bp sequence which is conserved in the promoters of several yeast genes encoding glycolytic enzymes and occurs at positions -486 to -480 of the TDH3 promoter. This DNA sequence represents a novel promoter element: it contains no UAS activity itself, yet potentiates the activity of a GRF1 UAS. The potentiation of the GRF1 UAS by this element occurs when placed upstream from the TATA box of either the TDH3 or CYC1 promoters. The characteristics of this element (termed GPE for GRF1 site potentiator element) indicate that it represents a binding site for a different yeast protein which increases the promoter activation mediated by the GRF1 protein. Site-specific deletion and promoter reconstruction experiments suggest that the entire activation potential of the -676 to -381 region of the TDH3 gene promoter may be accounted for by a combination of the GRF1 site and the GPE.

Base Sequence↗
PubMed1985

Conserved sequences upstream of yeast ribosomal protein genes.

Computer analysis has previously revealed the presence of a 12-nucleotide common sequence element (AACATCTGCATGACA; HOMOL1) in the upstream regions of several yeast ribosomal protein genes. By extending the sequence analysis of the 5'-flanking regions of a number of other ribosomal protein genes (including those encoding S10-1, S10-2, S33 and L16-2) we could establish that HOMOL1 occurs upstream of most but not all yeast ribosomal protein genes. Apart from HOMOL1 an additional conserved sequence (ACCCATACATTTA; RPG-box) was detected in front of nearly all yeast ribosomal protein genes, although in some cases it is present in the opposite orientation in the other strand. There seems to be no correlation between the occurrence of one box and that of the other. However when both boxes are present the RPG-box is always located 3' to the HOMOL1-sequence mostly at a distance of only a few nucleotides. A further one-to-one comparison of the upstream regions of several yeast ribosomal protein genes revealed extensive additional sequence homologies that are suggested to be involved in the coordinate control of ribosomal protein gene expression in yeast.

Base Sequence↗
PubMed1995

Sequence analysis of the upstream region of dhlB, the gene encoding haloalkanoic acid dehalogenase of Xanthobacter autotrophicus GJ10.

The DNA sequence upstream of the dhlB gene encoding the haloalkanoic acid dehalogenase of Xanthobacter autotrophicus GJ10 was determined and contained an open reading frame, designated dhlC, which encoded a protein with a significant similarity with the family of Na(+)-dependent symport proteins. The dhlC gene was subcloned under control of a T7 promoter, and found to encode a polypeptide of 45 kDa on SDS-PAGE. Upstream of dhlC, a -24/-12 promoter sequence was found. Further upstream, in the opposite direction of transcription, another open reading frame, designated dhlR, with homology with the family of sigma 54-dependent transcriptional activator proteins was detected. The dhlR gene was cloned and expressed under the control of a T7 promoter and encoded a polypeptide of 51 kDa on SDS-PAGE. The genetic organization of the dhlB region suggested that the expression of dhlC and dhlB was controlled by the product of dhlR and sigma 54 which may explain the observed overexpression of the haloalkanoic acid dehalogenase under starvation conditions.

Amino Acid Sequence↗
PubMed1996

Localization of the sequence-determined DNA bending center upstream of the streptokinase gene skc.

DNA sequences upstream of the core promoter region of the streptokinase gene (skc) from Streptococcus equisimilis H46A increase skc transcription more than tenfold in vivo. This promoter upstream region contains a segment of intrinsically bent DNA, the precise location of which was determined experimentally by circular permutation analysis and theoretically by computer prediction. Electrophoretic analysis of circularly permuted upstream DNA fragments placed the bend center approximately at position -100 with respect to the major transcription initiation site of skc. This position was in excellent agreement with the center of maximum curvature predicted theoretically. Knowledge of the precise location of the bend center will be useful for future studies of the possible effect of DNA bending on skc transcription.

Base Sequence↗
PubMed2004

Identification of a second flagellin gene and functional characterization of a sigma70-like promoter upstream of a Leptospira borgpetersenii flaB gene.

Leptospira borgpetersenii, one of the causative agents of leptospirosis in both animals and humans, is a bacterial pathogen with characteristic motility that is mediated by the rotation of two periplasmic flagella (PF). The flaB gene coding for a core polypeptide subunit of PF was previously characterized by sequence analysis of its open reading frame (ORF) (M. Lin, J Biochem Mol Biol Biophys 2:181-187, 1999). The present study was undertaken to isolate and clone the uncharacterized sequence upstream of the flaB gene by using a PCR-based genome walking procedure. This has resulted in a 1470-bp genomic DNA sequence in which an 846-bp ORF coding for a 281-amino acid polypeptide (31.3 kDa) is identified 455 bp upstream from the flaB start codon. The encoded protein exhibits 72% amino acid identity to the deduced FlaB protein sequence of L. borgpetersenii and a high degree of sequence homology to the FlaB proteins of other spirochaetes. This has demonstrated for the first time that a second flaB gene homolog is present in a Leptospira species. The newly identified gene is designated flaB1, and the previously cloned flaB renamed flaB2. Within the intergenic sequence between flaB1 and flaB2, a potential stem-loop structure (12-bp inverted repeats) was identified 25 bp downstream of the flaB1 stop codon; this could serve as a transcription terminator for the flaB1 mRNA. Three E. coli-like promoter regions (I, II, and III) for binding Esigma(70), a regulatory sequence uncommonly found in flagellar genes, were predicted upstream of the flaB2 ORF. Only promoter region II contains a promoter that is functional in E. coli, as revealed at phenotypic and transcriptional levels by its capability of directing the expression of the chloramphenicol acetyltransferase (CAT) gene in the promoter probe vector pKK232-8. These observations may suggest that flaB1 and flaB2 are transcribed separately and do not form a transcriptional operon controlled by a single promoter.

Bacterial Proteins↗
PubMed2001

Comparative analysis of human, bovine, and murine Oct-4 upstream promoter sequences.

The Oct-4 gene encodes a transcription factor that is specifically expressed in embryonic stem cells and germ cells of the mouse embryo. Cells that differentiate into somatic tissues lose Oct-4 expression. Regulation of Oct-4 gene transcription involves a TATA-less minimal promoter and two upstream elements: the proximal (PE) and distal enhancers (DE). We report here the nucleotide sequence of the 5' upstream regulatory regions of the human and murine Oct-4 genes. A comparative alignment analysis between these regions and those of the bovine Oct-4 ortholog reveals four conserved regions of homology (CR 1 to 4) between these species (66-94% conservation). The 1A sequence within the mouse PE is located approximately half-way between CR 2 and CR 3. A putative Sp1/Sp3 binding site and the overlapping hormone responsive element (HRE) in CR1 are identical in all three species. A high number of CCC(A/T)CCC motifs exhibit various levels of homology in these upstream regions. We discuss the importance of these and other sequences and present candidate factors that may bind and regulate Oct-4 gene expression.

Animals↗
PubMed2005

The contamination of wild watercress with Fasciola hepatica in central France depends on the ability of several lymnaeid snails to migrate upstream towards the beds.

As most natural watercress beds in central France are located upstream of the permanent habitats of two lymnaeid species, Galba truncatula and Omphiscola glabra, field investigations were made from 1999 to 2004 on 67 beds to determine why the contamination of watercress with Fasciola hepatica is irregular over time in these sites, while definitive hosts, especially lagomorphs, are regularly found infected around them. Snails are able to migrate upstream in winter and spring towards the beds, and a 4-year survey demonstrated the existence of annual variation in the colonization of these sites by snails. In the 45 beds irregularly contaminated with F. hepatica over time, 37.7-62.2%, according to the year, were not populated by lymnaeids, in spite of the presence of snail populations downstream. G. truncatula was found alone in 8.8-13.3% of sites and was the first colonizing snail in 24.3-33.3% when the two lymnaeid species successively settled in these waterholes. The colonizing ability of O. glabra was more limited, as it was observed alone in 2.2% of beds and was the first colonizing snail in only 2.2-20% of them. The distances covered by these snails significantly increased with increasing migration time. After migration, a few overwintering snails (3.8% of G. truncatula and 6.8% of O. glabra) are able to colonize the beds, but their numbers decreased when the distance of migration was greater. O. glabra migrated more quickly and reached more watercress beds than G. truncatula. However, this did not influence the natural infections of snails, which were more frequent in the F1 of G. truncatula. The variability observed in the colonization of beds by snails might explain the regular or irregular contamination of wild watercress by metacercariae of F. hepatica. One of the factors which may explain this variability is the occurrence of showers in spring, so that a few snails are able to reach watercress beds after their upstream migration.

Animals↗
PubMed1997

Characterization of upstream activating sequences involved in activation and regulation of pho4 expression in Schizosaccharomyces pombe.

In this study, a short region of the pho4 promoter located just upstream of the TATA box, called the upstream activating sequence (UAS), was shown to be responsible for both activation and regulation of pho4 expression in Schizosaccharomyces pombe. This cis-acting sequence is able to activate transcription when placed upstream of the minimal promoter of the constitutively expressed adh gene, and to ensure regulation by thiamine. The organisation of the pho4 promoter is remarkable in that UAS and TATA box are very closely associated. This proximity appears to be essential for efficient transcriptional regulation. Indeed, only slight variations in transcript levels were observed under derepression conditions when the UAS was moved away from the TATA box, while regulation of transcript levels appeared to be strongly affected. This observation suggests the existence of a negative regulatory element whose action requires very close association of UAS and TATA box. Surprisingly, when UAS was moved about 40 bp away from the adh TATA box, residual but very significant repression of gene expression by thiamine, which apparently does not result from transcriptional regulation, was revealed by measuring protein production. These data prompted us to hypothesize the existence of two distinct and cooperative mechanisms of regulation involving specific factor binding to the UAS, the first one controlling transcript levels and the second acting posttranscriptionally.

Amino Acid Sequence↗
PubMed1997

The regulatory protein MucR binds to a short DNA region located upstream of the mucR coding region in Rhizobium meliloti.

The Rhizobium meliloti MucR protein is known to regulate the biosynthesis of the two exopolysaccharides, succinoglycan and galactoglucan. The mucR gene was successfully overexpressed in Escherichia coli BL21 cells by heat shock induction using a two-plasmid system. Cell extracts of the production strain contained about 20% of a polypeptide of 17 kDa apparent molecular mass, corresponding to the size expected for MucR. As shown by an electrophoretic mobility shift assay, these extracts were active in the specific retardation of a 219-bp DNA fragment including 134-bp of the non-coding region upstream of the mucR gene. Primer extension analysis showed that this DNA fragment was located within the transcribed region upstream of the mucR gene. Competition experiments revealed that a 44-bp sequence present within the 134-bp upstream of the mucR gene contained the MucR binding site. Although binding of MucR to this site exhibited a moderate dissociation constant of Kd approximately 1.4 x 10(-7) M, the reaction was highly specific since fragments containing binding sites for the homologous Ros protein from Agrobacterium tumefaciens were not able to compete for MucR binding.

Bacterial Proteins↗
PubMed2001

Association analysis of polymorphisms in the upstream region of the human dopamine D4 receptor gene (DRD4) with schizophrenia and personality traits.

The human dopamine D4 receptor (DRD4) is of major interest in molccular studies of schizophrenia and personality traits. We examined the association of schizophrenia and polymorphisms in the upstream region of the DRD4 gene (-768G>A in the negative modulator region; -521C>T, -376C >T, and -291C>T in the cell type-specific promoter region; and -616C>G between the two regions) in 208 schizophrenic patients and 210 normal controls. No significant difference in genotype and allele frequencies was observed between the two groups, indicating that these polymorphisms do not make a major contribution to the pathogenesis of schizophrenia. We also studied the association of polymorphisms in the upstream region and a 48-bp repeat polymorphism in exon III of the DRD4 gene with personality traits in 173 Japanese individuals who completed the temperament and character inventory (TCI). The -768G>A polymorphism was significantly associated with reward dependence (P= 0.044), while no significant association was observed between novelty seeking and polymorphisms in the upstream region or the exon III repeat polymorphism of the DRD4 gene.

Base Sequence↗
PubMed1990

The gene for human acidic fibroblast growth factor encodes two upstream exons alternatively spliced to the first coding exon.

We have isolated two cDNA clones encoding human acidic fibroblast growth factor (aFGF) which represent the utilization of alternative upstream exons in aFGF mRNA. Isolation and sequence analysis of genomic clones spanning the first coding exon and each of the upstream sequences confirms that the divergent 5' sequences are separate exons, spliced alternatively to the first coding exon 34 nucleotides upstream of the initiator AUG codon. Restriction mapping of the genomic clones provides a minimum size estimate of 45 kilobase pairs for the aFGF locus.

Base Sequence↗
PubMed1985

Genomic organization and nucleotide sequence of two distinct histone gene clusters from Xenopus laevis. Identification of novel conserved upstream sequence elements.

We have performed a detailed analysis of the genomic organization and the nucleotide sequence of two distinct Xenopus laevis histone gene clusters totaling approximately 23.5 X 10(3) base-pairs. Each cluster contains at least one copy of each of the five histone genes. However, these genes are present in different arrangements within each cluster and different H1A, H2A and H2B proteins (variants) are encoded by the respective genes of each cluster. Southern blot analysis of genomic X. laevis DNA indicates that each cluster is a member of a distinct family of tandemly repeated histone gene clusters. A comparative analysis of the nucleotide sequences flanking the histone genes within these two clusters has revealed the presence of multiple conserved sequence elements that are specific for each histone gene class and located at preferred upstream positions. Several of these elements correspond to sequences that are known to be required for maximal transcription of the corresponding genes. Most of these sequence elements have not been identified previously, although we find that many of them are present at corresponding locations upstream of histone genes from other organisms. We suggest that the conserved upstream sequence elements may play an important role in the expression of histone genes in vivo.

Amino Acid Sequence↗
PubMed1985

A region flanking the GAL7 gene and a binding site for GAL4 protein as upstream activating sequences in yeast.

A region of DNA 116 to 271 base-pairs upstream from the GAL7 gene of Saccharomyces cerevisiae activates transcription from a heterologous promoter and does so in either orientation, showing that the GAL7 upstream region contains an upstream activating sequence (UAS). The level of transcription obtained with two GAL7 UAS's in tandem was only 1.3 times that with one. Previous studies of the GAL1-GAL10 intergenic region were indicative of two binding sites for the GAL4 positive regulatory protein; we find that a single (synthetic) site is capable of gene activation. The level of transcription obtained with the intact GAL1-GAL10 UAS was five times that with the single site.

Base Sequence↗
PubMed1986

Conserved sequences and cell-specific DNase I hypersensitive sites upstream from the co-ordinately expressed alpha I- and alpha II-globin genes of Xenopus laevis.

The globin gene family of Xenopus laevis comprises pairs of closely related genes that are arranged in two clusters, each pair of genes being co-ordinately and stage-specifically expressed. To get information on putative regulatory elements, we compared the DNA sequences and the chromatin conformation 5' to the co-ordinately expressed adult alpha-globin genes. Sequence analysis revealed a relatively conserved region from the cap site up to position -289, and further upstream seven distinct boxes of homology, separated by more diverged sequences or deletions/insertions. The homology boxes comprise 22 to 194 base-pairs showing 78 to 95% homology. Analysis of chromatin conformation showed that DNase I preferentially cuts the upstream region of both genes at similar positions, 5' to the T-A-T-A and the C-C-A-A-T boxes, only in chromatin of adult erythroblasts and erythrocytes, where adult globin genes are expressed, but not in chromatin of adult liver cells or larval erythrocytes, where these genes are silent. This suggests that cell- and stage-specific activation of these genes coincides with specific changes in chromatin conformation within the proximal upstream region. No difference was found in the nucleotide sequence within the DNase I hypersensitive region proximal to the adult alpha 1-globin gene in DNA from embryonic cells, in which this gene is inactive, and adult erythrocytes, expressing this gene.

Animals↗