Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The proximal 5'-flanking region of the alpha-subunit gene from humans and cattle confers pituitary-specific expression to heterologous reporter genes in transgenic mice. To investigate whether these promoter regions also contain the necessary regulatory elements for cell-specific expression and hormonal regulation, we used three independent lines of transgenic mice. Two lines of transgenic mice contained chimeric genes consisting of either 1.6 kilobasepairs (kbp) of human or 3 15 basepairs of bovine alpha-subunit proximal 5'-flanking sequence linked to the bacterial gene encoding chloramphenicol acetyltransferase (CAT). A third line of transgenic mice contained the proximal 1.6 kbp of 5'-flanking sequence of the human alpha-subunit gene linked to the bacterial lacZ gene encoding beta-galactosidase (beta gal; H alpha beta gal transgenic mice). Hormonal replacement paradigms indicate that both human and bovine alpha CAT transgenes are regulated by GnRH, suggesting that their expression occurs in gonadotropes. Thus, the proximal 5'-flanking regions of both the human and bovine alpha-subunit genes must contain regulatory elements that confer both gonadotrope-specific expression and responsiveness to GnRH. In contrast to the human alpha-subunit promoter, the bovine alpha-subunit promoter lacks a functional cAMP response element, suggesting that transduction of both cell-specific and GnRH transcriptional signals occurs through cAMP response element-independent pathways. Thyrotropes also express the glycoprotein hormone alpha-subunit gene. Yet, hormone replacement paradigms with propylthiouracil and T3 were ineffective in altering CAT activity in the pituitary of human or bovine alpha CAT transgenic mice. Because a thyroid hormone response element has been localized to the proximal 5'-flanking region of the human alpha-subunit gene, these data suggest that the alpha CAT transgenes lack sufficient information to direct expression to thyrotropes. Direct evidence for this possibility was obtained through immunocytochemical studies performed on pituitaries from H alpha beta gal transgenic mice. beta-Galactosidase activity appeared in gonadotropes, but not thyrotropes. We conclude, therefore, that distinct and separable regulatory elements mediate the expression of the alpha-subunit gene in gonadotropes and thyrotropes.
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PMID:Gonadotrope- and thyrotrope-specific expression of the human and bovine glycoprotein hormone alpha-subunit genes is regulated by distinct cis-acting elements. 128 Mar 29

Factor X is a vitamin K-dependent glycoprotein that plays an essential role in both the intrinsic and extrinsic pathways of blood coagulation. Studies on a recombinant lambda phage containing the 5'-flanking region of the human factor X gene showed that the factor X gene was linked to and was located at the 3' end of the factor VII gene: the initiation codon of the factor X gene was 2823 base pairs (bp) downstream from the polyadenylation site of the factor VII gene. This 2.8-kilobase intergenic region, and progressively deleted fragments of it, was fused to the chloramphenicol acetyltransferase gene, and transient expressions in HepG2 cells, human fibroblasts, and Chinese hamster ovary cells were measured. A liver-specific promoter element, FXP1-binding site, essential for hepatocyte-specific transcription was identified. This promoter sequence, further localized to -63 to -42 bp in DNase I footprint studies, was homologous to LF-A1 or hepatic nuclear factor-4 recognition sequence and was equally functional in the normal and inverse orientations. FXP1 site bound to nuclear protein(s) from HepG2 cells and complex formation was partially abolished by the presence of duplex oligonucleotides containing liver factor-A1 or hepatic nuclear factor-4-binding sequences. Two additional positive elements located upstream of the promoter region, spanning from -215 to -149 bp (FXP2 site), and -457 to -351 bp (FXP3 site), were also established by reporter gene assays.
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PMID:Liver-specific expression of the gene coding for human factor X, a blood coagulation factor. 131 96

Three transcripts map to the varicella-zoster virus (VZV) open reading frame (ORF) 67, which encodes glycoprotein IV (gpIV). All of these transcripts are polyadenylated and are transcribed from left to right towards the genomic terminal short repeats. Previous Northern (RNA) blot analyses suggested that the most abundant of these transcripts (1.65 kb) might code for gpIV. We performed S1 nuclease protection and primer extension assays and determined that the 5' terminus of the 1.65-kb transcript maps 91 bp upstream from the gpIV initiation codon. An AT-rich region (ATAAA), -28 bp from the cap site, is a potential TATA box, and at -71 bp there is a consensus CCAAT box motif. The 3' end of the 1.65-kb transcript is 20 bp downstream of two overlapping polyadenylation signals, AATAAA and ATTAAA, and just downstream of the 3' terminus is a GU-rich sequence. These results are reminiscent of data from our analysis of the VZV gpV gene, confirming that VZV appears able to use unusual TATA box motifs. Many canonical TATA sequences are present upstream from these VZV transcriptional start sites but, apparently, are not used. We tested sequences upstream from the gpIV cap site for promoter activity in transient expression experiments by cloning a DNA fragment (+63 to -343 bp) into pCAT3M, which contains a chloramphenicol acetyltransferase reporter gene. This clone showed little constitutive promoter activity but was activated more than 200-fold by infection with VZV and 5-fold with herpes simplex virus. The two known VZV transactivating genes (those for ORF 4 and ORF 62) were tested for their abilities to activate expression from the gpIV promoter by using their cognate promoters. The ORF 4 gene was minimally active, whereas the ORF 62 gene gave twofold induction; both genes, acting together, gave fivefold induction. However, replacement of the IE62 promoter with the immediate-early cytomegalovirus promoter in the ORF 62 construct gave over 40-fold induction of chloramphenicol acetyltransferase activity under the gpIV promoter in the same assay.
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PMID:Transcription from varicella-zoster virus gene 67 (glycoprotein IV). 131 76

DNA sequence analysis and 5'-end mapping of mRNA were used to identify and clone DNA fragments which contain the presumptive promoter (Pgl) and poly(A) site (An) of the bovine herpesvirus 1 (BHV1) gl glycoprotein. To confirm the presence of these regulatory regions in the above fragments, they were cloned together with a chloramphenicol acetyltransferase (CAT) reporter gene into pUC19. The recombinant plasmid formed, pEC3, was capable of inducing CAT activity when transfected into bovine cells demonstrating that the Pgl-CAT-An sequence constituted a functional CAT expression cassette. The cassette was excised from pEC3, transferred to a plasmid insertion vector (pIV3A) and subsequently inserted into the thymidine kinase (tk) gene of BHV1. Insertion in either orientation, relative to the tk gene, gave rise to BHV1 recombinants which expressed CAT activity in infected cells. Analysis of RNA from infected cells indicated that CAT transcripts were present in multiple species of RNA. This unexpected result was found to reflect temporal shifts in promoter and poly(A) site usage during infection. Although the poly(A) site which forms part of the expression cassette was used extensively early in infection, most CAT transcripts synthesized at late times read through this promoter-proximal site and terminated at the distal site normally used for tk mRNA synthesis.
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PMID:Bovine herpesvirus 1 as a live virus vector for expression of foreign genes. 132 99

To investigate the cis-acting sequences involved in regulation of a herpes simplex virus gamma 1 gene, deletion analyses of the glycoprotein B (gB) gene promoter were performed. In transfection assays with gB-chloramphenicol acetyltransferase plasmids, high-level constitutive expression from the gB promoter was found with an 89-bp sequence (-69 to +20). Additional sequences in the 5'-transcribed noncoding leader region (+20 to +136) were required for full stimulation by herpes simplex virus infection. Plasmids with progressive deletions of the gB leader sequence demonstrated that chloramphenicol acetyltransferase expression in infected cells was proportional to the length of the leader region retained. In recombinant viruses containing a gB-gC gene fusion, a similar 83-bp (-60 to +23) region of the gB gene was found to promote accurately initiated gC mRNA from the viral genome with the same kinetics as the wild-type gB gene. Although the kinetics of expression remained the same, RNA abundance was greater with a 298-bp (-260 to +38) promoter than with the 83-bp promoter.
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PMID:Analysis of the gB promoter of herpes simplex virus type 1: high-level expression requires both an 89-base-pair promoter fragment and a nontranslated leader sequence. 132 69

Osteopontin (secreted phosphoprotein-1, Opn) is a phosphorylated glycoprotein expressed by transformed cells, macrophages, activated T-lymphocytes, specialized epithelial cells and bone cells that is characteristically enriched in milk and in the mineralized matrix of bone. The synthesis of Opn by bone cells is regulated by glucocorticoids and growth factors, which promote bone formation, and by the osteotropic hormone calcitriol (1,25-dihydroxycholecalciferol) and retinoic acid, which mediate bone resorption, indicating a bifunctional role for this protein in bone remodelling. To study the transcriptional regulation of the opn gene, two genomic clones (10 and 15 kb) encoding the opn gene were isolated from a porcine liver genomic library cloned into lambda phage. From the 15-kb clone a 4-kb EcoRI fragment containing the first two exons and 2.6 kb of the 5' flanking region of the opn gene was sequenced, and the transcriptional start site determined by primer extension analysis and S1 nuclease mapping. To identify the opn promoter, chimeric chloramphenicol acetyltransferase constructs were prepared using fragments from the first intron and the 5' flanking region of the opn gene. Transient transfection of porcine bone cells with these constructs showed strong promoter activity located within 74 bp upstream from the transcription initiation site. Within this region a TATA sequence, TTTAAA, was identified at positions -26 to -31. However, the highest transcription rate was observed in a construct extending 180 bp upstream that included a CCGCCC Sp1 binding sequence (-63 to -68), and an AP1 site (-74 to -80). Further upstream in the 5' flanking region and within the first intron of the opn, a number of consensus sequences could be identified. Chimeric constructs containing a GGGTCAtatGGTTCA direct repeat consensus sequence for a vitamin D3 response element located at nucleotides -2245 to -2259 responded to the addition of 0.1 microM calcitriol by a 2.5-fold stimulation of transcription, although a greater than 2-fold increase was also observed in shorter constructs -180 to -905 lacking such a consensus sequence. Promoter activity was also exhibited by a region containing a TTTAAA sequence in the first intron that corresponded to the putative promoter site reported for mouse opn in macrophages (Miyazaki, Y., Setoguchi, M., Yoshida, S., Higuchi, Y., Akizuki, S. & Yamamoto, S. (1990) J. Biol. Chem. 265, 14432-14438).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Characterization of the promoter region of the porcine opn (osteopontin, secreted phosphoprotein 1) gene. Identification of positive and negative regulatory elements and a 'silent' second promoter. 163 16

The extracellular glycoprotein cytotactin is expressed in a characteristic and complex spatiotemporal sequence during development of the chicken embryo. To identify the various control elements underlying its expression, the promoter region of the cytotactin gene has been isolated and characterized. Clones were isolated from genomic libraries by using a fragment near the 5' end of the cDNA sequence. The sequence of this cDNA fragment was found to be distributed over two exons separated by a large first intron. The site of transcription initiation was determined by S1 nuclease and primer-extension mapping. Sequencing of a 4.3-kilobase (kb) genomic DNA clone that contains 3986 base pairs (bp) upstream of the RNA start site, the first exon, and part of the first intron revealed a number of sequence motifs implicated in the regulation and expression of eukaryotic genes. These included CCAAT boxes, phorbol ester-responsive elements, enhancer elements, and a consensus TATA sequence located 24 bp upstream of the major RNA cap site. The flanking sequence also contained a number of regions of dyad symmetry and direct repeats unique to cytotactin, as well as an array of A + T-rich sequences that resemble engrailed elements. Constructs containing fragments of the upstream region of the cytotactin gene fused to a promoterless gene for chloramphenicol acetyltransferase were transiently transfected into chicken embryo fibroblasts to define functional promoter sequences. Although sequences from -721 to +121 exhibited minimal promoter activity, the entire region between -3986 to +374 was required to yield maximal expression in chicken embryo fibroblasts. Transfection of the -3986/+374 chloramphenicol acetyltransferase plasmid into the human U251MG astrocytoma cells but not HT1080 fibrosarcoma cells resulted in chloramphenicol acetyltransferase expression, consistent with the observed synthesis of cytotactin protein only by the U251MG cell line. These data indicate that the chicken cytotactin promoter can control expression in a cell type-specific fashion within cells of another species. These studies provide a basis for the dissection of cis elements and trans factors that govern the developmental expression of the cytotactin gene.
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PMID:Identification and characterization of the promoter for the cytotactin gene. 169 83

Chronic administration of estradiol inhibits transcription of the gene encoding the alpha-subunit of pituitary glycoprotein hormones. Here, we show, using transfection analyses and a filter binding assay, that 1500 basepairs of proximal 5' flanking sequence of the human alpha-subunit gene lack a functional estrogen response element when transfected into heterologous cell lines, and fail to bind estrogen receptor purified from calf uterus. Yet, this same region of the alpha-subunit gene confers estradiol responsiveness (transcriptional suppression) to the bacterial chloramphenicol acetyltransferase gene in transgenic mice. A smaller promoter fragment of the bovine alpha-subunit gene also confers responsiveness to estradiol in transgenic mice, suggesting that the same element may mediate the steroid responsiveness of both promoters. Furthermore, regulation by estradiol of the chimeric human or bovine alpha-chloramphenicol acetyltransferase genes is pituitary specific, underscoring the physiological significance of these studies. Based on these results, we conclude that estradiol regulates expression of the alpha-subunit gene in vivo through a mechanism that does not involve high affinity binding of estrogen receptor to the alpha-subunit gene. Whether this mechanism is manifest at the level of the pituitary or hypothalamus remains to be determined.
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PMID:Estradiol inhibits transcription of the human glycoprotein hormone alpha-subunit gene despite the absence of a high affinity binding site for estrogen receptor. 171 10

The putative promoter of the variant surface glycoprotein (VSG) gene of Trypanosoma brucei was cloned into a plasmid containing the chloramphenicol acetyltransferase (CAT) gene. After electroporation into trypanosomes, this construct directed the expression of the CAT reporter gene. The essential region for promoter activity was found to reside within 88 bp upstream of the putative transcription start site. Transcription of the CAT construct occurred at approximately the same level in both bloodstream and procyclic forms and was resistant to alpha-amanitin. However, CAT expression appeared to be modulated in the two forms of the parasite. Sequences 3' to the gene seemed to be important in this respect, as CAT activity in bloodstream forms was readily detectable only when the 3' region of a VSG cDNA was placed downstream of the CAT gene. Two separate VSG gene promoter sequences, both cloned from T. brucei AnTat 1.3A, were equally able to direct CAT expression, which suggests that there are a number of potential VSG gene promoters in the genome, although usually only one expression site is fully active at any one time.
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PMID:Transient activity assays of the Trypanosoma brucei variant surface glycoprotein gene promoter: control of gene expression at the posttranscriptional level. 198 30

The rate of transcription of the beta 2-adrenergic receptor gene is increased in response to beta-adrenergic agonist stimulation of the receptor at the cell surface. This effect is mediated by stimulation of adenylyl cyclase and elevation of intracellular cAMP levels. We have previously shown that this responsiveness to cAMP resides in the 5'-flanking region of the human beta 2-adrenergic receptor gene (Collins, S., Bouvier, M., Bolanowski, M. A., Caron, M. G., and Lefkowitz, R. J. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4853-4857). A 34-base pair sequence derived from the beta 2-adrenergic receptor promoter region (-70 to -37 base pairs), containing the sequence GTACGTCA, confers responsiveness to cAMP when present in either orientation 5' to the thymidine kinase promoter on the chloramphenicol acetyltransferase reporter gene. Overexpression of the catalytic subunit of protein kinase A fully substituted for forskolin in inducing expression through this sequence, indicating that the cAMP induction is mediated through this kinase. Mutations within the GTACGTCA sequence completely abolished the stimulation. A 43-kDa transcription factor (cAMP response element-binding protein) confers cAMP responsiveness through binding to specific sequences. In gel mobility shift assays, purified cAMP response element-binding protein bound to the 34-base pair oligonucleotide from the beta 2-adrenergic receptor gene with an affinity similar to that for the well-characterized cAMP response element from the human glycoprotein hormone alpha-subunit gene, and failed to bind to mutated elements. Thus, positive autoregulation of the beta 2-adrenergic receptor gene appears to occur through receptor-mediated stimulation of adenylyl cyclase, with consequent activation of cAMP response element-binding protein and stimulation of beta 2-adrenergic receptor gene transcription. These results demonstrate a novel mechanism by which a receptor (beta 2-adrenergic receptor) stimulatory for adenylyl cyclase can exert positive feedback regulation on its own expression.
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PMID:A cAMP response element in the beta 2-adrenergic receptor gene confers transcriptional autoregulation by cAMP. 217 52


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