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PubMed1993

Organization, sequence, and expression of the murine S100 beta gene. Transcriptional regulation by cell type-specific cis-acting regulatory elements.

The organization, sequence, and transcriptional regulation of expression of the murine S100 beta gene are reported. The gene is approximately 9 kilobase pairs in length and is composed of three exons and two introns. The deduced murine S100 beta protein sequence differs from the human S100 beta protein by only 1 amino acid. The murine S100 beta gene contains a TATA box (AATAA) and a reverse CCAAT box (ATTGG) located at 30 nucleotides and 92 nucleotides upstream of the cap site, respectively. A 149-base pair DNA fragment (-157/-9) spanning the TATA box and the reverse CCAAT box functions as a promoter. The murine S100 beta promoter drives a 4-fold higher level of transcription in glial (C6) than in non-glial (3T3) cells, suggesting the existence of a potential cell type-specific regulatory element within the promoter region. The 5'-flanking region suppresses transcription from the homologous S100 beta as well as the heterologous SV40 promoters in an orientation-independent fashion. However, the 5'-flanking region exhibits cell type specificity when suppressing the S100 beta promoter-dependent transcription, indicating its involvement in the cell type-specific expression of S100 beta gene. In order to map cell type-specific regulatory elements, transcription analyses of various deletions of the 5'-region were carried out in C6 and 3T3 cells. Two cell type-specific negative regulatory elements, one active in non-glial cells and another active in glial cells, were mapped to the regions -1552/-1234 and -1234/-551, respectively. A strong negative regulatory element and a relatively weak negative element were located in the regions -551/-157 and -1669/-1552, respectively. The murine S100 beta gene is under complex transcriptional regulation involving tonic negative control exerted by combination of multiple cis-acting regulatory elements including cell type-specific elements.

Amino Acid Sequence↗
PubMed1994

Transcription stimulation of the adenovirus type 12 E1a gene in vitro by the 266-amino-acid E1A protein.

We previously showed that the 13S but not the 12S mRNA product of the E1a gene of the highly oncogenic type 12 adenovirus (Ad12) stimulates the expression of its own gene. In this study, the mechanism for the autoregulation of the Ad12 E1a gene was investigated in vitro. The 266-amino-acid E1A protein of Ad12 was synthesized in yeast cells and purified as a 57-kDa polypeptide. The purified Ad12 E1A protein stimulated transcription from the proximal promoter of its own gene but had almost no effect on that from the distal promoter. A 35-bp upstream region including a TATA box for the proximal promoter seemed to be sufficient for transcription stimulation by the E1A protein. The Ad12 E1A protein formed a complex with a TATA box-binding protein (TBP), as does the E1A protein of nononcogenic Ad serotypes. Moreover, the E1A protein significantly reduced the binding of TBP to a TATA sequence, while it did not affect the DNA-binding activity of nuclear factor I, a stimulatory protein of the distal transcription of the Ad12 E1a gene. These results suggest that the 13S mRNA product of the Ad12 E1a gene regulates the transcription of its own gene by modulating the activity of TBP.

Adenovirus E1A Proteins↗
PubMed1990

Conservation of essential sequences in the major late promoter and tripartite leader of the simian adenovirus type 30.

We report here the cloning and sequencing of the major late promoter (MLP) and the tripartite leader (TPL) from simian adenovirus type 30 (sAd30) and the comparison of the sAd30 nucleotide (nt) sequence with that of human adenoviruses (hAd). The nt sequence homology between sAd30 and hAd2 is 75% from -66 to +190 relative to the cap site. This sAd30 MLP segment contains the upstream regulatory sequence element, TATA box, and downstream regulatory sequence elements that are homologous to hAd MLP. The sAd30 upstream regulatory sequence has a small palindromic DNA sequence GTCACGTGAC, and the TATA box contains the sequence of ATAAA instead of TATAAA. The sAd30 TPL was located on the sAd30 genome as identified by sequence homology with the hAd counterpart. The splice sites of TPL introns were confirmed by sequence analysis of cDNAs synthesized from sAd30-infected cells. There is a 74.2% nt sequence homology between the TPL of sAd30 and hAd2. The conservation of these sequence elements during evolution of Ad suggests that they are essential for the transcription and translation of Ad ML transcripts.

Adenoviruses, Human↗
PubMed1991

Cloning of the human gene for intercellular adhesion molecule 1 and analysis of its 5'-regulatory region. Induction by cytokines and phorbol ester.

Human intercellular adhesion molecule-1 (ICAM-1), a specific ligand for the lymphocyte function-associated Ag-1 (LFA-1), plays an important role in leukocyte-endothelial cell interactions. It is induced by proinflammatory cytokines such as IL-1, TNF-alpha, or IFN-gamma. However, little is known concerning the intracellular regulatory mechanisms which trigger ICAM-1 up-regulation. In order to study potential regulatory elements involved in ICAM-1 induction we have cloned the human ICAM-1 gene and 5 kb of its 5'-regulatory region. The sequence of the cDNA was found to be distributed over seven exons separated by six introns, whereby each of the five extracellular Ig-like domains of ICAM-1 is encoded by its own exon. The upstream sequence harbors a number of sequence motifs implicated in the regulation and expression of eukaryotic genes, including binding sites for the transcription factors SP-1, AP-1, and NF-kB. Primer extension and S1 nuclease analysis revealed two transcription initiation sites 319 bp and 41 bp upstream of the translation start site. Consensus TATA boxes were found at the expected positions about 25 bp upstream of both start sites. Reverse transcriptase polymerase chain reaction showed differential use of the two TATA boxes in A549 and HS913T cells. Both RNA seem to code for the same for of ICAM-1 protein. For regulation studies a 1.3-kb EcoRI/SalI fragment of the 5'-flanking region was used to promote transcription of a linked luciferase reporter gene in transient-transfection assays in A549 and HS913T cells. Treatment of A549 cells with IL-1 or TNF-alpha resulted in a two- or fourfold increase in luciferase activity. Furthermore, a sixfold induction could be achieved after treatment with the phorbol ester PMA. In contrast, agents that increase intracellular cAMP levels did not induce luciferase activity. Northern blot analysis was used to investigate the kinetics of ICAM-1 mRNA synthesis upon induction with TNF-alpha and PMA. These data suggest that the up-regulation of ICAM-1 by cytokines occurs at least partly at the transcriptional level. Deletion analysis of the 1.3-kb fragment of the 5'-flanking region revealed sequences responsible for promotion and inhibition of transcription. In particular, two functionally distinct regions have been characterized: a short fragment containing an NF-kB binding site has been shown to function as an activator, followed immediately downstream by a sequence acting as a silencer element. Therefore, ICAM-1 gene expression seems to be modulated by multiple cis-acting elements.

Base Sequence↗
PubMed1992

The structure of the mouse parvalbumin gene.

Parvalbumin (PV) is a calcium-binding protein of the EF-hand family, expressed mainly in fast contracting/relaxing muscles of vertebrates. We have isolated five overlapping genomic PV clones which overall span 28 kilobase pairs (kb) around the Pva locus on mouse Chromosome (Chr) 15. The positions of four introns were determined by DNA sequencing. They interrupt the coding sequences at positions corresponding to those in rat and human PV genes. The transcription start site, 25 bp downstream from the TATA-box, was mapped by oligonucleotide primer extension on poly(A)(+)-RNA. The analysis of 0.4 kb promoter sequence of the mouse PV gene revealed CCAAT- and TATA-box sequences and a 59 bp GC-rich stretch between positions -59 and -118. Similar motifs have been found in the parvalbumin genes of rat and human. A perfect 11-bp repeat upstream to positions -149 and -163 respectively is homologous only to the rat promoter. These results will be related to tissue and species differences in PV expression.

Amino Acid Sequence↗
PubMedDate not supplied

Nucleotide sequence of bovine parathyroid hormone messenger RNA.

The sequence of bovine parathyroid hormone mRNA has been determined by sequence analysis of near full-length cloned DNA complementary to the mRNA. Restriction fragments hybridized to the mRNA and extended toward the 5' terminus with reverse transcriptase were analyzed to derive the sequence not present in cDNA. The reverse transcripts were heterogeneous in length with three major stopping points within 8 nucleotides of each other and a minor stop about 30 bases further toward the 5' terminus of the mRNA. The sequence of the gene corresponding to the minor reverse transcript begins with the sequence 5' XXXATATATAAAA which contains the consensus sequence for a TATA box, a putative eukaryotic promoter sequence. Assuming that the major reverse transcriptase stop nearest the 5' terminus of the mRNA, which is 24 bases downstream from the TATA box, represents the beginning of bovine PTH mRNA, the mRNA contains 672 nucleotides, 100 in the 5' noncoding region, 348 in the coding region and 224 in the 3' noncoding region. Bovine PTH mRNA contains 38% G and C bases. The 3' noncoding region is particularly rich in A and U bases with the last 100 nucleotides of the molecules containing 46% U and 32% A. As with other mRNAs, the sequences CG and UAG occur much less than expected. The 5' noncoding region does not contain an AUG before the initiator codon and contains two potential regions that could base-pair with sequences near the 3' terminus of 18S ribosomal RNA. The sequence AAUAAA is present 14 nucleotides from the polyadenylic acid at the 3' terminus. Bovine PTH mRNA exhibits extensive homology with human PTH mRNA.

Animals↗
PubMed2006

Quantitative analysis of in vivo initiator selection by yeast RNA polymerase II supports a scanning model.

Initiation of transcription by RNA polymerase II (RNAP II) on Saccharomyces cerevisiae messenger RNA (mRNA) genes typically occurs at multiple sites 40-120 bp downstream of the TATA box. The mechanism that accommodates this extended and variable promoter architecture is unknown, but one model suggests that RNAP II forms an open promoter complex near the TATA box and then scans the template DNA strand for start sites. Unlike most protein-coding genes, small nuclear RNA gene transcription starts predominantly at a single position. We identify a highly efficient initiator element as the primary start site determinant for the yeast U4 small nuclear RNA gene, SNR14. Consistent with the scanning model, transcription of an SNR14 allele with tandemly duplicated start sites initiates primarily from the upstream site, yet the downstream site is recognized with equivalent efficiency by the diminished population of RNAP II molecules that encounter it. A quantitative in vivo assay revealed that SNR14 initiator efficiency is nearly perfect (approximately 90%), which explains the precision of U4 RNA 5' end formation. Initiator efficiency was reduced by cis-acting mutations at -8, -7, -1, and +1 and trans-acting substitutions in the TFIIB B-finger. These results expand our understanding of RNAP II initiation preferences and provide new support for the scanning model.

Alleles↗
PubMed1986

Sequence divergence and selection of cap sites in the rat gamma-crystallin gene family.

The transcription initiation sites of the six rat gamma-crystallin genes were mapped by combining the results of primer extension and S1 nuclease mapping experiments. To obtain more accurate results from the S1 nuclease mapping experiments, intron-deleted clones were constructed by a novel and efficient modification of existing methods involving the use of primer extension products to seal the exons. Four of the six gamma-crystallin genes have multiple transcription start sites. The major and most of the minor transcripts start with an adenosine. Analysis of the 5' flanking sequences of the gamma-crystallin genes shows that the sequence determining the position of the cap site is merely -CA- and that its optimal distance from the first T of the TATA box is 32 base pairs. Our data further suggest that an A to G transition in the first two base pairs of the Goldberg/Hogness box of one the genes does not affect the position of its major cap site. This, together with the fact that most minor transcription start sites are located upstream from the major cap sites, suggests that in the long TATA boxes of the rat gamma-crystallin genes the major RNA polymerase 'trap site' is not directly at the beginning of the TATA sequence.

Adenosine↗
PubMed1987

Structure of the human oestrogen-responsive gene pS2.

The human pS2 gene, whose expression is restricted to breast cancer cells, and whose transcription is induced by oestrogen in the human breast cancer cell line MCF-7, has been cloned from both placental and MCF-7 cell DNA. The exon-intron organization has been established by electron microscopy using genomic DNA-cDNA or -mRNA hybrid duplexes and by sequencing the exons and exon-intron junctions. The overall organization within and around the pS2 gene is the same in placental and MCF-7 cell DNA and the exonic sequences are identical to those previously determined from the cDNA. The 5'-flanking region of the pS2 gene is also identical (with the exception of two base transitions) in the two tissues. Thus no gene rearrangement nor sequence modification has occurred in the pS2 gene of the malignant and polyploid MCF-7 cells. A TATA-box, a CAAT-box and a GC-rich motif are present in the 5'-flanking region of the pS2 gene, but the latter motif is unusually located between the TATA-box and the capsite. No significant homology could be detected between the 5' flanking sequences of the pS2 gene and those of other oestrogen-responsive genes from different species.

Amino Acid Sequence↗
PubMed1988

Molecular characterization of beta-globin gene mutations in patients with beta-thalassaemia intermedia in south China.

We have studied the spectrum of mutations producting beta-thalassaemia intermedia in South China. The methods of mutation detection include oligonucleotide analysis, polymerase chain reaction amplification of the beta-globin gene and direct genomic sequencing. The mutations have been identified in 22 beta-globin genes from the patients in 11 unrelated families. Seven different mutations have been identified and the A to G substitution in the TATA box of the beta-globin gene accounts for 42% of these mutant beta-globin genes. Most patients have a beta(+) thalassaemia and one copy of the TATA box mutation. In two patients with beta(0) thalassaemia intermedia the mild phenotype may be explained in one by the presence of the - + - + + 5' beta-globin gene cluster haplotype which contains the Xmn I site -158 nt to the G gamma-globin gene or in the other by the number of alpha-globin genes present.

Base Sequence↗
PubMedDate not supplied

Activating protein factor binds in vitro to upstream control sequences in heat shock gene chromatin.

DNA sequences, important for the control of Drosophila heat shock gene expression, are packaged in chromatin in a nuclease hypersensitive configuration. Recently, two protein-binding (exonuclease-resistant) sites which cover the TATA box sequence and an upstream control element were shown to occur in vivo amidst the 5' terminal hypersensitive regions of several heat shock genes. Protein-binding at the TATA box is independent of heat shock, but the binding at the upstream element is heat shock dependent, and it was proposed that a heat shock activator protein, HAP, positively regulates the genes. Here, I report the detection of HAP activity in heat shocked cell extracts by reconstituting specific binding to hsp82 gene chromatin in vitro. Inhibition of the binding by free DNA from the 5' region of heat shock genes implies a coordinate regulation of the gene family through HAP interaction with the upstream heat shock consensus sequence. Furthermore, the special ease of induction of the hsp82 gene over other heat shock genes can be explained in molecular terms by the higher affinity of HAP for the hsp82 binding site, which contains a 28 base sequence with almost perfect dyad symmetry, GAAGCCTCTAGAAG/TTTCTAGAGACTTC.

Animals↗
PubMed1995

The mouse APLP2 gene. Chromosomal localization and promoter characterization.

Senile plaques are primarily comprised of deposits of the beta-amyloid precursor-like proteins APLP1 and APLP2. proteins (APPs). APP is a member of a gene family, including amyloid precursor-like proteins APLP1 and APLP2. Using interspecific mouse backcross mapping, we localized the mouse APLP2 gene to the promixmal region of mouse chromosome 9, syntenic with a region of human 11q. We cloned an approximately 1.2-kilobase mouse genomic fragment containing the APLP2 gene promoter. The APLP2 promoter lacks a typical TATA box, is GC-rich, and contains several sequences for transcription factor binding. S1 nuclease protection analysis revealed the presence of multiple transcription start sites. The lack of a TATA box, the presence of a high GC content, and multiple transcription start sites place the APLP2 promoter in the class of promoters of "housekeeping genes." Regulatory regions within the promoter were assayed by transfection of mouse N2a and Ltk- cells with constructs containing progressive 5'-deletions of the APLP2 promoter fused to the bacterial chloramphenicol acetyl transferase (CAT) reporter gene. A minimal region that includes sequences 99 bp upstream of the predominant transcription start site of the APLP2 promoter was sufficient to direct high levels of CAT expression.

Alzheimer Disease↗
PubMed1990

Functional domains and upstream activation properties of cloned human TATA binding protein.

The TATA binding protein, TFIID, plays a central role in the initiation of eukaryotic mRNA synthesis. Here, we present a human cDNA clone for this factor. Comparison of its predicted protein sequence with those from Drosophila and yeast reveals a highly conserved carboxyl-terminal 180 amino acids. By contrast, the amino-terminal region of TFIID has diverged in both sequence and length. A striking feature of the human protein is a stretch of 38 glutamine residues in the NH2-terminal region. Expression of human TFIID in both Escherichia coli and HeLa cells produces a protein that binds specifically to a TATA box and promotes basal transcription; the conserved COOH-terminal portion of the protein is sufficient for both of these activities. Recombinant TFIID forms a stable complex on a TATA box either alone or in combination with either of the general transcription factors, TFIIA or TFIIB. Full-length recombinant TFIID is able to support Sp1 activated transcription in a TFIID-depleted nuclear extract, while a deletion of the NH2-terminal half of the protein is not. These results indicate the importance of the NH2-terminal region for upstream activation functions and suggest that additional factors (co-activators) are required for mediating interactions with specific regulators.

Amino Acid Sequence↗
PubMed1991

Structure of the 5'-flanking regulatory region and gene for the human growth factor-activatable Na/H exchanger NHE-1.

The amiloride-sensitive, growth factor-activatable Na/H exchanger (NHE-1) is a ubiquitous mammalian protein that is involved in the regulation of intracellular pH and cell volume. We have determined the intron/exon boundaries and the transcription initiation sites and have characterized a portion of the 5'-flanking regulatory region of the human NHE-1 gene. The Na/H exchanger gene spans approximately 70 kilobases. The coding region is divided into 12 exons and 11 introns, one of which is 41.5 kilobases in length. The first exon contains the entire 5'-noncoding region, which is 786 bases long, and 352 bases of the coding sequence. Primer extension identified two discrete start sites for RNA polymerase. 1377 bases of the 5'-regulatory region were sequenced. The promoter/enhancer region is characterized by a TATA box, four GC boxes, two CAAT boxes, five CACCC boxes, three Ap-1 sites, a cyclic AMP response element, and four partial glucocorticoid response elements. Promoter activities of a 313- and a 1441-base pair fragment containing the TATA box were demonstrated by their ability to direct chloramphenicol acetyltransferase expression when transiently expressed in fibroblasts.

Animals↗
PubMed1990

Conserved enhancer and silencer elements responsible for differential Adh transcription in Drosophila cell lines.

The distal promoter of Adh is differentially expressed in Drosophila tissue culture cell lines. After transfection with an exogenous Adh gene, there was a specific increase in distal alcohol dehydrogenase (ADH) transcripts in ADH-expressing (ADH+) cells above the levels observed in transfected ADH-nonexpressing (ADH-) cells. We used deletion mutations and a comparative transient-expression assay to identify the cis-acting elements responsible for enhanced Adh distal transcription in ADH+ cells. DNA sequences controlling high levels of distal transcription were localized to a 15-base-pair (bp) region nearly 500 bp upstream of the distal RNA start site. In addition, a 61-bp negative cis-acting element was found upstream from and adjacent to the enhancer. When this silencer element was deleted, distal transcription increased only in the ADH+ cell line. These distant upstream elements must interact with the promoter elements, the Adf-1-binding site and the TATA box, as they only influenced transcription when at least one of these two positive distal promoter elements was present. Internal deletions targeted to the Adf-1-binding site or the TATA box reduced transcription in both cell types but did not affect the transcription initiation site. Distal transcription in transfected ADH- cells appears to be controlled primarily through these promoter elements and does not involve the upstream regulatory elements. Evolutionary conservation in distantly related Drosophila species suggests the importance of these upstream elements in correct developmental and tissue-specific expression of ADH.

Alcohol Dehydrogenase↗
PubMed1983

Transient expression of a mouse alpha-fetoprotein minigene: deletion analyses of promoter function.

The constitutive transcription of a mouse alpha-fetoprotein (AFP) minigene was examined during the transient expression of AFP-simian virus 40-pBR322 recombinant DNAs introduced into HeLa cells by Ca3(PO4)2 precipitation. We tested three constructs, each of which contains the AFP minigene and pBR322 DNAs inserted in the late region of simian virus 40 and found that the relative efficiency of AFP gene expression was dependent on the arrangement of the three DNA elements in the vector. The transcripts begin at the authentic AFP cap site and are properly spliced and polyadenylated. To define a sequence domain in the 5' flanking region of the AFP gene required for constitutive expression, sequential 5' deletion mutants of the AFP minigene were constructed and introduced into HeLa cells. All AFP deletion mutants which retained at least the TATA motif located 30 base pairs upstream from the cap site were capable of directing accurate and efficient AFP transcription. However, when the TATA sequence was deleted, no accurately initiated AFP transcripts were detected. These results are identical to those obtained from in vitro transcription of truncated AFP 5' deletion mutant templates assayed in HeLa cell extracts. The rate of AFP transcription in vivo was unaffected by deletion of DNA upstream of the AFP TATA box but was greatly affected by the distance between the simian virus 40 control region and the 5' end of the gene. The absence of any promoter activity upstream of the TATA box in this assay system is in contrast to what has been reported for several other eucaryotic structural genes in a variety of in vivo systems. A sequence comparison between the 5' flanking region of the AFP gene and these genes suggested that the AFP gene lacks those structural elements found to be important for constitutive transcription in vivo. Either the AFP gene lacks upstream promoter function in the 5' flanking DNA contained within the minigene, or the use of a viral vector in a heterologous system precludes its identification.

Animals↗
PubMed1990

Transcription initiated by RNA polymerase II and purified transcription factors from liver. A complex set of promoter sequences governs formation of the initial complex.

Using a transcription system derived from rat liver, we have investigated the function of the TATA-box and cap site/initiator regions in assembly of stable intermediates on the pathway leading to formation of a functional RNA polymerase II preinitiation complex. Our findings argue that neither the TATA-box region nor the cap site/initiator region functions independently in the binding of transcription factors during formation of these intermediates, but, rather, the two sequence elements function cooperatively to direct assembly of a stable preinitiation complex at the promoter.

Animals↗
PubMed2006

Positioned nucleosomes due to sequential remodeling of the yeast U6 small nuclear RNA chromatin are essential for its transcriptional activation.

Transcription from the yeast SNR6 (U6 small nuclear RNA) chromatin, a gene transcribed by the enzyme RNA polymerase III, depends on its transcription factor IIIC (TFIIIC) and the promoter elements (the intragenic box A and box B located downstream to its terminator) to which TFIIIC binds. The genes transcribed by polymerase III generally lack the upstream promoter elements where TFIIIC is known to recruit the transcription initiation factor TFIIIB. The TFIIIC-dependent chromatin remodeling of the gene in vitro that involves translational positioning of a nucleosome between boxes A and B is found to be essential for its transcriptional activation. We show here that the role of TFIIIC is not limited to the recruitment of TFIIIB on chromatin templates. The pre-binding of TFIIIB to the SNR6 TATA box in the upstream gene region does not alleviate TFIIIC requirement for transcriptional activation of the chromatin. Binding of TFIIIC to an array of pre-positioned nucleosomes results in an upward shift of the single nucleosome between boxes A and B. The approximately 40-bp shift of this nucleosome in the 3' to 5' direction leads to increased nuclease sensitivity of the approximately 40-bp DNA 3' to the upstream TATA box. Further chromatin remodeling accompanies the binding of TFIIIB in the next step. This two-step remodeling mechanism using the basal factors of the gene yields high transcription levels and generates a chromatin structure similar to that reported for the gene in vivo.

Adenosine Triphosphate↗