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Analysis of the selenocysteine tRNA[Ser]Sec gene transcription in vitro using Xenopus oocyte extracts.

An in vitro transcription system was devised using a Xenopus oocyte S150 extract for analyzing expression of the Sec tRNA[Ser]Sec gene. The activator element, located at about -200 that is required for maximal expression of the tRNA[Ser]Sec gene in vivo, had no effect on tRNA[Ser]Sec transcription in vitro. In vitro transcription of this gene even tolerated mutations within or deletion of the PSE, showing marked contrast with the requirements observed in vivo. However, the TATA box was indispensable for basal level expression of the gene both in vivo and in vitro. The region spanning from the TATA box to the transcription start point (-33 to -1) was sufficient for Pol III to recognize the tRNA[Ser]Sec gene promoter in vitro.

Animals↗

Molecular cloning of the mouse gene coding for carbonic anhydrase IV.

Carbonic anhydrase IV (CA IV) is expressed on apical surfaces of renal tubular epithelium and endothelium of specialized capillary beds. It plays a key role in bicarbonate reabsorption in kidney and in CO2 transport in other tissues. The human cDNA and genomic sequences have been cloned and characterized. Here we report the cloning and characterization of the entire mouse CA IV gene (contained in two overlapping lambda clones), which should enable generation of targeting constructs for disrupting the mouse CA IV gene to produce mouse models for in vivo analysis of CA IV gene function. The gene is approximately 8.2 kb long and contains eight exons ranging from 54 to 434 bp in length. The first exon (exon 1a) encodes the signal sequence. Exons 1b through 7 encode the remaining coding sequences. Exon 7 encodes the C terminus of the membrane-associated protein, as well as the 242-bp 3' untranslated sequence. The nucleotide sequence alignment between mouse and rat CA IV cDNAs reveals 84% identity. The nucleotide sequence alignment between mouse and human CA IV shows 69% identity in the coding region and all of the exon-intron boundaries are conserved, as are the sizes of the introns. The corresponding mouse and human exons are similar, except for the length of the untranslated regions in exons 1a and 7 and two small insertion/deletion events in exons 1a and 4. The 5' flanking region of the mouse gene (-300 to -1) is GC rich and contains 16 CpG dinucleotides. A TATA box sequence and several transcription factor binding sequences are identified upstream of exon 1a. Comparison of the nucleotide sequences surrounding the TATA box (-300 to -1) between mouse and human CA IV genes revealed 70% identity, indicating that regulatory sequences are as highly conserved as coding sequences between mouse and human CA IV genes.

Animals↗

Transcriptional functions of a new mammalian TATA-binding protein-related factor.

A mammalian protein highly homologous to TATA-binding protein (TBP) has been identified and cloned. The recombinant mammalian TBP-related factor binds to the TATA box of the Ad-MLP and forms stable complexes with TFIIB on the promoter DNA. The mammalian TBP-related factor is able to substitute for TBP in supporting transcription by RNA polymerase II in an in vitro reconstituted system.

Amino Acid Sequence↗

Identification of a functional TATA element in the STA2 glucoamylase gene promoter from Saccharomyces cerevisiae.

Accurate transcription by RNA polymerase II is usually dependent on the presence of a TATA element, and/or an initiator element, in the promoters of protein-encoding genes. The STA1-3 genes, encoding three glucoamylase isozymes (Sta1p, Sta2p and Sta3p, respectively) responsible for starch hydrolysis in the yeast Saccharomyces cerevisiae, have been shown to contain long and complex promoters with several regulatory regions. These promoters are also virtually identical to the yeast MUC1 gene promoter; this gene encodes a mucin-like protein and is evolutionary linked to, and transcriptionally co-regulated with, STA1-3. The STA1-3 genes contain two putative TATA sequences; one conforming to the typical TATA box sequence, TATAAA, and another with the sequence of TATAAT. Here we present a study into the functional relevance of these putative TATA sequences and their effects on the transcription of the STA2 gene (as a representative model of the STA1-3 multigene family) and, by analogy, the MUC1 gene. We show that the TATAAA motif is the functional TATA box for STA2 and influences transcript levels, transcript initiation sites, and glucoamylase activities.

Enzyme Activation↗

TATA elements direct bi-directional transcription by RNA polymerases II and III.

Eukaryotic promoter elements specify the direction and efficiency of transcription, as well as the type of RNA polymerase to be used. One such element, the TATA box, is thought to participate in determining the direction of transcription and can function within promoters for RNA polymerase II or III, depending on the sequence context. In this report the ability of four different TATA boxes to support transcription in vitro was determined. It was found that TATA elements are not directional. However, they support transcription by RNA polymerases II and III. An upstream activating sequence was found to stimulate downstream transcription by RNA polymerase II and to inhibit upstream transcription by RNA polymerases II and III. Thus a promoter necessarily consists of a TATA element and upstream sequences in order to specify the direction of transcription and the type of polymerase to be used.

Acanthamoeba↗

Islet amyloid polypeptide: structure and upstream sequences of the IAPP gene in rat and man.

Islet amyloid polypeptide (IAPP) or amylin is a pancreatic islet hormone which was first found in amyloid in insulinomas and in pancreases of patients with type 2 diabetes. In rat a similar polypeptide occurs; however, pancreatic amyloid in this species has not been described. Here we report the structure of the rat and human IAPP gene. Both consist of three exons and two introns which are very similar. The upstream sequence of the rat IAPP gene contains a TATA-box, a CCAAT-sequence and a GT-element, whereas the upstream sequence of the human IAPP gene contains a TATA-box and a rat insulin enhancer-like sequence. This suggests that the rat and human IAPP gene may be controlled differently at the transcriptional level.

Amyloid↗

Herpes simplex virus transactivator ICP4 operationally substitutes for the cellular transcription factor Sp1 for efficient expression of the viral thymidine kinase gene.

The herpes simplex virus type 1 (HSV-1) ICP4 protein is a transcriptional activator of many eucaryotic RNA polymerase II promoters. The HSV-1 thymidine kinase gene (tk) promoter is induced by ICP4 and contains binding sites for the cellular transcription factors TFIID, Sp1, and CCAAT-binding proteins, each of which affects expression of the tk gene. In this study, the effects of mutations in these sites on the transcription of tk in the presence and absence of ICP4 were determined during viral infection. Only the TATA box was necessary for efficient expression in the presence of ICP4; however, ICP4 apparently can still induce tk transcription even when the TATA box is disrupted. Alteration of the Sp1 sites had a minor effect on ICP4-induced expression in comparison to a large effect in the absence of ICP4, indicating that ICP4 can operationally substitute for the function of the transcription factor Sp1. In addition, tk was still expressed with the kinetics of an early gene in the absence of binding sites for Sp1 and CCAAT-binding proteins.

Animals↗

Identification of human monoamine oxidase (MAO) A and B gene promoters.

The promoter of human monoamine oxidase (MAO) A and B genes have been identified. The core promoter region of MAO A is comprised of two 90 bp repeats each of which contains two Sp1 elements and lacks a TATA box. The MAO B core promoter region contains two sets of overlapping Sp1 sites which flank a CACCC element all upstream of a TATA box. The different organization of the MAO A and B promoters may underlie their different cell and tissue specific expression.

Base Sequence↗

Cloning and chromosomal mapping of a human immunodeficiency virus 1 "TATA" element modulatory factor.

A critical regulatory element in many promoters transcribed by RNA polymerase II is the "TATA" box, which is located 25-30 nucleotides upstream of the transcription initiation site. TFIID is a biochemically defined HeLa cell nuclear fraction containing a transcription factor activity that binds specifically to the TATA box and is critical in determining both basal and regulated promoter activity. Recently, the gene for a TATA-binding protein was cloned and found to bind to various TATA elements and to substitute for TFIID in stimulating basal gene expression in in vitro transcription systems. However, it is possible that additional cellular factors can bind to the TATA element and influence the level of gene expression. By using lambda gt11 expression cloning with oligonucleotides corresponding to the human immunodeficiency virus 1 TATA element, we report the identification of a cellular protein with a calculated molecular mass of 123 kDa that we designate TATA element modulatory factor (TMF). TMF binds to the human immunodeficiency virus 1 TATA element in gel-retardation assays and inhibits activation of the viral long terminal repeat by the TATA-binding protein in in vitro transcription assays. TMF contains leucine-zipper amino acid motifs and exhibits homology in its DNA binding domain with the phage-encoded DNA binding protein Ner. Chromosomal mapping localizes the TMF gene to human chromosome 3p12-p21, which is a site of frequent rearrangements in lung and renal carcinomas. Thus, TMF is a transcription factor that likely regulates the expression of both viral and cellular genes.

Amino Acid Sequence↗

Several different upstream promoter elements can potentiate transactivation by the BPV-1 E2 protein.

The enhancer and upstream promoter regions of RNA polymerase II transcribed genes modulate the rate of transcription initiation and establish specific patterns of gene expression. Both types of region consist of clusters of DNA binding sites for nuclear proteins. To determine how efficiently the same factor can activate transcription when acting as an enhancer or promoter factor, we have studied transactivation by the BPV-1 E2 protein, a papillomavirus transcriptional regulator. By cotransfecting a BPV-1 E2 expression vector and a series of reporter plasmids containing well-defined chimeric promoters we have found that whilst E2 can strongly stimulate complex promoters such as that of the HSV tk gene, it does not efficiently activate constructions containing only a TATA box and initiation site. We show that insertion of upstream promoter elements, but not of spacer DNA, between E2 binding sites and the TATA box greatly increases E2 activation. This effect was observed with more than one type of upstream promoter element, is not related to the strength of the promoter and is unlikely to result from co-operative DNA binding by E2 and the transcription factors tested. These results would suggest that E2 has the properties of an enhancer rather than promoter factor and that in certain cases promoter and enhancer factors may affect different steps in the process of transcriptional activation.

Base Sequence↗

Characterization of the mouse type X collagen gene.

Type X collagen, a homotrimer of alpha 1 (X) polypeptide chains, is specifically expressed by hypertrophic chondrocytes in regions of cartilage undergoing endochondral ossification. We have previously characterized a genomic clone containing the major part of the mouse type X collagen gene (col10a1) and assigned the locus for col10a1 to mouse chromosome 10 (Apte et al., Eur. J. Biochem. 224: 217-224, 1992). In this paper, through additional characterization of cDNA and genomic clones, we describe the complete organization of the col10a1 gene. The col10a1 gene is 7.2 kb in size and, as in the chick, col10a1 gene transcripts are generated from only three exons. Exon 1 encodes only the 5' untranslated (5' UT) region of the mRNA and is separated from exon 2 by an intron 562 bp in length. Exon 2 (169 bp) encodes 15 bp of 5' UT message, the translation start plus 17-amino acid residues of the putative signal peptide, and 33 1/3 codons of the putative N-terminal non-collagenous (NC2) domain of type X collagen. Exon 3 is 2854 bp in size and encodes 4 2/3 codons of the NC2 domain, the entire collagenous domain of 463 residues (COL), the entire C-terminal non-collagenous (NC1) domain (161 amino acid residues) and the entire 965 bp 3' untranslated (3' UT) sequence of the mRNA. Two TATA boxes are present in tandem in the col10a1 promotor. Both TATA boxes are active in transcription, generating two populations of transcripts with different 5'-termini; the longer transcript is of low abundance and is detectable only by PCR in newborn mice. The col10a1 promotor contains a CCAAT box as well as other consensus sequence elements required for binding of potential transcription factors. Characterization of the col10a1 gene provides data essential for studies of the regulation of type X collagen expression during mammalian endochondral bone growth and development. Knowledge of the complete structure of the mouse type X collagen gene will also be useful for the investigation of type X collagen gene abnormalities in murine chondrodysplasias and for the generation of transgenic mice.

Amino Acid Sequence↗

Cell-specific activation of the glial-specific JC virus early promoter by large T antigen.

JC virus causes the human demyelinating disease progressive multifocal leukoencephalopathy by selective infection of glial cells. This cell specificity results from glial-specific expression of viral early genes (large and small T antigens). Analysis of transcriptional regulation by the MH1 JC virus early promoter demonstrates that glial specificity is directed by the basal promoter. Because T antigen regulates the basal region of several viral and cellular promoters, we investigated whether it controls the JC virus basal promoter in a glial-specific manner. A JC virus T antigen expression plasmid generated a 95-kDa protein which exhibited nuclear localization and physical association with p53. T antigen repressed the JC virus and SV40 early promoters 4- to 5-fold in glioma cells. Conversely, T antigen induced 100- to 200-fold activation of the JC virus early promoter in nonglial cells, whereas the SV40 promoter was repressed. Activation required the JC virus TATA box sequence and a pentanucleotide repeat immediately upstream of the TATA box, but was independent of the upstream enhancer region. These data demonstrate that the JC virus basal promoter is responsible for glial-specific gene expression and suggest a mechanism for this regulation.

Antigens, Polyomavirus Transforming↗

RAP30/74: a general initiation factor that binds to RNA polymerase II.

We have previously shown by affinity chromatography that RAP30 and RAP74 are the mammalian proteins that have the highest affinity for RNA polymerase II. Here we show that RAP30 binds to RAP74 and that the RAP30-RAP74 complex (RAP30/74) is required for accurate initiation by RNA polymerase II. RAP30/74 is required for accurate transcription from the following promoters: the adenovirus major late promoter, the long terminal repeat of human immunodeficiency virus, P2 of the human c-myc gene, the mouse beta maj-globin promoter (all of which have TATA boxes), and the mouse dihydrofolate reductase promoter (which lacks a TATA box). RAP30/74 is not required for initiation by RNA polymerase III at the adenovirus virus-associated RNA promoters. Therefore, RAP30/74 is a general initiation factor that binds to RNA polymerase II.

Carrier Proteins↗

Identification of an upstream activation sequence and other cis-acting elements required for transcription of COX6 from Saccharomyces cerevisiae.

Transcription of Saccharomyces cerevisiae COX6, the nuclear gene for subunit VI of cytochrome c oxidase, is activated in heme-proficient cells, requires the HAP2 gene, and is subject to glucose repression. In this study, by deletion mutagenesis of the COX6 promoter, we identified two regions that are important for transcription. The first was an upstream activation site, UAS6. It was found to be contained within an 84-base-pair (bp) sequence, between bp -256 and -340 of the COX6 translational initiation codon, and to contain sequences required for activation by heme and HAP2 and for release from glucose repression. When located upstream of a CYC1-lacZ fusion gene, deleted for both of its UASs, this segment functioned as a UAS element. Although UAS6 could promote expression in either orientation, it showed a marked orientation dependence in its response to HAP2 and the carbon source. The second region lay between bp -255 and -91. It contained two of the three major 5' termini of COX6 mRNAs and a putative TATA box. Deletion analysis of this region demonstrated that the putative TATA box is not required for transcription and that this region is separable into two redundant domains.

Chromosome Deletion↗

Core promoter-selective RNA polymerase II transcription.

The initiation of mRNA synthesis in eukaryotic cells is a complex and highly regulated process that requires the assembly of general transcription factors and RNAP II (RNA polymerase II; also abbreviated as Pol II) into a pre-initiation complex at the core promoter. The core promoter is defined as the minimal DNA region that is sufficient to direct low levels of activator-independent (basal) transcription by RNAP II in vitro. The core promoter typically extends approx. 40 bp up- and down-stream of the start site of transcription and can contain several distinct core promoter sequence elements. Core promoters in higher eukaryotes are highly diverse in structure, and each core promoter sequence element is only found in a subset of genes. So far, only TATA box and INR (initiator) element have been shown to be capable of directing accurate RNAP II transcription initiation independent of other core promoter elements. Computational analysis of metazoan genomes suggests that the prevalence of the TATA box has been overestimated in the past and that the majority of human genes are TATA-less. While TATA-mediated transcription initiation has been studied in great detail and is very well understood, very little is known about the factors and mechanisms involved in the function of the INR and other core promoter elements. Here we summarize our current understanding of the factors and mechanisms involved in core promoter-selective transcription and discuss possible pathways through which diversity in core promoter architecture might contribute to combinatorial gene regulation in metazoan cells.

Animals↗

A sequence that directs transcriptional initiation in yeast.

While RNA polymerase II of the yeast Saccharomyces cerevisiae initiates transcription at discrete sites, these sites are located over a wide range of distances from the TATA box for different genes. This variability has led to a number of proposals for consensus sequences located at the initiation site which, in conjunction with the TATA box, would direct initiation. We tested this hypothesis via oligonucleotide-directed mutagenesis, by placing the sequence CAAG, a member of one of these consensus sequences, upstream of the coding sequence of the CYC7 gene at a site at which initiation does not occur. The distance between the TATA sequence and this putative initiation site was varied by inserting it into the wild-type gene and three deletion mutants. The results demonstrated that this sequence can serve as an initiation site when located 49, 77, or 106 bp from the TATA sequence, but not when located 30 bp away.

Base Sequence↗

Cloning of the gene coding for a human receptor for formyl peptides. Characterization of a promoter region and evidence for polymorphic expression.

Recently we reported that, in HL-60 cells, transcription of the formyl peptide receptor (FPR) gene can be up- and downregulated by agents that induce differentiation of HL-60 cells into neutrophils. To begin studying the mechanisms involved in regulation of FPR gene expression, we cloned two human cDNAs and the gene coding for FPR. The genomic clone (pINF14) contained a 14.5-kb insert. A 2.7-kb EcoRI fragment was obtained from pINF14 that hybridized with an FPR open reading frame probe. The EcoRI fragment was sequenced and found to contain an intronless FPR open reading frame. Sequence alignment of the EcoRI genomic fragment with the FPR cDNA revealed that the first 31 bases of 5' untranslated FPR cDNA were not represented in the genomic fragment. Furthermore, a splicing consensus sequence was present in the genomic fragment at the site of divergence with the cDNA sequence. Restriction mapping and Southern blot analysis identified a 121-bp fragment that contained the sequence corresponding to the first 31 bases of 5' untranslated FPR cDNA. An additional (previously undescribed) 15-bp cDNA sequence in the 5' end of FPR were identified using an anchored polymerase chain reaction. This sequence was also contained in the genomic 121-bp fragment. This 121-bp fragment was located 5.2 kb (intron) upstream of the FPR open reading frame. It contained an unusual TATA box and displayed transcriptional activity in vitro and in vivo. Potential binding sites for AP-1 and glucocorticoid receptor were identified upstream of the putative TATA box.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

Structure and regulation of rat liver microsomal stearoyl-CoA desaturase gene.

The cosmid library of rat genomic DNA was screened with the coding region of microsomal stearoyl-CoA desaturase (SCD) cDNA as the probe. Two independent clones having 36 kb (SCDI) and 31 kb (SCDII) of inserts were isolated. Southern mapping analyses revealed that SCDI codes for SCD and SCDII for an SCD-homologue, and that the genes differ markedly in the upstream regulatory regions. Nucleotide sequence analysis of SCDI revealed that the SCD gene is organized with 6 exons and 5 introns spanning about 15 kb, thus coding an mRNA with an open reading frame of 1,074 bp of the desaturase. Transcription starts at 114 by upstream of the translation initiation codon, and 32 bp upstream of the transcription initiation site there is a TATA box. A sequence similar to Fat Specific Element 2 (FSE2), the negative regulatory element of adipocyte differentiation, was present at 45 bp upstream of the TATA box. The tissue distribution and induction due to dietary manipulation of SCDI and SCDII mRNAs as revealed by Northern hybridization were markedly different from each other.

Adipose Tissue↗