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PubMed1990

Activity and enhancer binding factors for JC virus regulatory elements in differentiating embryonal carcinoma cells.

We have studied cell-type-specific expression by JC virus (JCV) DNA regulatory sequences using embryonal carcinoma (EC) cells as a model system. In transient transfection assays, JCV enhancer demonstrated activity in retinoic acid-differentiated neuronal type cells but not in undifferentiated or DMSO-differentiated muscle type cells. To correlate in vivo activity with the binding of transcription factors, we performed DNasel footprinting experiments. Retinoic acid-treated EC cell extracts provided three completely protected regions, each containing sequences with homology to nuclear factor 1 (NF1) binding motifs and the partially protected TATA box. Oligonucleotide competition studies suggest that all three NF1 binding motifs are bound by the same factors but with different affinities and that there are cooperative interactions between NF1 proteins binding to adjacent regions. No protected region other than the partially protected TATA box was detected in undifferentiated and DMSO-differentiated EC cells in which JC regulatory sequences were not expressed.

Animals↗
PubMed2004

Truncation and mutagenesis analysis of the human X-arrestin gene promoter.

X-arrestin (arrestin-3) is an arrestin present specifically in the outer segments of red-, green-, and blue-cone photoreceptors. The X-arrestin gene is on Xcen-q22, and consists of 17 exons with a promoter containing a TATA box and elements important for photoreceptor expression, including three CRX and one PCE-1-like element. In order to delineate the promoter structure necessary for the pan-cone-specific expression of X-arrestin, the expression of the gene in retinoblastoma cell lines was investigated, and a structure-function analysis of the promoter was conducted in the appropriate cellular substrate. Expression of X-arrestin was detected at a low level in the Y79 retinoblastoma cell line but not in the WERI retinoblastoma cell line. Truncation and expression analysis of the X-arrestin promoter in Y79 showed maximal activity in the proximal 378-bp region containing the CRX and PCE-1-like elements upstream of the TATA and CAAT boxes and a negative regulator in the distal 1-2-kbp region. Mutagenesis of the three CRX and PCE-1-like elements and expression analysis demonstrated complete elimination of the promoter activity. Mutagenesis of the TATA box and PCE-1-like element individually resulted in similar decrease in promoter activity, but the decrease in the promoter activity was greater when the CRX elements were mutagenized with a 5' to 3' spatial gradient in the negative effect, suggesting a cooperative effect of the three CRX elements. The regulation of expression from this promoter may involve the binding of a multi-protein enhanceosome complex at the CRX triplet and the PCE-1-like element, resulting in the recruitment and activation of the RNA polymerase II complex at the downstream TATA box.

Arrestins↗
PubMed1992

During negative regulation of the human papillomavirus-16 E6 promoter, the viral E2 protein can displace Sp1 from a proximal promoter element.

The principal early promoter of human papillomaviruses (HPVs), designated P97 in the case of HPV-16, contains four characteristically aligned cis-responsive elements, namely one binding site for Sp1, two for the viral E2 proteins, and the TATA box. The Sp1 binding site is needed to mediate activation of P97 by the remote epithelial-specific enhancer, and the two E2 binding sites contribute to a negative feedback-loop of viral gene expression. The Sp1 consensus motif and the TATA-box distal E2 binding site are spaced in all genital papillomaviruses by a single nucleotide. We show here that at physiological concentrations, the binding of E2 proteins and Sp1 are mutually exclusive events, since a bandshift analysis with nuclear extracts from ID13, a mouse cell line transformed by BPV-1, showed only the E2 or the Sp1 bandshift, but no complex indicative of the concomitant binding of both factors. Increasing concentrations of in vitro translated E2 protein compete efficiently with the Sp1 factor for binding to an oligonucleotide containing both binding sites. Interference between Sp1 and E2 protein binding is apparently relevant for P97 repression in vivo, since a mutational analysis revealed that both E2 binding sites are necessary for negative transcriptional regulation: Alone, neither the distal site, where E2 protein can induce Sp1 displacement, nor the proximal site, where E2 protein interferes with formation and function of the pre-initiation complex, have a significant effect, but two functional E2 binding sites lead to repression of P97.

Animals↗
PubMed1986

Nucleotide sequence of a portion of the Autographa californica nuclear polyhedrosis virus genome containing the EcoRI site-rich region (hr5) and an open reading frame just 5' of the p10 gene.

The nucleotide sequence of a 1587 bp region lying within the HindIII-Q fragment of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV) DNA has been determined. It begins in the EcoRI-S-EcoRI-X region, continues to the HindIII-P/Q boundary and contains an open reading frame that codes for a polypeptide of 240 amino acids (p26). This open reading frame is also included in the 1100 and 1500 base transcripts previously mapped to this region. The sequence reveals that the 5' ends of the 1100 and 1500 base transcripts are located 20 bp downstream from the end of a putative TATA box (TAATTAAAT) and 19 bp upstream from the translation start codon (ATG) of the p26 open reading frame. The translation termination codon (TAA) falls in the immediate 5' flanking region of the major late p10 gene of AcMNPV, 3 bp downstream from the putative TATA box. The probable polyadenylation site for the 1100 base transcript lies 23 bp downstream from the cap site for the 750 and 2500 base transcripts encoding the p10 protein. The 5' flanking region of the p26 open reading frame contains the EcoRI site-rich region, hr5, whose sequence is included here. The EcoRI site-rich region, hr5, consists of six imperfect tandem repeats of a sequence that includes the EcoRI recognition site. These direct repeats also include many inverted repeats.

Amino Acid Sequence↗
PubMed1992

A protein-binding site in the c-myc promoter functions as a terminator of RNA polymerase II transcription.

Termination of transcription not only allows polymerases that have completed RNA synthesis to recycle, but it also has important functions in transcriptional regulation and in preventing promoter interference. The molecular basis for termination by RNA polymerase II (pol II) is unclear, however. We have identified a termination site in the promoter region of the c-myc gene, whose function correlates with DNA binding by a nuclear factor. When the c-myc gene was transcribed in injected Xenopus oocytes or a HeLa nuclear extract, a fraction of RNA initiated at the first promoter, P1, terminated at two positions, T1A and T1B, which flank the TATA box of the second promoter, P2. T1B is a T-rich sequence that resembles previously identified attenuation sites, but T1A appears to represent a different class of termination site. T1A is situated approximately 10 bases upstream of an element that overlaps the P2 TATA box. Mutagenesis of this element affected both the efficiency and the position at which termination occurred. A 28-base sequence including this element caused a low level of termination when inserted into the alpha-globin gene in either orientation. This sequence bound a factor called TBF I (terminator-binding factor), whose binding specificity correlated with T1A terminator function. We suggest that TBF I may function as a pol II termination factor.

Animals↗
PubMed1996

Identification of regulatory sequences of juvenile hormone-sensitive and -insensitive serum protein-encoding genes.

The promoters of three juvenile hormone (JH)-sensitive, and one JH-insensitive hexamerin-encoding genes (Hex) were isolated from Trichoplusia ni, and sequences necessary for, or affecting, transcriptional activity were identified by biochemical and functional methods. The transcription start points (tsp) for each of the four Hex were determined biochemically, by both primer extension and the sequencing of multiple, independent full-length cDNA clones. The function of each inferred tsp, as an actual tsp, was confirmed by in vitro transcription assay. The transcription initiator sequence, GNACAGT, was identical for three of the Hex, while the fourth used a divergent motif. Using the in vitro transcription system, a minimal core promoter of 60 bp (bp -34 to +24) of the BJHSP1 (basic JH suppressible protein 1) gene, containing a single TATA box motif approximately 30 bp upstream of the tsp, was functionally sufficient to support alpha-amanitin-sensitive transcription. The same construct was also transcriptionally functional in a homologous cell line transfection assay. The corresponding region of the other Hex also contains a similarly positioned TATA box motif, and promoter constructs for each, that included that included the tsp, initiator and inferred basal transcription apparatus binding site, were all transcriptionally functional in a cell line transfection assay. The action of sequences 5' to the minimal promoter region in modulating the rate of transcription was shown by a cell line transfection assay of a nested deletion series of the promoter for the BJHSP1 gene, in which the results identified a strongly suppressive element between positions -160 and -109. This system of Hex genes, including those sensitive to JH and one not sensitive, should be useful in a comparative approach toward identifying those regulatory motifs that are functionally necessary to transduce the regulatory action of JH.

Animals↗
PubMed2006

Comparative analysis of core promoter region: information content from mono and dinucleotide substitution matrices.

We have studied the core promoter region in five sets of promoter sequences by calculating the average mutual information content H (relative entropy). We have used specially constructed substitution matrices to calculate mono and dinucleotide replacements in a given block of aligned sequences. These substitution matrices use log-odds form of scores, which are in bits of information. Here, we constructed and applied nucleotide substitution matrices for the core promoter region to calculate the information content to study the Transcription Start Site (TSS), TATA-box and downstream regions. As expected, the information content decreases with increasing block size. This clearly implies that the TSS region is likely to be 5-10 bases in size (length). We also notice that both in the case of mouse and humans, both TATA-boxes and TSS regions are likely to play important roles in proper transcriptional initiation.

Amino Acid Substitution↗
PubMed1997

Transcriptional regulation of the CYP11A1 and ferredoxin genes.

The first step in the synthesis of all steroids is the cleavage of cholesterol side chain, catalyzed by an electron transport system located in mitochondria consisting of ferredoxin reductase, ferredoxin, and cytochrome P450scc. These proteins are present in adrenal, gonad, placenta, and some parts of the brain. In addition, ferredoxin and ferredoxin reductase are also found in the kidney and liver. Whereas ferredoxin reductase levels remain constant in the cell, ferredoxin and P450scc levels are stimulated by trophic hormones using cAMP as an intracellular messenger. The ferredoxin promoter is relatively simple, consisting of a TATA box and two Sp1-binding sites. This simple module is enough to direct cAMP-dependent transcription in a steroidogenic cell-specific fashion. The regulatory region for the P450scc gene is more complex, containing many protein binding sites for different regulation purposes. Its TATA box directs cAMP-dependent transcription in a cell-type-specific manner. A transcription factor, steroidogenic factor 1 (SF1), activates P450scc gene expression. The tissue-specific expression of the P450scc gene is probably accomplished through the interaction of SF1 with other protein factors located further upstream of the control region. SF1 may also be involved in the cAMP response. An upstream region binding to cAMP-Responsive Element Binding Protein CREB and AP1 can respond to cAMP for gene activation. These analyses of regulatory elements provide the structural architecture for transcriptional regulation of the ferredoxin and the CYP11A11 gene.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1994

Cloning and characterization of the human thromboxane synthase gene promoter.

A genomic phage clone hybridized to the 5' end of human thromboxane synthase (TS) cDNA was isolated. Sequencing analysis of a 1.7 kb subfragment revealed that it contained the entire 5' untranslated region and 46 bp of the coding sequence of TS cDNA, an upstream canonical TATA box (TATAAA), and several binding sites for transcription factors (AP1, PEA3, PU.1, and GR), indicative of a promoter/first exon region of the TS gene. RNase protection assay mapped the transcription start site of the human TS gene to the nucleotide A 30 bp downstream from the TATA box. The authenticity of the promoter was further confirmed by its ability to direct expression of a CAT reporter gene in transfected HL60 cells.

Base Sequence↗
PubMed1983

The primary structure and expression of four cloned human histone genes.

The complete nucleotide sequence of four human histone genes has been determined. Each gene codes for a core histone protein which is very homologous with the corresponding calf thymus of rat histones. The 5' and 3' flanking regions of the human histone genes contain previously identified concensus sequences: the TATA box, the GACTTC element; the CCAAT sequence; the 3' terminal dyad symmetry element thought to be involved in transcription termination; and a recently identified H2b specific upstream sequence. A putative H2a specific upstream sequence 5'-TTCTTGGACTCCTCTTTTC-3' is present approximately 40 base pairs upstream from the TATA box in the human H2a gene promoter. Nuclease S1 analysis of the human histone mRNAs encoded within each of these clones demonstrates that the mRNA terminii map to the expected positions relative to the known concensus sequences, and that the abundance of each mRNA is regulated during the HeLa cell cycle. Finally, in contrast to the H2b, H3 and H4 mRNAs encoded within clones pHh 4A/pHh4C, pHh5B and pHu4A, respectively, the H2a mRNA encoded by Hh5G is not present in human placental RNA.

Amino Acid Sequence↗
PubMed1992

Transcription in vivo and in vitro of the histone-encoding gene hmfB from the hyperthermophilic archaeon Methanothermus fervidus.

Immediately upstream of the hmfB gene, in a DNA fragment cloned from Methanothermus fervidus, are two identical tandemly repeated copies of a 73-bp sequence that contain the sequence 5'TTTATATA, which conforms precisely to the consensus TATA box element proposed for methanogen promoters. By using this duplicated region as the template DNA and a cell-free transcription system derived from Methanococcus thermolithotrophicus, transcription in vitro was found to initiate at two identical sites 73 bp apart, each 25 bp downstream from a TATA box, thus providing strong evidence for the functional conservation of this transcriptional signal in two phylogenetically very diverse methanogens. Transcription of the hmfB gene in vivo in M. fervidus was found to occur at only one of these sites, and consistent with this observation, recloning and sequencing of this intergenic region after its amplification by the polymerase chain reaction demonstrated that the genome of M. fervidus contains only one copy of the 73-bp sequence upstream of the hmfB gene. Since the second copy of the 73-bp sequence, presumably generated artifactually during the original hmfB cloning, functioned equally well as a promoter in the M. thermolithotrophicus transcription system, all information needed by the heterologous RNA polymerase to initiate transcription accurately in vitro must be present within this sequence. The hmfB gene encodes HMf-2, one of the two subunits of HMf, an abundant DNA binding protein in M. fervidus which binds to DNA molecules in vitro, forming nucleosomelike structures. Cell-free transcription was inhibited by adding HMf or eucaryotic core histones at protein-to-DNA mass ratios of 0.3:1 and 1:1, respectively, whereas the archael histonelike protein HTa from Thermoplasma acidophilum inhibited transcription in vitro only at much higher protein-to-DNA mass ratios and the bacterial histonelike protein HU from Escherichia coli had no detectable effect on transcription.

Archaeal Proteins↗
PubMed1993

Characterization of a structurally tricistronic gene of human cytomegalovirus composed of U(s)18, U(s)19, and U(s)20.

A tricistronic gene mapped between 0.91 and 0.93 map units within the EcoRI D fragment of the human cytomegalovirus unique short region (U(s)) has been cloned, sequenced, and expressed in vitro. Cloned cDNAs of 2.3, 1.8, and 1.1 kb derived from this region were isolated from a lambda gt11 cDNA library made from virus-infected fibroblasts and used for this study. Two major classes of 3'-coterminal mRNAs, 2.8 and 1.1 kb, were transcribed from this region. Sequence analysis of the cDNAs and the upstream genomic DNA revealed three open reading frames (ORFs), U(s)18, U(s)19, and U(s)20, and a common polyadenylation signal located 15 bases upstream of the poly(A) tail of both the 2.85- and 1.1-kb mRNAs. Protein structure analyses predicted the existence of multiple hydrophobic moieties, suggesting that the U(s)18, U(s)19, and U(s)20 polypeptides were transmembrane proteins. The major transcription initiation site, determined by primer extension and S1 nuclease mapping, for the 2.85-kb transcript was located right at the first initiation codon of the U(s)20 ORF. There was no typical TATA box or CAAT box upstream of the 2.85-kb mRNA cap site except for a TATAAGA sequence that was found about 210 bp downstream from the major cap site. The 1.1-kb transcript was initiated 33 bp upstream of the U(s)18 translation initiation site, and an atypical TATA box sequence (GATAAGA) was found 22 bp upstream of the transcription start site. Differences in transcription kinetics and sensitivities to metabolic inhibitors suggest that they were regulated by different mechanisms; the 2.85-kb mRNA belongs to the early (beta) class of transcripts, while the 1.1-kb mRNA is a late (gamma) message. Subgenomic DNA segments derived from the U(s)18, U(s)19, and U(s)20 ORFs were subcloned and overexpressed in Escherichia coli as fusion proteins with glutathione-s-transferase. Western immunoblot analysis with antibodies against the U(s)18, U(s)19, and U(s)20 fusion proteins detected virus-specific polypeptides with molecular sizes of 36, 32, and 43 kDa, respectively. All three antibodies also exhibited a positive immunofluorescence reaction with human cytomegalovirus-infected cells harvested at late stages of infection.

Amino Acid Sequence↗
PubMed2001

Use of Saccharomyces cerevisiae in the identification of novel transcription factor DNA binding specificities.

Members of the steroid/hormone nuclear receptor superfamily regulate target gene transcription via recognition and association with specific cis-acting sequences of DNA, called hormone response elements (HREs). The identification of novel HREs is fundamental to understanding the physiological function of nuclear receptor-mediated signalling pathways. A number of these receptors are transcriptionally active, or can be induced to an active state, when expressed in the yeast strain Saccharomyces cerevisiae. This aspect of nuclear receptor activity was used to screen random rat genomic DNA fragments for their ability to function as a HRE for the farnesoid X-activated receptor (FXR). An isolated genomic fragment mediated FXR transcriptional activation without the co-expression of the retinoid-X receptor (RXR), a receptor previously thought to be an obligate heterodimer partner for FXR function. This genomic sequence of DNA contained a pair of highly conserved HRE half-sites arranged in an everted orientation and separated by 3 bp (ER3). Furthermore, it was located 240 bp from a highly conserved TATA box motif. A minimal ER3 sequence of DNA was further demonstrated to function as a FXR HRE and was bound in vitro by FXR-expressing yeast extracts. Using RT-PCR, an expressed mRNA fragment was identified within an 8 kb region downstream of the putative TATA box motif. This sequence of DNA was observed to bear homology to a cDNA found in mouse blastocyst. These findings define a novel FXR DNA binding specificity but, more importantly, these data suggest that this strategy might be universally applied to any transcription system that can be reconstituted in yeast.

Amino Acid Sequence↗
PubMed2001

Effect of deletion and the site of insertion in double copy anti-tat retroviral vectors: viral titres and production of anti-tat mRNA.

In attempts to further develop murine leukemia virus (MLV) based retroviral vectors for gene therapy, we investigated vector production and antisense expression from retroviral constructs with U3 deletions or insertions. Promoter elements in the U3 region of the 3' LTR of the vector pLXSN were deleted and replaced with DNA encoding the HIV anti-tat gene under control of the tRNAmet promoter to produce a double copy self inactivating vector (DC-SIN). DC-SIN constructs were compared to vectors containing the anti-tat cassette inserted at 5 different sites of the U3 region (DC-insertions). Titres of DC-SIN and DC-insertion vectors were similar but approximately 10 fold lower than parental pLXSN. Cells transduced with DC-SIN and DC-insertion vectors all expressed anti-tat mRNA. Transcripts from the MLV-LTR were detected in cells transduced with DC-insertion but not DC-SIN vectors or a vector with the anti-tat cassette between CAAT and TATA boxes of the promoter, indicating inactivation of the viral promoter in the latter vectors. Cells transduced with constructs of either design showed comparable efficacy of protection against HIV challenge. Thus, no U3 insertion site was preferred for virus production. Insertion of a tRNA promoter between CAAT and TATA boxes and the DC-SIN design which would not introduce an active RNA pol II promoter into the genome are attractive for further development of safe gene therapy agents.

Genes, tat↗
PubMed1984

Activation of a heterologous promoter in response to dexamethasone and cadmium by metallothionein gene 5'-flanking DNA.

Human metallothionein-IIA (hMT-IIA) gene expression is regulated by heavy metals and glucocorticoids. When the cloned hMT-IIA gene or its 5'-flanking DNA structure fused to herpes simplex virus thymidine kinase (HSV-TK) structural gene sequences were transferred into TK- Rat 2 fibroblasts, both genes were inducible by Cd++ and/or dexamethasone. Placement of the hMT-IIA gene 5'-flanking region, either intact of deleted in its TATA box and cap site, upstream of the HSV-TK gene promoter rendered the latter both glucocorticoid- and heavy metal-inducible. Thus the structure that mediates both Cd++ and glucocorticoid responsiveness is present in the hMT-IIA gene 5'-flanking DNA, does not require its TATA box or cap site, and can activate a heterologous promoter.

Animals↗
PubMed1985

Transcription of the triose-phosphate-isomerase gene of Schizosaccharomyces pombe initiates from a start point different from that in Saccharomyces cerevisiae.

Gene tpi, encoding the glycolytic enzyme triose phosphate isomerase (TPI) from the fission yeast Schizosaccharomyces pombe was cloned by complementation of a Saccharomyces cerevisiae tpil mutant. Nucleotide sequence analysis of the cloned gene revealed a single open reading frame (ORF) encoding a protein 59% homologous to S. cerevisiae TPI. The gene has a very high codon usage bias. Messenger RNA synthesis initiates at two points located 38 and 44 nucleotides downstream from a TATA box promoter sequence. In S. cerevisiae, transcription of this S. pombe gene initiates about 26 nucleotides downstream from the S. pombe start points. This observation indicates that the two yeasts have diverged in the mechanism which determines the 5' end of the messenger RNA relative to the TATA box. It appears that in some respects the transcription initiation mechanism of S. pombe more closely resembles that of higher eukaryotes than does the S. cerevisiae mechanism.

Amino Acid Sequence↗
PubMed1988

Transcription elements and factors of RNA polymerase B promoters of higher eukaryotes.

The promoter for eukaryotic genes transcribed by RNA polymerase B can be divided into the TATA box (located at -30) and startsite (+1), the upstream element (situated between -40 and about -110), and the enhancer (no fixed position relative to the startsite). Trans-acting factors, which bind to these elements, have been identified and at least partially purified. The role of the TATA box is to bind factors which focus the transcription machinery to initiate at the startsite. The upstream element and the enhancer somehow modulate this interaction, possibly through direct protein-protein interactions. Another class of transcription factors, typified by viral proteins such as the adenovirus EIA products, do not appear to require binding to a particular DNA sequence to regulate transcription. The latest findings in these various subjects are discussed.

Animals↗
PubMed1994

Primary sequence and functional analysis of the bovine galanin gene promoter in human neuroblastoma cells.

Galanin (GAL) is a biologically active neuropeptide that has been suggested to play a role in stress-induced inhibition of insulin secretion, in dementia of the Alzheimer's type, and in the regulation of growth hormone secretion. We report here the isolation of a bovine genomic clone containing more than 5-kb 5'-flanking sequences. Partial sequence analysis of the genomic clone revealed an atypical TATA-box in the promoter (ATAAATA) and several consensus sequences that typically bind transcription factors, including those that bind NF kappa B, Sp1, and AP-2. Primer extension and RNase protection analyses revealed that transcription is initiated at two sites, 28 and 31 bp, respectively, downstream from the TATA-box. To locate functionally active regulatory elements on the GAL gene, we first identified a neural crest-derived human neuroblastoma cell line, SK-N-SH subclone SH-SY5Y, that expressed easily detectable levels of endogenous GAL mRNA. We then constructed plasmids containing various lengths of bovine GAL 5'-flanking sequences and the first exon fused to a reporter plasmid encoding luciferase. Transfection of these plasmids into the SH-SY5Y cells and analysis by transient expression indicated that 131 bp of 5' gene sequence was sufficient to obtain maximal basal expression. Further, expression was suppressed 16-fold when 5 kb were included, suggesting the presence of a distal repressor element(s). In another set of experiments, we found that GAL mRNA levels could be induced more than 10-fold by 20-hr treatment with phorbol 12-myristate 13-acetate (PMA). In cells transfected with the same plasmids, luciferase activity was also induced by PMA, but the degree of induction did not significantly differ among the deletion constructions (varying from six- to eight-fold), suggesting that elements conferring PMA induction and/or RNA stabilization may be located within 131 bp of the transcriptional start site, in the first exon, or on gene sequences not studied here.

Animals↗