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PubMed1988

Cloning and characterization of the human gene for the alpha-subunit of Gi2, a GTP-binding signal transduction protein.

We isolated and characterized human genomic clones encompassing the gene for the alpha-subunit of Gi2, a GTP-binding signal transduction protein abundantly expressed in myeloid cells. The gene is divided into 9 exons and spans 23.5 kb. Exons 2, 6 and 7 encode putative guanine nucleotide-binding domains that are highly conserved among GTP-binding proteins. A polyadenylation signal located within exon 9 predicts an mRNA size (approximately 2.3 kb) several hundred bases longer than that of published cDNAs, and consistent with the size seen on RNA blot hybridization. Primer extension and S1 nuclease analysis determined a major and several minor transcriptional start sites. The first exon and 5' flanking region are highly G + C rich, contain several GC boxes (SP1 transcription factor binding sites), a CAAT box, and lack a TATA box. The presumptive promoter region is thus similar to that of ras and other widely expressed genes.

Amino Acid Sequence↗
PubMed1989

Structure, evolution and anaerobic regulation of a nuclear gene encoding cytosolic glyceraldehyde-3-phosphate dehydrogenase from maize.

A nuclear gene encoding cytosolic glyceraldehyde-3-phosphate dehydrogenase from maize (subunit GAPC1, gene Gpc1) and 2.2 x 10(3) base-pairs of its 5' flanking region have been cloned and sequenced. The structure of the maize Gpc1 gene (10 introns) is different from that of the maize gene encoding subunit GAPA of chloroplast glyceraldehyde-3-phosphate dehydrogenase (1 intron) and relatively similar to that of the chicken gene (11 introns). Introns in the Gpc1 gene show a positional polarity; the more 3' their position, the more they are displaced relative to introns in the chicken gene. The Gpc1 gene and other nuclear genes from maize are associated with CpG islands, the relative size of which determines the degree of codon bias in the gene. The promoter of the maize Gpc1 gene contains an anaerobic regulatory element and a pyrimidine box upstream from the TATA box and within intron 1. Southern blotting analyses and Northern hybridizations suggest that there are three functional Gpc genes in maize whose transcript levels are controlled differentially by anaerobiosis. In spite of its "typical" anaerobic promoter, the Gpc1 gene does not seem to be an anaerobic gene in vivo.

Anaerobiosis↗
PubMed1994

The structure, organization and differential expression of the gene encoding rat heme oxygenase-2.

Overlapping phage lambda clones were utilized to determine the complete nucleotide (nt) sequence of the rat gene encoding HO-2, the major heme oxygenase isozyme in the brain. This isozyme is the constitutive cognate of HSP32 (HO-1). The 12,563-bp gene consists of five exons and four introns, the first two exons are separated by a large intron of 8429 nt. The minus strand of intron 1 contains a nested sequence of 1046 nt with 87% identity to the cDNAs encoding the mouse and human non-histone chromosomal protein, HMG-17. In addition to the coding region, the similarity includes 40 bp upstream from the putative start codon and 800 bp of 3' untranslated sequence. The HO-2 gene lacks a conventional TATA box, but a TATA-like sequence (TAACTA) is found 26 nt upstream from the major transcription start point (tsp), as determined by primer extension. Upstream of the tsp, only a glucocorticoid-response element is found. The structure of the regulatory region is consistent with the previously demonstrated refractory nature of this isozyme to common inducers of gene expression and its apparent response to developmental changes in the adrenal steroid hormone profile. HO-2 is encoded by two transcripts (approx. 1.3 and approx. 1.9 kb), the larger of which is translated less efficiently than the smaller. Presently, we show that the transcripts are the products of a single gene and differ in the use of the polyadenylation signal.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2006

Studies on the activity of a bidirectional promoter of Mungbean yellow mosaic India virus by agroinfiltration.

The AV promoter expressing AV1 and AV2 genes and AC1 promoter expressing AC1 gene are present in opposite orientation in the intergenic region of Mungbean yellow mosaic India virus (MYMIV). Transient Agrobacterium-mediated delivery of putative promoter constructs into Nicotiana benthamiana and different legumes, followed by reporter gene (beta-d-glucuronidase, GUS) assay, identified the promoter region of both AC1 and AV genes that is necessary for transcriptional initiation. Transcription activator protein-independent activity of AV promoter and differential regulation of AC1 promoter are unique to MYMIV. The AV promoter is a composite core promoter having both TATA box and Initiator elements (TATA(+)Inr(+)). Many transcription factor binding sites were identified in the upstream promoter sequences of both virion and complementary sense genes, which might be used in the transcription regulation studies of the host plant as well as the virus.

Artificial Gene Fusion↗
PubMed1997

Cloning and expression of the 11beta-hydroxysteroid dehydrogenase type 1 gene in the baboon.

In the baboon, the 11beta-hydroxysteroid dehydrogenase (11beta-HSD)-catalyzed metabolism of maternal cortisol and cortisone by the placenta is an important component in the sequence of events regulating the function of the fetal pituitary-adrenocortical axis. The present study was designed to isolate and sequence the promoter region of the baboon 11beta-HSD-1 gene. The 11beta-HSD-1 genomic DNA was isolated from a baboon kidney genomic library using a human 11beta-HSD-1 cDNA as a probe. The sequence of a 1.7 kb fragment of the 5'-flanking region showed extensive homology (> 95) to that published by others for human 11beta-HSD-1 particularly in exons I and II (> 95%) and in the proximal promoter ( > 98%). Using total RNA from adult baboon liver annealed with a 22 bp antisense primer located at the 3' end of Exon I, parallel genomic sequencing reactions with the same primer confirmed that the baboon 11beta-HSD-1 gene has two transcriptional start sites 93 nucleotides apart and that both start sites are preceded by a CAAT box but not a TATA box. RNase protection assays confirmed that both transcription start sites are utilized in liver and near term baboon placenta and that transcripts emanating from the downstream start site dominate in the placenta in contrast to the preferential utilization of the upstream start site in adult liver.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed2001

Cloning and characterization of human cathepsin L promoter.

Cathepsin L is a lysosomal cysteine protease, which is over-expressed and secreted by malignant cells. It is very potent in degrading collagen, elastin, laminin and other components of the basement membrane and, therefore, has been implicated in tumor invasion and metastasis. The structural portion of the human cathepsin L (hCATL) gene was cloned to elucidate its genomic organization (Chauhan et al., J. Biol. Chem. 218 (1993) 1039). In the present study, a 1.90 kb DNA fragment, containing 1825 bp of the 5' upstream region of hCATL and 75 bases of the first exon of the hCATL, was amplified by PCR from an adaptor ligated placental genomic library. This fragment has been demonstrated to exhibit promoter activity by luciferase reporter assays. Sequence analysis of this fragment revealed the presence of approximately 29 different putative transcription factor binding sites. Several of them like AP-4, GATA-1, Lmo2, CEBPB, MZF-1, NF-AT, etc. were present more than once in this region. However, a consensus CAAT box but no consensus TATA box was found within the 1.0 kb upstream of exon 1. The transcription initiation site of hCATL, using placental total RNA, was mapped to a single adenine residue 289 bases upstream of the ATG codon.

5' Flanking Region↗
PubMed2002

Genomic structure, organization, and promoter region analysis of murine guanylyl cyclase/atrial natriuretic peptide receptor-A gene.

We have determined the complete genomic nucleotide sequence and analyzed the promoter region of murine guanylyl cyclase/natriuretic peptide receptor-A gene (Npr1,coding for NPRA). The gene spans about 17.8 kb and contains 22 exons interrupted by 21 introns. All the exon-intron boundaries possess the consensus GT/AG splice junctions. Four different types of short interspersed nuclear elements (ten mouse B1 elements, seven mouse B2-B4 elements, one ID and one MIR element) and one medium reiteration frequency repeats have been found in the non-coding regions of the gene. Eleven tandem repeats, including three in the promoter region of the gene, have been identified. The transcription start site, 362 bp upstream from the start codon, was determined by 5'- rapid amplification of cDNA ends. The 1.98 kb 5'-flanking region contains three potential SP1 binding sites and one inverted CCAAT box but lacks the TATA box. This region also contains several putative cis-acting motifs known to bind kidney specific nuclear protein HFH-3, cAMP-responsive element binding protein (CREB) and AP-4. In addition, the binding sites for a variety of transcription factors: AML-1 alpha, SRY, Nkx-2.5, LyF-1, p300, GATA-1/2, HNF-3 beta, c/EBP alpha/beta and USF have been localized in the promoter region of Npr1 gene. The analyses and characterization of the genomic structure of murine Npr1 gene should yield important insights into the species-specific regulation of this important gene family.

5' Flanking Region↗
PubMed2003

Genome-wide analysis of CREB target genes reveals a core promoter requirement for cAMP responsiveness.

We have employed a hidden Markov model (HMM) based on known cAMP responsive elements to search for putative CREB target genes. The best scoring sites were positionally conserved between mouse and human orthologs, suggesting that this parameter can be used to enrich for true CREB targets. Target validation experiments revealed a core promoter requirement for transcriptional induction via CREB; TATA-less promoters were unresponsive to cAMP compared to TATA-containing genes, despite comparable binding of CREB to both sets of genes in vivo. Indeed, insertion of a TATA box motif rescued cAMP responsiveness on a TATA-less promoter. These results illustrate a mechanism by which subsets of target genes for a transcription factor are differentially regulated depending on core promoter configuration.

Animals↗
PubMed2001

Molecular cloning and structural analysis of human sterol C5 desaturase.

Sterol C5 desaturase (SC5D) converts lathosterol to 7-dehydrocholesterol in cholesterol biosynthesis. In this study, we investigated the genome structure of SC5D and transcription of the human SC5D gene to try to elucidate the mechanism by which cholesterol synthesis is regulated. The SC5D gene had a structural gene from a single copy from genome DNA that contained five exons and four introns. The human SC5D was found to be located in chromosome 11q24.2-24.3 by fluorescence in situ hybridization mapping. Human SC5D mRNA transcripts, of which the major transcript was a 2 kb and the minor transcripts were 8 kb and 1.4 kb mRNA transcripts, were detected in almost all of the tissues examined. The human SC5D gene contained a GC box instead of a TATA box upstream of the transcript start sites. Human SC5D transcription started from several transcription start sites, and the first start site was located 31 bp upstream of the translation start site (ATG). The expression level of SC5D mRNA extracted from human liver carcinoma cells decreased as the amount of cholesterol added to the culture medium was increased. Inhibition of SC5D transcription was ascribed to the suppression of promoter activity of SC5D.

Acetyl Coenzyme A↗
PubMed2003

Finding signals for plant promoters.

The strongest signal of plant promoter is searched with the model of single motif with two types. It turns out that the dominant type is the TATA-box. The other type may be called TATA-less signal, and may be used in gene finders for promoter recognition. While the TATA signals are very close for the monocot and the dicot, their TATA-less signals are significantly different. A general and flexible multi-motif model is also proposed for promoter analysis based on dynamic programming. By extending the Gibbs sampler to the dynamic programming and introducing temperature, an efficient algorithm is developed for searching signals in plant promoters.

Algorithms↗
PubMed1994

Transcriptional regulation of basic fibroblast growth factor gene by p53 in human glioblastoma and hepatocellular carcinoma cells.

Mutations of the p53 gene are found in various human cancers. The frequency of its mutation is reported to increase during tumor progression in most tumors. In human gliomas, mutations of the p53 gene are found in about one-third of the malignant forms and in few of the benign ones, indicating their possible involvement in tumor progression. On the other hand, we have recently shown that basic fibroblast growth factor (basic FGF) plays a crucial role in tumor progression as an autocrine growth factor in tissues of human gliomas. Therefore, we hypothesized that p53 might regulate the promoter activity of the basic FGF gene, which has several GC boxes and no typical TATA box. In this study, cotransfection assays using human glioblastoma and hepatocellular carcinoma cells and establishment of stable cell lines expressing mutant-type p53 were performed. The basic FGF gene promoter was demonstrated to be regulated by p53 at the transcriptional level and its basal core promoter was found to be responsive to p53. Expression of endogenous basic FGF was also demonstrated to be activated by mutant type p53. Wild-type p53 repressed gene expression of the basic FGF and its mutant activated it in vitro, implying one of the possible pathways in tumor progression.

Carcinoma, Hepatocellular↗
PubMed1995

Promoter binding factors regulating cyclin B transcription in the sea urchin embryo.

Cyclin B is a key regulatory protein of the cell cycle, central to the control of the G2/M transition. In the developing sea urchin embryo, the cyclin B gene is transcriptionally regulated in concert with changing patterns of cell division. In an effort to understand the mechanism controlling cyclin B expression during development, we have conducted an analysis of the Strongylocentrotus purpuratus cyclin B gene promoter. DNase I foot-printing of the cyclin B upstream region revealed eight binding regions within 435 bp of the start of transcription; seven of these sites were within 215 bp. Found within these regions were consensus sequences for two CCAAT boxes, TATA, and E-boxes and sequences with some similarity to E2F and octamer binding motifs. Upstream sequences were functionally defined by generating cyclin B-CAT fusion genes, containing deletions and base specific mutations, and testing for relative levels of expression by gene transfer. Both CCAAT boxes were found to be essential for maximal levels of expression. A third binding site (PR7) with no recognizable consensus sequence was also found to act as a positive element. Our results suggest that protein binding to the E2F-like sequences may act to reduce expression. Protein binding was further characterized by gel mobility-shift and methylation interference. The CCAAT boxes were found to bind similar, if not identical, proteins. Sequence comparisons and methylation interference data indicate that the likely protein binding these CCAAT sequences is the characterized CCAAT-binding protein CP1. A probe containing site PR7 formed multiple gel shift complexes that, by methylation interference, appeared to be interrelated. One major complex was formed with an oligonucleotide containing the two E2F-like sequences. Protein binding to this probe was specific and required bases within the E2F-like sequences. Our results indicate that cyclin B is subject to positive and negative regulation, involving multiple factors that bind between -200 and -90 bp from the start of transcription.

Animals↗
PubMed1999

Characterization of the structure and regulation of the murine gene encoding gut-enriched Krüppel-like factor (Krüppel-like factor 4).

Gut-enriched Krüppel-like factor (GKLF, KLF4) is an epithelial-specific transcription factor whose expression is associated with growth arrest. In order to understand the mechanisms regulating expression of the gene encoding GKLF, we isolated a genomic clone containing murine GKLF. The gene spans 5.3 kb and contains four exons. A major start site of transcription was mapped to an adenine residue 601 nt 5' of the translation initiation codon. An additional 1 kb of the 5'-flanking region was sequenced and found to contain multiple cis -elements homologous to the binding sites of several established transcription factors including Sp1, AP-1, Cdx, GATA, and USF. In particular, three closely spaced GC-boxes 5' of the TATA box resemble the established binding site for GKLF. DNase I protection and electrophoretic mobility shift assays verified that recombinant GKLF bound to each of the three GC-boxes. In co-transfection experiments, GKLF transactivated a reporter gene linked to the GKLF 1 kb 5'-flanking region, as did Sp1, Sp3 and Cdx-2. Mutations of one or both of the first and second GC-boxes in the promoter resulted in diminished transactivation by GKLF. These results demonstrate that the 5'-flanking sequence of the mouse GKLF gene functions as a promoter and is subject to autoregulation by its own gene product.

Animals↗
PubMed2000

Structure of the pig sterol 14alpha-demethylase (CYP51) gene and its expression in the testis and other tissues.

A cDNA coding sterol 14alpha-demethylase (CYP51), which was isolated from a pig liver cDNA, contained a 1,512 bp open reading frame and a 758 bp 3'-untranslated region. The deduced amino acid sequence was 94% identical to those of human and rat CYP51s. The pig CYP51 gene spanned about 21 kb and was divided into 10 exons. The sites of exon-intron junctions were completely identical to those in the human and rat CYP51 genes. Five GC boxes, but not a TATA box, were found in the 5'-flanking region of the gene, and cyclic AMP and sterol responsive elements were also found in this region. The main transcription start site determined with the 5'-RACE method with poly(A)(+) RNA from the liver and testis was located at 143 nucleotides upstream from the initiation codon in both tissues. Northern blot analysis revealed that an approximately 2.4 kb mRNA, which is produced through the use of a polyadenylation signal (AATAAA) located at 740 nucleotides downstream of the stop codon, was expressed in all the tissues examined in pigs: The mRNA levels were much higher in the liver and testis than in the kidney, lung, and epididymis. Furthermore, after the onset of spermatogenesis, a smaller size of mRNA (about 1.8 kb) was found in the testis but not in the epididymis. The 1.8 kb mRNA was produced through the use of an unusual polyadenylation signal (AAGAAA) located at 28 nucleotides downstream of the stop codon.

3' Untranslated Regions↗
PubMed2006

Characterization of the 5'-regulatory regions of the rat and human apelin genes and regulation of breast apelin by USF.

Apelin, a peptide widely expressed in the body, is the endogenous ligand for the APJ receptor. To investigate how the apelin gene is regulated transcriptionally, we cloned and characterized approximately 3000 and approximately 4000 bp 5'-upstream fragments of the rat and human apelin genes. Putative CAAT-like box, but not TATA-box sites were identified. The rat (-207/-1 bp) and human (-100/+74 bp) core promoter sequences contain putative binding sites for upstream stimulatory factor (USF)-1/-2. Mutagenesis and overexpression assays showed that USF up-regulates basal and inducible apelin transcription. EMSA and supershift experiments indicated binding of USF-1/-2 to the rat (-114/-109 bp) and human (-84/-79 bp) apelin promoters. ChIP experiments show that USF is recruited to the putative USF binding site in the human apelin promoter in cultured breast cells. In concert with increased breast apelin expression during pregnancy and lactation in rats, EMSAs demonstrate an elevated binding of pregnant and lactating rat breast nuclear proteins to a consensus USF oligonucleotide. In vivo ChIP assays verified increased USF binding to the apelin promoter in breast of lactating rats. Together, our findings show that USF exerts a stimulatory role in regulation of breast apelin expression during pregnancy and lactation.

5' Flanking Region↗
PubMed2006

Vaccinia virus intermediate and late promoter elements are targeted by the TATA-binding protein.

Vaccinia virus replicates in the cytoplasm of the host cell and encodes its own RNA polymerase and transcription factors. The proteins that target the poxvirus RNA polymerase to intermediate- and late-class promoters have not been identified. In this study, representatives of the intermediate and late promoters were characterized at the nucleotide level to identify essential motifs. Both intermediate and late viral promoters are shown to have an essential element suggestive of TATA boxes, which are potential targets for the TATA-binding protein (TBP). Several approaches were used to test for TBP requirement in vaccinia virus transcription, including overexpression of TBP, expression of a dominant negative mutant of TBP, RNA interference, and expression of adenovirus E1A protein, which inactivates TBP. In each case, the results support an essential role for TBP in vaccinia virus intermediate- and late-gene transcription. These findings indicate that poxviruses have integrated TBP as a central feature into an otherwise heterologous transcription system. A model for transcriptional switching, in which both intermediate and late promoter elements are targeted by TBP that recruits viral transcription factors to assemble a functional complex on their cognate promoters and a dysfunctional, repressed complex on the other class, is proposed.

Adenovirus E1A Proteins↗
PubMed1999

Transcriptional activation signals found in the Epstein-Barr virus (EBV) latency C promoter are conserved in the latency C promoter sequences from baboon and Rhesus monkey EBV-like lymphocryptoviruses (cercopithicine herpesviruses 12 and 15).

The Epstein-Barr virus (EBV) EBNA2 protein is a transcriptional activator that controls viral latent gene expression and is essential for EBV-driven B-cell immortalization. EBNA2 is expressed from the viral C promoter (Cp) and regulates its own expression by activating Cp through interaction with the cellular DNA binding protein CBF1. Through regulation of Cp and EBNA2 expression, EBV controls the pattern of latent protein expression and the type of latency established. To gain further insight into the important regulatory elements that modulate Cp usage, we isolated and sequenced the Cp regions corresponding to nucleotides 10251 to 11479 of the EBV genome (-1079 to +144 relative to the transcription initiation site) from the EBV-like lymphocryptoviruses found in baboons (herpesvirus papio; HVP) and Rhesus macaques (RhEBV). Sequence comparison of the approximately 1,230-bp Cp regions from these primate viruses revealed that EBV and HVP Cp sequences are 64% conserved, EBV and RhEBV Cp sequences are 66% conserved, and HVP and RhEBV Cp sequences are 65% conserved relative to each other. Approximately 50% of the residues are conserved among all three sequences, yet all three viruses have retained response elements for glucocorticoids, two positionally conserved CCAAT boxes, and positionally conserved TATA boxes. The putative EBNA2 100-bp enhancers within these promoters contain 54 conserved residues, and the binding sites for CBF1 and CBF2 are well conserved. Cp usage in the HVP- and RhEBV-transformed cell lines was detected by S1 nuclease protection analysis. Transient-transfection analysis showed that promoters of both HVP and RhEBV are responsive to EBNA2 and that they bind CBF1 and CBF2 in gel mobility shift assays. These results suggest that similar mechanisms for regulation of latent gene expression are conserved among the EBV-related lymphocryptoviruses found in nonhuman primates.

Animals↗
PubMed1988

Factors involved in specific transcription by mammalian RNA polymerase II: purification, genetic specificity, and TATA box-promoter interactions of TFIID.

Selective and accurate transcription of purified genes by RNA polymerase II requires multiple factors. The factor designated TFIID was purified extensively from HeLa cell nuclear extracts by using a simple and novel complementation assay. Thus, TFIID was preferentially inactivated by mild heat treatment of a nuclear extract, and supplementation of the heat-treated extract with TFIID-containing fractions restored adenovirus major late (ML) promoter-dependent transcription. By using this assay, TFIID was purified approximately 300-fold by conventional chromatographic methods. The most purified TFIID fraction was demonstrated to be required for transcription of a number of other cellular and viral class II genes. This factor showed specific interactions with both the adenovirus ML promoter and a human heat shock 70 (hsp-70) promoter. On the ML promoter, the DNase I-protected region extended from around position -40 to position +35, although some discontinuities (and associated hypersensitive sites) were apparent near the initiation site and near position +27; the upstream and downstream boundaries of the TFIID-binding site were also confirmed by exonuclease III digestion experiments. In contrast to these results, the DNase I-protected regions on the human hsp-70 promoter were confined to a smaller area that extended from positions -35 to -19. DNase I hypersensitive sites were observed in both the adenovirus ML and hsp-70 promoters, most notably in the region at position -47. These results indicate either that there are different forms of TFIID or that a single TFIID can interact differently with distinct promoters.

Adenoviridae↗