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)

Three positive transcriptional control regions have been identified in the promoter of the human heart-skeletal muscle adenine nucleotide translocator gene (ANT1). By transfecting promoter-chloramphenicol acetyltransferase fusion constructs into C2C12 myogenic cells, each positive region was found to increase transcription 2-3-fold. The first region spans from -123 to -674 base pairs (bp), the second from -2.6 to -3.1 kilobases, and the third from -3.1 to -8.8 kilobases. Linker-scanning mutants generated using the polymerase chain reaction and modified oligonucleotides have identified the OXBOX (5'-GGCTCTAAAGAGG) as the positive element within the -123 to -674-bp region. This element enhances transcription in muscle cells but not in HeLa cells, suggesting that it is muscle-specific. Gel retardation experiments have revealed a factor from C2C12 cells which specifically binds to a 40-bp piece of the ANT1 promoter containing the OXBOX. Since the OXBOX is also found in the promoter of the human ATP synthase beta subunit gene, it is the first tissue-specific element identified which could coordinately regulate mitochondrial oxidative phosphorylation genes.
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PMID:OXBOX, a positive transcriptional element of the heart-skeletal muscle ADP/ATP translocator gene. 224 5

The rat pyruvate kinase L (PKL) gene produces the L- and R-type isozymes by alternative transcription that is regulated in a tissue-specific manner. To investigate which DNA elements are involved in hepatocyte-specific expression of the L-type isozyme, we performed transient DNA transfer experiments with PKL/chloramphenicol acetyltransferase fusion genes. We found three positive regulatory regions required for expression of the L-type isozyme in adult rat hepatocytes by functional analyses of a series of 5' and internal deletion constructs of the fusion genes. These regions, designated as PKL-I, PKL-II, and PKL-III, were located between nucleotides -76 and -94, -126 and -149, and -150 and -170, respectively. PKL-I showed enhancer-like activity alone, whereas PKL-II and PKL-III did not have any independent effect. Combinations of L-I + L-II and L-II + L-III, but not of L-I + L-III, showed synergistic enhancer activities when oriented in the same direction. The inclusion of all three elements oriented in the same direction had the maximum synergistic effect, indicating that these elements function as a unit. This unit enhanced expression from heterologous as well as homologous promoters in a manner that was independent of its orientation and position relative to the cap site. The activity of the unit was not detected in HeLa cells or K562 erythroleukemia cells, suggesting that this unit possessed cell-type specificity. PKL-I consists of a palindrome sequence 5'-CTGGTTATACTTTAACCAG-3', which contain a sequence homologous to the LF-B1-binding site. PKL-II contains the sequence 5'-TTCCTGGACTCTGGCCCCCAGTGT-3', which is similar to that of the LF-A1-binding site. PKL-III contains a palindrome sequence 5'-CCACGGGGCACTCCCGTGG-3', which include a sequence homologous to the binding site of the adenovirus major late transcription factor. Gel retardation assay indicated that the different trans-acting factors interacted with three elements and that the transacting protein bound to PKL-I was in fact LF-B1. However, the trans-acting factors bound to PKL-II and PKL-III were different from LF-A1 and major late transcription factor, respectively. Thus, we conclude that three cis-acting elements are very important for specific expression of the PKL gene in hepatocytes and that LF-B1 and two unknown factors bound to these elements interact with each other to cause a synergistic effect.
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PMID:Identification and characterization of hepatocyte-specific regulatory regions of the rat pyruvate kinase L gene. The synergistic effect of multiple elements. 224 64

Nucleotide sequences that direct transcription of the human 5-lipoxygenase gene have been examined by ligation to the chloramphenicol acetyltransferase gene and determination of chloramphenicol acetyltransferase activity in transfected HeLa and HL-60 cells. Various lengths of 5'-flanking sequences up to 5.9 kilobase pairs 5' of the transcriptional initiation sites were tested. Two positive and two negative apparent regulatory regions were seen. Part of the promoter sequence (-179 to -56 from ATG), which includes five repeated GC boxes (the putative Sp1 binding sequence) was essential for transcription in both HeLa and HL-60 cells. Gel-shift assays (using the DNA fragment -212 to -88) revealed that the transcriptional factor Sp1 could bind to this region of the 5-lipoxygenase promoter. Furthermore, HL-60 nuclear extracts contained specific nuclear factor(s) binding to 5-lipoxygenase promoter DNA, which could not be detected in HeLa cell nuclear extracts.
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PMID:Characterization of the human 5-lipoxygenase gene promoter. 225 Dec 50

Collagen II, the major component of cartilage, is synthesized primarily by chondrocytes and by certain cells in the eye. Previously, we have studied the regulatory regions of the collagen II gene by DNA transfection assays (Horton, W., Miyashita, T., Kohno, K., and Yamada, Y. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8864-8868). These studies show that both the promoter and an enhancer sequence in the first intron are required for high transcriptional activity in chondrocytes. These elements do not show significant activity in cells which do not synthesize collagen II, such as in muscle cells and fibroblasts. In this report, we have constructed plasmids containing various deletions of the promoter of the collagen II gene, fused to a reporter gene for chloramphenicol acetyltransferase (CAT) and transfected them into both chick embryonic fibroblasts and HeLa cells. We have found that silencer elements in the collagen II promoter region reduce CAT activity 11-fold in fibroblasts, while not affecting the enhancer-mediated transcription in chondrocytes. Deletions in the promoter showed that most of the silencing activity was localized in two sites, between -360 and -460 base pairs and between -620 and -700 base pairs. Furthermore, a fragment containing these two sequences in a thymidine kinase promoter CAT construct reduced the activity of the promoter in an orientation independent fashion. Sequence analysis revealed that the two silencer regions are homologous and contain consensus motifs for silencer elements found in other genes. Gel retardation experiments showed that nuclear factors from HeLa cells bind specifically to a DNA fragment containing the silencer, whereas chondrocyte nuclear extracts did not show any activity. Thus, our study indicates that the expression of the collagen II gene is controlled by both negative and positive elements to ensure that the gene is only expressed in suitable cells.
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PMID:Two silencers regulate the tissue-specific expression of the collagen II gene. 232 96

Transcription of the chicken ovalbumin gene is induced both in vivo and in vitro by four classes of steroid hormones. Recent experiments identified a steroid-dependent regulatory element (SDRE) in the 5'-flanking region of the ovalbumin gene between -900 and -521. To characterize the regulatory properties of the SDRE more precisely, additional mutations were created in this region, and fusion genes prepared by linking the ovalbumin 5'-flanking region and promoter to the chloramphenicol acetyltransferase structural gene. When the ovalbumin-chloramphenicol acetyltransferase fusion genes were transfected into steroid-responsive primary oviduct cells, mutants lacking sequences between -900 and -732 were no longer responsive to estrogen, corticosterone, progesterone, or dihydrotestosterone. The SDRE did not confer steroid-dependent expression on the heterologous thymidine kinase promoter by itself but did in conjunction with the negative regulatory element identified between -350 and -100. This suggests that the two elements act as a single functional entity and that the SDRE is not behaving as a typical steroid response element. Gel shift analyses revealed that two SDRE.protein complexes were formed when nuclear protein extracts were derived from estrogen-treated chicken oviduct but that only one complex was formed with extracts from estrogen-withdrawn oviduct or from other tissues. Neither an estrogen response element oligomer nor a glucocorticoid/progesterone response element oligomer competed for either of the DNA.protein complexes. Partially purified progesterone receptor also did not bind to the SDRE. These data indicate that induction of the ovalbumin gene by steroid hormones requires complex interactions involving both the SDRE and the negative regulatory element.
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PMID:The steroid-dependent regulatory element in the ovalbumin gene does not function as a typical steroid response element. 233 44

The minimal DNA sequence required for glucocorticoid induction of the phosphoenolpyruvate carboxykinase (PEPCK) gene in H4IIE rat hepatoma cells was defined. This novel glucocorticoid response unit (GRU) spans about 110 base pairs (bp) and includes two receptor-binding elements plus two accessory factor-binding elements. Purified glucocorticoid receptor bound to two regions (GR1 and GR2) between -395 and -349 bp relative to the transcription start site. Factors in crude rat liver nuclear extract bound to DNA in the regions -455 to -431 and -420 to -403 bp, which are designated accessory factor 1 (AF1) and accessory factor 2 (AF2) elements, respectively. Gel retardation analysis revealed that at least two proteins bound to AF1 and that they were distinct from the protein(s) that bound to AF2. Various combinations of GR1, GR2, AF1, and AF2 were fused to the chloramphenicol acetyltransferase (CAT) reporter gene and cotransfected with a glucocorticoid receptor expression plasmid (pSVGR1) into H4IIE cells to identify the functional GRU. Neither the glucocorticoid receptor binding region nor the accessory factor binding region alone was sufficient to confer glucocorticoid responsiveness. The two components of the glucocorticoid receptor binding region functioned independently, and each accounted for half of the maximal response, provided the accessory factor elements were present. Similarly, deletion of either AF1 or AF2 diminished glucocorticoid induction of the PEPCK gene to approximately half of the maximum. We propose that the complex PEPCK gene GRU provides the stringent regulation required of this critical enzyme in liver.
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PMID:Characterization of a complex glucocorticoid response unit in the phosphoenolpyruvate carboxykinase gene. 238 23

The DNA sequences required for expression of the ribosomal protein gene rpL32 were identified by transient-expression assays of chimeric rpL32-chloramphenicol acetyltransferase genes. These studies showed that maximal rpL32 expression requires sequences in a 150- to 200-base-pair region spanning the transcriptional start site. Three discrete regions of importance were identified: one between positions -79 and -69 and two others located downstream of the transcriptional start site. Progressive 5' or 3' deletions caused stepwise decreases in expression, which suggested a complex interplay of redundant or compensatory elements. Gel mobility shift assays were used to identify trans-acting nuclear factors which bind to segments of the rpL32 promoter that are known to be important for transcription. Evidence for several distinct nuclear factors is presented. The binding sites for these factors were localized to the following regions: -79 to -69, -36 to -19, -19 to +11, +11 to +46 in exon I, and within the first 31 base pairs of intron 1. One of these factors may bind to multiple sites within the promoter region. Interestingly, the factor that binds to a sequence motif in the first exon also binds to similar motifs in a comparable region of the c-myc gene.
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PMID:Localization of transcriptional regulatory elements and nuclear factor binding sites in mouse ribosomal protein gene rpL32. 254 59

The present experiments show that the single gene for the lens-specific protein alpha A-crystallin of chickens and mice uses a different subset of cis- and trans-acting regulatory elements for expression in transfected embryonic chicken lens epithelial cells. A chicken alpha A-crystallin-chloramphenicol acetyltransferase (CAT) fusion gene required 162 base pairs whereas the murine alpha A-crystallin-CAT fusion gene required only 111 base pairs of 5'-flanking sequences for efficient tissue-specific expression in the transfected chicken lens cells. Gel retardation and competition experiments were performed using embryonic chicken lens nuclear extract and oligodeoxynucleotides identical to the 5'-flanking region of the chicken (-170/-111) and murine (-111/-88 and -88/-55) alpha A-crystallin gene. The results indicated that these homologous promoters use different nuclear factors for function. Methylation interference analysis identified a dyad of symmetry (CTGGTTCCCACCAG) at position -153 to -140 in the chicken alpha A-crystallin promoter which binds one or more lens nuclear factors. Gel mobility shift experiments using nuclear extracts of brain, reticulocytes, and muscle of embryonic chickens or HeLa cells suggested that the factor(s) binding to the chicken alpha A-crystallin gene promoter sequences are not lens specific. Despite differences in the functional and protein-binding properties of the alpha A-crystallin gene promoter of chickens and mice, expression of the chicken alpha A-crystallin-CAT fusion gene in transgenic mice was lens specific, consistent with a common underlying mechanism for expression of the alpha A-crystallin gene in chickens and mice.
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PMID:Tissue-specific expression of the chicken alpha A-crystallin gene in cultured lens epithelia and transgenic mice. 258 97

A series of constructs that links the rat muscle creatine kinase promoter to the bacterial chloramphenicol acetyltransferase gene was generated. These constructs were introduced into differentiating mouse C2C12 myogenic cells to localize sequences that are important for up-regulation of the creatine kinase gene during myogenic differentiation. A muscle-specific enhancer element responsible for induction of chloramphenicol acetyltransferase expression during myogenesis was localized to a 159-base-pair region from 1,031 to 1,190 base pairs upstream of the transcription start site. Analysis of transient expression experiments using promoters mutated by deletion indicated the presence of multiple functional domains within this muscle-specific regulatory element. A DNA fragment spanning this region was used in DNase I protection experiments. Nuclear extracts derived from C2 myotubes protected three regions (designated E1, E2, and E3) on this fragment from digestion, which indicated there may be three or more trans-acting factors that interact with the creatine kinase muscle enhancer. Gel retardation assays revealed that factors able to bind specifically to E1, E2, and E3 are present in a wide variety of tissues and cell types. Transient expression assays demonstrated that elements in regions E1 and E3, but not necessarily E2, are required for full enhancer activity.
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PMID:The upstream muscle-specific enhancer of the rat muscle creatine kinase gene is composed of multiple elements. 276 36

Expression of human immunodeficiency virus type 1 (HIV-1) can be activated in a chronically infected T-cell line (ACH2 cells) by a cytokine, human tumor necrosis factor alpha (TNF-alpha). TNF-alpha treatment of ACH2 cells resulted in an increase in steady-state levels of HIV RNA and HIV transcription. Gel mobility shift assays demonstrated that the transcriptional activation of the HIV long terminal repeat (LTR) by TNF-alpha was associated with the induction of a nuclear factor(s) binding to the NF-kappa B sites in the LTR. Deletion of the NF-kappa B sites from the LTR eliminated activation by TNF-alpha in T cells transfected with plasmids in which the HIV LTR directed the expression of the bacterial chloramphenicol acetyltransferase gene. Thus, TNF-alpha appears to activate HIV RNA and virus production by ACH2 cells through the induction of transcription-activating factors that bind to the NF-kappa B sequences in the HIV LTR.
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PMID:Tumor necrosis factor alpha activates human immunodeficiency virus type 1 through induction of nuclear factor binding to the NF-kappa B sites in the long terminal repeat. 276 7


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