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PubMed1990

Characterization of human 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase gene and its expression in mammalian cells.

Three beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) catalyze the oxidative conversion of delta 5-3 beta-hydroxysteroids to the delta 4-3-keto configuration and is therefore essential for the biosynthesis of all classes of hormonal steroids, namely progesterone, glucocorticoids, mineralocorticoids, androgens, and estrogens. Using human 3 beta-HSD cDNA as probe, a human 3 beta-HSD gene was isolated from a lambda-EMBL3 library of leucocyte genomic DNA. A fragment of 3 beta-HSD genomic DNA was also obtained by amplification of genomic DNA using the polymerase chain reaction. The 3 beta-HSD gene contains a 5'-untranslated exon of 53 base pairs (bp) and three successive translated exons of 232, 165, and 1218 bp, respectively, separated by introns of 129, 3883, and 2162 bp. The transcription start site is situated 267 nucleotides upstream from the ATG initiating codon. DNA sequence analysis of the 5'-flanking region reveals the existence of a putative TATA box (ATAAA) situated 28 nucleotides upstream from the transcription start site while a putative CAAT binding sequence is located 57 nucleotides upstream from the TATA box. Expression of a cDNA insert containing the coding region of 3 beta-HSD in nonsteroidogenic cells shows that the gene encodes a single 42-kDa protein containing both 3 beta-hydroxysteroid dehydrogenase and delta 5-delta 4-isomerase activities. Moreover, all natural steroid substrates tested are transformed with comparable efficiency by the enzyme. In addition to its importance for studies of the regulation of expression of 3 beta-HSD in gonadal as well as peripheral tissues, knowledge of the structure of the human 3 beta-HSD gene should permit investigation of the molecular defects responsible for 3 beta-HSD deficiency, the second most common cause of adrenal hyperplasia in children.

3-Hydroxysteroid Dehydrogenases↗
PubMed1997

cis elements that contribute to geminivirus transcriptional regulation and the efficiency of DNA replication.

The A genomic component of the geminivirus tomato golden mosaic virus (TGMV) contains a 5' intergenic sequence that includes the overlapping AL61 promoter and positive-strand origin of DNA replication. The TGMV AL1 protein negatively regulates its own transcription and mediates origin recognition by binding to a repeated motif shared by the AL61 promoter and the viral origin. We examined a series of truncated or mutated 5' intergenic regions in transient expression and replication assay to identify other DNA sequences that contribute to TGMV promoter and origin function. These experiments revealed that negative regulation of the AL61 promoter is complex, involving multiple cis-acting sequences and the AL1 and AL4 proteins, which acted through different DNA elements. We also found that mutation of the TATA box motif in the AL61 promoter reduced overall transcriptional activity and AL1-mediated repression, confirming the importance of this sequence in promoter function. Mutation of a G-box consensus sequence was highly detrimental to AL61 transcription and abolished AL1 sensitivity, suggesting that AL1 interferes with transcriptional activation. Cotransfection experiments showed that the TATA box and G-box motif mutations also impaired viral DNA replication in the presence of a wild-type origin but had no effect in its absence, demonstrating that these transcriptional motifs also function as replication efficiency elements.

Base Sequence↗
PubMed1998

Complex organisation of the 5'-end of the human glycine tRNA synthetase gene.

Glycine tRNA synthetase (glyRS) catalyses the addition of the amino acid glycine to its cognate tRNA molecules. In the silk moth worm Bombyx mori, this gene is subject to complex transcriptional regulation because of the predominance of glycine in silk. In vertebrates, glycine is a major constituent of collagen but there have been no studies of glyRS regulation. In this study we have isolated and mapped a genomic clone containing the 5'-end of glyRS. Primer extension studies identified only one transcriptional start point (TSP) in three different cell lines. Expression of the transcript identified may be regulated translationally because it contains five potential initiation codons, three of which are in good context for initiation. The most 3' of the potential initiation codons has previously been predicted to be the initiating codon for cytoplasmic glyRS. Two of the upstream codons are in-frame with this codon, and both are predicted to extend the N-terminus of glyRS to include a mitochondrial targeting sequence. Sequencing of genomic DNA surrounding the TSP showed features common to the promoters of housekeeping genes, as well as a canonical TATA box at the unusual position of +9. Surprisingly, promoter activity in vitro was not specified by a 1.9 kb genomic fragment containing the TSP and TATA box, but by a contiguous 0.4 kb fragment immediately downstream. These studies suggest that the transcription of glyRS from a single start point requires downstream promoter elements.

Animals↗
PubMed1989

Transcriptional regulatory sequences of the housekeeping gene for human triosephosphate isomerase.

To examine the functional organization of the human triosephosphate isomerase (TPI) promoter, deletion, insertion, and linker scanning mutations were introduced into the TPI promoter of hybrid TPI/beta-globin genes. These genes were transiently expressed in mouse L and human HeLa cells, and the effect of each mutation on the frequency and position of transcription initiation was assayed by S1 nuclease transcript mapping. Multiple positive regulatory elements reside between positions -595 and +1 in L cells and -920 and -7 in HeLa cells and coordinately promote maximum hybrid gene transcription. These elements include an array of GC boxes (positions -126 to -48) that variably conform to the consensus Sp1-binding site, and a canonical TATA box (positions -27 to -21) that is essential for detectable levels of transcription. In an additive yet position-dependent fashion, the GC boxes function in cis to the TATA box to control both the frequency and position of transcription initiation. Additional positive elements reside upstream of position -131 and are required for full promoter function. Also, an inhibitory element(s) residing between position -7200 and either -2800 in L cells or -920 in HeLa cells reduces transcription approximately 7-fold relative to the level of transcription achieved with the maximally active promoter.

Animals↗
PubMed2004

Human replication-dependent histone H3 genes are activated by a tandemly arranged pair of two CCAAT boxes.

We have analysed the transcriptional regulation of the human histone H3 genes using promoter deletion series, scanning mutagenesis, specific mutagenesis and electrophoretic mobility-shift assay experiments. The promoters of five of the six examined histone H3 genes showed near-maximal activity at lengths of 133-227 bp: H3/d 198 bp, H3/h 147 bp, H3/k 133 bp, H3/m 227 bp, H3/n 140 bp (exception H3/i). To search for functional cis-elements within these regions, we performed scanning mutagenesis of the two histone H3 promoters H3/k and H3/m. Mutagenesis revealed that the functional framework of the histone H3 promoters consists of a TATA box and two tandemly arranged CCAAT boxes in relatively fixed positions. Alterations of the distance between the CCAAT boxes and of the distance between the CCAAT boxes and the TATA box resulted in significant loss of activity. In electrophoretic mobility-shift assay experiments, the factor CBF (CCAAT-binding factor)/NF-Y (nuclear factor-Y) bound to isolated CCAAT boxes of the H3/k promoter. This suggests that an initiation complex is formed on the histone H3 promoter that has a defined structure and limited flexibility, consisting of two molecules of CBF/NF-Y and further (general or specific) transcription factors.

Base Sequence↗
PubMed2006

Core promoter elements recognized by transcription factor IIB.

The general transcription factor TFIIB (transcription factor IIB) plays a critical role in the assembly of the RNA polymerase II pre-initiation complex. TFIIB can make sequence-specific DNA contacts both upstream and downstream of the TATA box. This has led to the definition of two core promoter BREs (TFIIB-recognition elements), one upstream [BRE(u) (upstream BRE)] and one downstream of TATA box [BRE(d) (downstream BRE)]. TFIIB-BRE(u) and TFIIB-BRE(d) contacts are mediated by two independent DNA-recognition motifs within the core domain of TFIIB. Both the BRE(u) and the BRE(d) modulate the transcriptional potency of a promoter. However, the net effect of the BREs on promoter activity is dependent on the specific blend of elements present within a core promoter.

Binding Sites↗
PubMed2000

The 5'-end of the porcine perinatal myosin heavy chain gene shows alternative splicing and is clustered with repeat elements.

The porcine perinatal myosin heavy chain (MyHC) is a major isoform in foetal skeletal muscles. We report here on its cDNA and genomic isolation, molecular characterisation and expression. Exon 2 and the first 4 bases of exon 3 of the perinatal MyHC gene. both part of the 5'-end untranslated region, showed differential splicing. About 2% of all perinatal MyHC transcripts of a 50-day-old foetus were without exon 2 and about half were without the 4 bases at the 5'-end of exon 3. Perinatal MyHC mRNA was expressed in all hind limb muscles of a 45-day-old foetus along with the slow and embryonic MyHC isoforms in the same fibres. Unlike other sarcomeric MyHCs reported to date, the porcine perinatal promoter is clustered with repeat elements (4 SINEs and 1 microsatellite) and is without a consensus TATA box at the predicted site upstream of exon 1. Nonetheless, in reporter gene transfections, its promoter was found to be highly muscle-specific. The absence of a TATA box may point to a fundamental difference in the regulatory function between the perinatal and adult MyHC isoforms.

Alternative Splicing↗
PubMed1996

Structure and genomic organization of the human B1 receptor gene for kinins (BDKRB1).

Two subtypes of mammalian bradykinin receptors, B1 and B2 (BDKRB1 and BDKRB2), have been defined based on their pharmacological properties. The B1 type kinin receptors have weak affinity for intact BK or Lys-BK but strong affinity for kinin metabolites without the C-terminal arginine (e.g., des-Arg9-BK and Lys-des-Arg9-BK, also called des-Arg10-kallidin), which are generated by kininase I. The B1 receptor expression is up-regulated following tissue injury and inflammation (hyperemia, exudation, hyperalgesia, etc.). In the present study, we have cloned and sequenced the gene encoding human B1 receptor from a human genomic library. The human B1 receptor gene contains three exons separated by two introns. The first and the second exon are noncoding, while the coding region and the 3'-flanking region are located entirely on the third exon. The exon-intron arrangement of the human B1 receptor gene shows significant similarity with the genes encoding the B2 receptor subtype in human, mouse, and rat. Sequence analysis of the 5'-flanking region revealed the presence of a consensus TATA box and of numerous candidate transcription factor binding sequences. Primer extension experiments have shown the existence of multiple transcription initiation sites situated downstream and upstream from the consensus TATA box. Genomic Southern blot analysis indicated that the human B1 receptor is encoded by a single-copy gene.

Animals↗
PubMed1990

Characterization of the 5'-flanking region of the human and rat Na,K-ATPase alpha 3 gene.

Genomic clones containing the 5'-flanking region and exon 1 of the human and rat Na,K-ATPase alpha 3 isoform gene have been isolated and characterized. The nucleotide sequences of 1.6 kb of the rat gene and 2.8 kb of the human gene in the 5'-flanking region were determined. Mapping of transcription initiation sites by primer extension and S1 nuclease protection analyses indicates that transcription is initiated in the same region in both genes although the rat gene has a greater number of initiation sites. Neither gene has a canonical TATA box, having instead a ATAT sequence preceding the transcription initiation sites. There is a perfect CCAAT sequence, in the reverse orientation, approximately 30 bp upstream of the potential TATA box in both genes. We have identified potential binding sites for transcription factors Sp-1, AP-1, AP-2, and AP-4, as well as for glucocorticoid and thyroid hormone receptors in the 5'-flanking regions. These are conserved in both human and rat alpha 3 isoform genes.

Animals↗
PubMed1995

Stimulation of RNA polymerase II transcription initiation by recruitment of TBP in vivo.

Eukaryotic transcriptional activators may stimulate RNA polymerase II activity by promoting assembly of preinitiation complexes on promoters through their interactions with one or more components of the basal machinery. On the basis of its central role in initiating transcription-complex formation upon binding to the TATA box, the general transcription factor TFIID, which includes the TATA-binding protein (TBP) and several TBP-associated factors, has been implicated as a target for activators. Consistent with this idea, an increasing number of activators have been reported to bind directly to TBP. To assess the functional importance of these in vitro interactions for transcriptional regulation in vivo, we made use of a novel strategy in yeast to show that a physical interaction with TBP is sufficient for a sequence-specific DNA-binding protein to increase initiation of transcription by RNA polymerase II. These results imply that binding of TFIID to promoter elements is a limiting step in transcription complex assembly in vivo.

DNA-Binding Proteins↗
PubMed2001

DNA sequence-dependent differences in TATA-binding protein-induced DNA bending in solution are highly sensitive to osmolytes.

The complex formed between the TATA-binding protein (TBP) and the "TATA box" of eukaryotic class II promoters is the foundation for assembly of the complex to which RNA polymerase II is ultimately recruited. TBP binds productively to canonical and diverse variant TATA sequences with >100-fold differences in transcription efficiency. Co-crystals of canonical sequences and >11 variant sequences bound to various TBP molecules all have approximately 80 degrees bends. In contrast, the bend angles for TBP-TATA complexes in solution, derived from distance distributions, are approximately 80 degrees for a canonical sequence but range from 30 degrees to 62 degrees for five variant sequences. We show in this study that the osmolytes used to crystallize TBP-TATA complexes induce profound increases in the DNA bends of two transcriptionally active TBP-bound variant sequences to a common angle of approximately 80 degrees but have little effect on a transcriptionally inactive variant. The effect of osmolyte on the TBP-induced DNA bend of a variant TATA box sequence is also manifest in the kinetics of association, demonstrating a functional consequence of an osmolyte-induced structural change.

DNA↗
PubMed2005

A highly distinctive mechanical property found in the majority of human promoters and its transcriptional relevance.

A recent study revealed that TATA boxes and initiator sequences have a common anomalous mechanical property, i.e. they comprise distinctive flexible and rigid sequences when compared with the other parts of the promoter region. In the present study, using the flexibility parameters from two different models, we calculated the average flexibility profiles of 1004 human promoters that do not contain canonical promoter elements, such as a TATA box, initiator (Inr) sequence, downstream promoter element or a GC box, and those of 382 human promoters that contain the GC box only. Here, we show that they have a common characteristic mechanical property that is strikingly similar to those of the TATA box-containing or Inr-containing promoters. Their most interesting feature is that the TATA- or Inr-corresponding region lies in the several nucleotides around the transcription start site. We have also found that a dinucleotide step from -1 to +1 (transcription start site) has a slight tendency to adopt CA that is known to be flexible. We also demonstrate that certain synthetic DNA fragments designed to mimic the average mechanical property of these 1386 promoters can drive transcription. This distinctive mechanical property may be the hallmark of a promoter.

Animals↗
PubMed2005

Genomic organization and functional analysis of the gene encoding the Krüppel-like transcription factor KLF6.

The Krüppel-like transcription Factor 6 (KLF6) is regulated during cell proliferation and differentiation events like mammalian development and tissue regeneration, while its aberrant expression is associated with tumor formation. To investigate KLF6 transcriptional control, the genomic organization of human KLF6 together with its cis-regulatory region was analyzed. A high sequence homology of KLF6 regulatory regions was found in mammals, which in turn predicts a high degree of evolutionary conserved transcriptional mechanisms. A transcription start site was identified at the first nucleotide downstream of a potential initiator element. Also, the role of KLF6 regulatory regions was determined by transfection experiments. A minimal promoter region lacking a TATA-box yet containing an Initiator was identified and found to be active in all cells analyzed. In addition, two strong activating sequences were located between positions -407/-344 and -307/-207, where the latter contained Sp1 and CAAT-box sites. Furthermore, ectopic expression of Sp1 increased the transcriptional activity of the KLF6 promoter. In conclusion, our data revealed that KLF6 gene transcription is under control of a TATA-box independent initiation mechanism together with an evolutionary conserved array of positive cis-acting elements.

Animals↗
PubMed1985

Sequence structures of a mouse major urinary protein gene and pseudogene compared.

Laboratory mouse strains carry approximately 35 major urinary protein (MUP) genes per haploid genome, tightly clustered together on chromosome 4. Most belong to two main groups (Groups 1 and 2). The available evidence strongly suggests that the Group 1 genes are active while the Group 2 genes are pseudogenes. Here we present the complete sequence of a Group 1 gene and a Group 2 gene and 700 bp of flanking sequence. The sequence of the Group 1 gene is consistent with its being active. The Group 2 gene contains two stop codons and a frame-shift mutation in the reading frame defined by the Group 1 gene, and would code for a signal peptide 25 rather than 19 amino acids long. The Group 2 gene differs from the Group 1 gene in other ways: a deletion upstream of the TATA box and another in intron 3, a base change in the TATA box itself, a 2 bp duplication at the splice acceptor boundary of intron 6, an altered poly(A) addition signal and a 1-base deletion 5' to the initiation codon. Some of these differences may explain the 10- to 20-fold higher level of Group 1 mRNA in mouse liver, and the fact that Group 1 and Group 2 transcripts are mainly spliced differently. The presence of the stop codon means that the Group 2 gene is a pseudogene in the context of the Group 1 gene. However, there is some evidence that the mature hexapeptide that it would code for may have biological activity. The 12 acceptor splice sites of the two genes all contain the identical sequence ACAG at the exon boundary.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1999

Properties of the adenovirus type 40 E1B promoter that contribute to its low transcriptional activity.

The adenovirus type 5 (Ad5) E1B promoter contains two elements essential for maximal activity, a TATA box and a GC box. The enteric adenovirus type 40 (Ad40) E1B promoter has a TATA box sequence identical to that of Ad5 and a GC box that fits the Sp1 binding site consensus. Nevertheless, Ad40 E1B RNA synthesis is severely impaired in HeLa cells, attributable in part at least to the weak transactivating activity of Ad40 E1A. However, the responsiveness of Ad40 early promoters to E1A transactivation has not been directly demonstrated. Using a transient expression assay with a chloramphenicol acetyl transferase (CAT) reporter gene, the Ad40 E1B promoter was very poorly transactivated by E1A of both Ad40 and Ad5 and showed only a limited response to the promiscuous varicella zoster virus transactivator p140. Construction of Ad5 recombinant viruses expressing the CAT gene under the control of the Ad5 or Ad40 E1B promoter allowed detection and measurement of expression from the Ad40 E1B promoter in a well-defined background and showed that overall activity is some 100-fold lower than for the Ad5 E1B promoter. Deletion analysis revealed that sequences upstream of the Sp1 binding site down-modulated Ad40 E1B promoter responsiveness, and two protein binding sites, identified by DNase footprinting and gel retardation assay, may be implicated in this effect. Gel shift analysis also showed that the Ad40 Sp1 binding site had a reduced affinity for Sp1 protein, relative to the Ad5 site, and that the context as well as the core sequence had an influence on Sp1 recognition.

Adenovirus E1B Proteins↗
PubMed1993

Two histone H1-encoding genes of the green alga Volvox carteri with features intermediate between plant and animal genes.

Southern hybridization indicated the presence of at least two and possibly four histone H1-encoding genes occurring as singlets in the Volvox carteri genome. Two of these genes, H1-I and H1-II, have been cloned and characterized. Their coding sequences are each interrupted by three introns, but only the position of the second intron is identically conserved in both H1-I and H1-II. The encoded 260-amino-acid (aa) (H1-I) and 240-aa (H1-II) polypeptides possess the typical tripartite organization of animal H1 histones, with variable N- and C-terminal domains flanking a conserved 'globular' DNA-binding domain. Extensive differences in their variable regions suggest that H1-I and H1-II (62% identity) represent two isotypes with different functions. A prominent KAPKAP-KAA motif in the H1-I N-terminal region, similarly seen in single H1 variants of a mosquito and a nematode, has a putative function in packing condensed subtypes of chromatin. Different from higher plants, but like animals, the H1 genes of V. carteri possess a typical 3' palindrome for mRNA processing, resulting in non-polyadenylated mRNAs. Transcription initiates 33 nucleotides (nt) (H1-I) and 26 nt (H1-II) downstream of typical TATA boxes. A putative 20-bp conserved enhancer element upstream of each TATA box closely resembles the consensus sequence associated with the nucleosomal histone-encoding genes in V. carteri [Müller et al., Gene 93 (1990) 167-175] and suggests stringent regulation. Accordingly, transcription of H1 was shown to be restricted to late embryogenesis, when new flagella are produced. We discuss the inferred accessory role of histone H1 proteins in stabilizing axonemal microtubules, as has been recently observed in sea urchin flagella [Multigner et al., Nature 360 (1992) 33-39].

Amino Acid Sequence↗
PubMed1998

Functional analysis of the human annexin I and VI gene promoters.

To gain insight into the molecular basis of annexin gene expression we have analysed the annexin I and VI gene promoters. A previously described 881 bp sequence immediately upstream of the annexin I transcription start site and a similar size fragment proximal to the annexin VI transcription start site both drove expression of the luciferase reporter gene in fibroblasts and epithelial cells. Neither promoter displayed any sensitivity to dexamethasone, suggesting that the putative glucocorticoid response element in the annexin I promoter is non-functional. Consistent with this, endogenous annexin I gene expression was unaffected by dexamethasone at the mRNA and protein levels in A431 cells. A series of 5' deletions of the two promoters were examined to define the minimal active sequences. For annexin I this corresponded to a sequence approx. 150 bp upstream of the transcription start site that included CAAT and TATA boxes. Unexpectedly, the annexin VI promoter, which also contains CAAT and TATA boxes, was fully active in the absence of these elements, a 53 bp sequence between these boxes and the transcription start site having maximal activity. Electrophoretic mobility-shift assays with nuclear extracts from A431 and HeLa cells with probes corresponding to this region revealed an SP1-binding site. These results show that the annexin I and VI genes have individual modes of transcriptional regulation and that if either annexin I or annexin VI has an anti-inflammatory role, then this is in the absence of steroid-induced gene expression.

Annexin A1↗
PubMed2000

Conserved protein motifs and structural organization of a fish gene homologous to mammalian apolipoprotein E.

Apolipoprotein E (apoE) plays a central role in lipid metabolism from its ability to interact with lipoprotein receptors. Besides its role in cardiovascular diseases, apoE polymorphism contributes to susceptibility to neurodegenerative diseases, such as Alzheimer's disease. The statistical significance of the combined match scores obtained after apoE motif-based protein sequence database searches, the structural features of the deduced protein, and the phylogenetic analysis, support the evidence that a homologue to mammalian apoE can be found in teleost fish. Isolation and characterization of the first nonmammalian APOE revealed that the zebrafish gene spans 2555/2692 bp instead of 3597 bp in human and has the same splice junctions and exon/intron organization as found in mammals, except that there is an additional intron that splits the last exon (exon 4) into two exons (exons 4 and 5). Enlargement of APOE size in the mammalian lineage occurs mainly by Alu repeats insertion. The additional intron found in zebrafish gene was also identified at the same splicing site in trout APOE and is located in the corresponding linker region following the conserved low density lipoprotein receptor binding domain. Primer extension and reverse transcriptase PCR (RT-PCR) assays demonstrated that two transcription start sites are located 26 and 28 bp upstream of the first intron and 22 or 24 bp downstream from a canonical TATA box. Sequence inspection of the 5'-flanking region upstream of the TATA box revealed potential regulatory DNA elements. These results will serve as a basis for comparative studies on transcriptional and post-transcriptional mechanisms of APOE regulation in vertebrates.

Amino Acid Motifs↗