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 aldolase C gene product is a glycolytic isoenzyme specifically detected in brain. We have previously defined a short 115-base pair promoter fragment able to confer on a reporter chloramphenicol acetyltransferase (CAT) gene a specific expression in brain of transgenic mice. In this promoter fragment, two GC-rich regions (A/A' and B boxes) were detected by in vitro DNase1 footprinting experiments with brain, fibroblast, or liver nuclear extracts. Both A/A' and B boxes, sharing structural homology, are able to interact with Sp1, Krox20/Krox24 factors and with other proteins (Thomas, M., Makeh, I., Briand, P., Kahn, A., and Skala, H. (1993) Eur. J. Biochem. 218, 143-151). In this paper, we describe a new ubiquitous factor termed Ub able to bind the A/A' box. We also delimit a third element (box C) binding a hepatocyte-enriched protein displaced by a hepatocyte nuclear factor 3-specific oligonucleotide. The functional involvement of each binding site in brain-specific transcription of the aldolase C gene has been tested in transgenic mice carrying different mutant promoters cloned in front of the CAT gene. A promoter containing only box C was totally inactive, suggesting an essential role of the region containing A/A' and B boxes. However, mutations or deletions of either the A/A' or the B box have no significant effect on the CAT gene expression. We therefore hypothesize that the A/A' and B sites may be functionally redundant. Indeed, constructs harboring only one of these two boxes (A/A' or B) linked to the C box displayed a brain-specific CAT activity similar to that obtained with the wild-type promoter. Furthermore, a transgene with disruption of the C box, keeping intact the A/A' and B boxes, was totally inactive, suggesting a crucial role of the hepatocyte nuclear factor 3 binding site in activation of the aldolase C gene.
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PMID:Functional dissection of the brain-specific rat aldolase C gene promoter in transgenic mice. Essential role of two GC-rich boxes and an HNF3 binding site. 765 3

Decay accelerating factor (DAF) is a complement regulatory protein that protects host tissue from complement-mediated damage by preventing the assembly and/or promoting the dissociation of C3 and C5 convertases. To identify and analyze the DNA sequence elements responsible for controlling DAF expression, the 5'-flanking region of the human DAF gene was cloned. Sequencing of 880 nucleotides upstream from the ATG codon revealed the absence of classic TATA or CAAT boxes. RNase protection and primer extension assays revealed a series of transcription start sites in a 10 nucleotide region located 86 nucleotides upstream from the ATG (the first of these start sites is numbered +1). Using HeLa, K562, EBV, and Molt 4 cells, DAF mRNA and protein were analyzed by Northern and Western blot. The DAF mRNA and protein levels roughly correlated, suggesting transcriptional control of gene expression. K562 and HeLa expressed high levels, EBV expressed intermediate levels, and Molt 4 expressed essentially no detectible DAF mRNA or protein. Regions of DAF 5'-flanking DNA were subcloned into plasmids containing the chloramphenicol acetyltransferase reporter gene and tested in transient transfection assays. The construct extending from -206 to +84 (-206/+84) had transcriptional activity in the DAF-positive HeLa, K562, and EBV lines, but no activity in the DAF-negative Molt 4 line. In the three DAF-positive lines, major enhancer activity was demonstrated between -206 and -77, and between -77 and -54 (containing cAMP responsive element and AP-1 binding site). Additional deletion of the region between -54 and -34 (containing an Sp1 binding site) reduces chloramphenicol acetyltransferase activity further but the low numerical values preclude statistical significance. The identification of transcription start sites and enhancer regions in the DAF gene will be important for studies of the mechanisms whereby cytokines and other factors may modulate DAF expression.
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PMID:Identification of 5'-flanking regions affecting the expression of the human decay accelerating factor gene and their role in tissue-specific expression. 767 27

We have isolated lambda-phage clones containing the human arylsulfatase B gene region from a genomic lambda 47.1 library. The human arylsulfatase B gene comprises 8 exons interrupted by 7 introns. DNA sequences of all intron-exon boundaries and the 5' flanking region of the gene were determined. All intron-exon splice junctions conformed to the GT/AG consensus sequence. Primer extension analysis revealed multiple start sites 1 to 135 nucleotides 5' of the ATG translational start codon. A 398 bp DNA-fragment of the 5' flanking region exhibits promotor activity when transiently expressed in BHK-21 cells using the bacterial chloramphenicol acetyltransferase gene as a reporter gene. This putative promotor region is located in a CpG island and contains potential Sp1 and AP2 binding sites but lacks typical TATA and CAAT box motifs.
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PMID:Structure of the human arylsulfatase B gene. 768 47

We have determined by deletion analysis that the most proximal region of the Pdha-2 promoter between nucleotide position -187 to +22 harbors a transcriptionally active core. This "core" promoter directs high levels of CAT (chloramphenicol acetyltransferase) reporter gene transcription in HeLa cells. DNase I footprinting of the proximal promoter revealed four regions of protection. One of these contains the consensus sequence for the Sp1 binding site and another the ATF/CREB binding site. The cis-sequences of the remaining two protected regions (designated MEP-2 and MEP-3; Mouse E1 alpha Promoter site) show no apparent consensus homology with cis-elements of other known transcription factors. Results of electrophoretic mobility shift assays confirm that the ATF/CREB and MEP binding sites interact in a characteristic and specific manner with factors present in nuclei of both testis and somatic tissue. The factor which recognizes the MEP-3 motif appears to be ubiquitous, whereas the MEP-2-protein complexes were tissue-specific. Interestingly, formation of a complex involving MEP-2 and a putative testis-specific binding factor (tau-MEP-2BF) is first observed in the testis of 2-week-old mice, this correlates with the expression of Pdha-2. In contrast, the formation of complexes between the MEP-2 binding site and a somatic variant of MEP-2BF (sigma-MEP-2BF) decreases in the testis as spermatogenesis proceeds. Our results suggest that 1) the MEP-2 binding factors are temporally regulated during spermatogenesis, and 2) interactions involving these factors with the MEP-2 cis-element may be important for modulating Pdha-2 expression.
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PMID:Temporal and tissue-specific interactions involving novel transcription factors and the proximal promoter of the mouse Pdha-2 gene. 769 72

Transcobalamin II (TCII) is a plasma protein that binds vitamin B12 (cobalamin; Cbl) and facilitates the cellular uptake of the vitamin by receptor-mediated endocytosis. In genetic disorders that are characterized by congenital deficiency of TCII, intracellular Cbl deficiency occurs, resulting in an early onset of megaloblastic anemia that is sometimes accompanied by a neurologic disorder. To define the genetic basis for TCII deficiency, we have cloned and characterized the human gene that encodes this protein. The gene spans a minimum of 18 kbp and contains nine exons and eight introns, with a polyadenylation signal sequence located 509 bp downstream from the termination codon and a transcription initiation site beginning 158 bp upstream from the ATG translation start site. The 5' flanking DNA does not have a TATA or CCAAT regulatory element, but a 34-nucleotide stretch beginning just upstream of the CAP site contains four tandemly organized 5'-CCCC-3' tetramers. This sequence is a motif for a trans-active transcription factor (ETF) that regulates expression of the epidermal growth factor receptor gene (EGFR), which also lacks TATA and CCAAT regulatory elements. A GC-rich sequence that binds the SP1 protein is located 356 nucleotides upstream from the first of the series of CCCC tetramers. Although this GC sequence is at an unusual location with respect to the CAP site, a 507-bp fragment containing this GC box drives the chloramphenicol acetyltransferase (CAT) reporter gene after transient transfection into NIH 3T3 cells. No CAT activity was observed when a 420-bp fragment lacking this GC box but containing the ETF-binding domains was similarly transfected into this cell line. One consensus and two atypical motifs for the c-myc ligand are located downstream and upstream, respectively, of the GC box, and this could explain the elevated plasma TCII observed in some patients with multiple myeloma, as the c-myc product is overexpressed in some myeloma cells. Restriction endonuclease digestion of genomic DNA from eight normal subjects with Taq I, Hinfl, Msp I, and Bgl I identified three patterns of restriction fragment length polymorphism (RFLP). A number of the exon/intron splice junctions of human TCII, TCI, and IF genes are located in homologous regions of these proteins, providing evidence that these genes have evolved by duplication of an ancestral gene. This characterization of the TCII gene and the RFLP should facilitate the identification of the mutation(s) responsible for the genetic abnormalities of TCII expression.
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PMID:The cloning and characterization of the human transcobalamin II gene. 774 31

We have isolated a genomic DNA clone covering the coding and 14 kb upstream region of the rat light neurofilament (NF-L) gene and sequenced 2.3 kb of its promoter. DNase I hypersensitive sites have been mapped in PC12 cells. For functional analysis of the NF-L promoter, constructs carrying 38, 97, 407, 564, 650, 1,099, 1,660, 2,003 base pairs (bp) upstream region in front of the chloramphenicol acetyltransferase (CAT) reporter gene were tested for their capability to direct CAT expression after transient transfection into various cell lines. Similar CAT activities were recorded both in rat pheochromocytoma (PC12) and mouse neuroblastoma N115 cells and also in several nonneural cell lines (HeLa, C127, NIH 3T3). Regions responsible for the basic promoter activity were located between -407 and +75 bp from the transcription initiation site. The NGF-responsive element was located between -38 and +75 bp, and sequence -97 to -38 was found to contain a functional cAMP-responsive element. In PC12 cells in which nerve growth factor (NGF) induces neurite outgrowth and NF-L transcription, NF-L promoter-driven CAT expression was stimulated up to 12-fold within three days of NGF treatment, whereas epidermal growth factor (EGF) had no effect. Rat NF-L promoter contained Sp1, AP-2 and CGCCCCCGC elements. In PC12 cells, NGF transiently induced the binding of transcription factors to the deoxyoligonucleotide probes containing the binding sites of these elements. The role of these factors in NF-L gene transcriptional induction by NGF in PC12 cells is discussed.
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PMID:Characterization of the rat light neurofilament (NF-L) gene promoter and identification of NGF and cAMP responsive regions. 774 11

E1a adenoviral oncoproteins have been known to modulate genes important for the growth and differentiation of cells. Our laboratory is interested in understanding how insulin promotes the growth and proliferation of cells. In this report, we have examined the ability of E1a to modulate the insulin receptor gene expression. In HepG2 cells, expression of the 243-amino acid E1a protein stimulated expression of the chloramphenicol acetyltransferase reporter under the control of the insulin receptor promoter. 5'-Deletion analysis of the insulin receptor promoter indicated that the region between -630 and -607 is important for regulation by E1a. This region contains two GA and four overlapping GC boxes that are putative Sp1-binding sites. A DNA fragment containing these sites was used as a probe in gel retardation assays. Three specific protein-DNA complexes were detected with HepG2 nuclear extract. These complexes could be competed partially by the DNA fragments with mutations in either the GA or GC boxes, but not by the DNA fragment with a mutation in both the GA and GC boxes. In addition, mutation of each of these sites lowered the basal activity of the promoter and partially reduced transactivation by E1a. Simultaneous mutation in both GA and GC boxes further reduced the basal activity and abrogated transactivation by E1a. Taken together, these results indicate that the loss of binding ability of Sp1 (or Sp1-like factors) is concomitant with reduction of the basal activity and the loss of E1a inducibility of the gene.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:E1a activation of insulin receptor gene expression is mediated by Sp1-binding sites. 777 68

The biosynthesis of estrogens is catalyzed by aromatase P450 (P450arom), the product of the CYP19 gene. The tissue-specific expression of the CYP19 gene is regulated by means of tissue-specific promoters through the use of alternative splicing mechanisms. Thus, transcripts containing various 5'-untranslated termini are present in human placenta and other fetal tissues, ovary, brain, and adipose stromal cells. Sequence corresponding to untranslated exon 1.4 is present in 5'-termini of transcripts expressed in adipose tissue and fetal liver, as well as adipose stromal cells in primary culture in the presence of dexamethasone and fetal calf serum (FCS). Identification of hormone-responsive, tissue-specific promoter regions, as well as growth factor-response elements upstream of exon 1.4, may provide insight into the regulation of estrogen biosynthesis in adipose tissue, which is implicated in the development of breast and endometrial cancer. The goals of the present study were to define the 1.4 promoter region with respect to the start of transcription and to characterize the region(s) responsible for conferring glucocorticoid responsiveness on aromatase expression. The transcription initiation site was identified by means of primer extension and S1 nuclease protection analyses. No TATA-like sequence was evident upstream of this site. Various deletion mutations of the upstream flanking region of exon 1.4 and including part of exon 1.4 were made using polymerase chain reaction or restriction enzyme digestion. The genomic fragments were fused upstream of the chloramphenicol acetyltransferase (CAT) reporter gene. These constructs were transfected into adipose stromal cells and fetal hepatocytes in primary culture in medium containing FCS with or without dexamethasone. The -560/+10 base pair (bp) construct expressed CAT activity after a putative silencer element was deleted, and expression was induced by dexamethasone about 3-fold. Transfection of the -330/+170 bp construct, which contains an upstream glucocorticoid response element (GRE) as well as an Sp1-like sequence in untranslated exon 1.4, resulted in an 8-fold stimulation of expression of CAT activity by dexamethasone. The upstream GRE as well as the Sp1-like sequence in untranslated exon 1.4 were mutated separately, and together, to further confirm whether the GRE or Sp1 binding site play a role in the regulation of promoter 1.4-driven transcription. Mutation of either the GRE or Sp1 binding site, or both, in the -330/+170 bp construct, resulted in loss of dexamethasone-induced CAT reporter gene expression.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Characterization of the sequences of the human CYP19 (aromatase) gene that mediate regulation by glucocorticoids in adipose stromal cells and fetal hepatocytes. 777 80

Tissue factor (TF) is a cellular receptor and cofactor for factor VII/VIIa which initiates the blood coagulation cascade. We have investigated the role of 5'-flanking DNA sequences in regulating the expression of the human TF gene in human umbilical vein endothelial cells (HUVEC). Using a chloramphenicol acetyltransferase (CAT) reporter gene, we attempted to transfect primary cultured HUVEC (passage 3-4) with calcium phosphate coprecipitation, DEAE Dextran, lipopolyamine-coated DNA or electroporation. Electroporation in HEPES-buffered saline of 1 x 10(7) cells at 200V and 250 microF was found to be optimal. Using these conditions, varying lengths of TF 5'-flanking sequences coupled to the CAT reporter gene were tested in transient expression studies. CAT expression corrected for variation in transfection efficiency and cell viability revealed that the sequences between -111 and +14 base pairs are essential for minimal transcriptional activity. This region contains consensus sequences for a TATA box and three Sp1 binding sites. A domain from -382 to -111bp, which contains two AP-1 consensus elements, promoted high levels of gene expression. This transcriptional activity was repressed by 50% with constructs containing sequences between -550 and -382 bp. A further 2-fold drop in transcription activity was attributed to the region between -948 and -550 bp. These results suggest that the basal transcription of the human TF gene in HUVEC is mediated through at least two negative regulatory elements upstream of the proximal promoter domain. The proximal promoter region which contains two AP-1 sites is essential for efficient transcription.
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PMID:Efficient gene transfer into human umbilical vein endothelial cells allows functional analysis of the human tissue factor gene promoter. 780 34

A genomic clone (19 kb) harboring the intron-exon sequences and the promoter-regulatory region of the E1 beta gene of human pyruvate dehydrogenase complex was isolated by screening a placental genomic library. The nucleotide sequence of the promoter region (1245 bp) showed 18 differences (including mismatches, insertions, and deletions) as compared to the published sequence [Koike et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5594-5597]. The E1 beta promoter lacked a TATA box homology but contained initiator sequences (two) and Sp1 sites (three) which are frequently found in TATA-less promoters. The DNase I footprinting pattern of the promoter region with crude rat liver nuclear extracts showed at least seven regions of protein binding and nuclease protection (P1-P7). The DNase I protected regions contained consensus nucleotide sequences recognized by GATA-1, Sp1, IgNF-A, Lva, bicoid Q9, NF-kB, HNF-5, H4TF-1, WAP5, and ADH transription factors. Transient expression of chloramphenicol acetyltransferase (CAT) suggested the possible presence of negative elements located within the sequence from -2316 to -930, whereas deletion constructs containing -929 to +32 and -98 to +32 DNA sequences showed approximately 7- and 20-fold increases in CAT activity over the basal CAT activity. Additional studies indicated the presence of an orientation-dependent cis element (or elements) within the region from -282 to -397 that acts as an enhancer or a repressor upon a heterologous thymidine kinase promoter.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of the promoter regulatory region of the human pyruvate dehydrogenase beta gene. 782 76


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