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 regulation by tumor necrosis factor alpha (TNF) of its own promoter has been investigated by transient transfection and nuclear protein binding assays. In human K652 erythroleukemia cells TNF produced an 8-10-fold activation of the human TNF promoter linked to the chloramphenicol acetyltransferase gene. The TNF-responsive element was localized to the -125 to -82 region by examining the TNF activation in 5'-deletion or site-directed mutants of the TNF promoter and by demonstrating that the -125 to -82 fragment confers TNF responsiveness to the thymidine kinase promoter. This region contains a palindrome, 5' TGAGCTCA 3', that resembles the consensus binding sequences for the transcription factors, activator protein-1 (AP-1), cyclic AMP-responsive element binding protein (CREB), and activation transcription factor (ATF). An internal deletion in the palindrome abolished the TNF responsiveness, whereas known AP-1 and CREB/ATF elements were unresponsive to TNF. In band shift analyses a nuclear factor from U937 cells specifically bound to the -125 to -82 TNF-responsive fragment in or near the palindromic sequence. Oligonucleotides containing AP-1 or CREB/ATF sites did not effectively compete for the binding, indicating that the U937 cell factor is different from these factors. Anti-c-fos antiserum did not affect binding of the U937 cell factor, whereas anti-c-jun antiserum did block its binding, indicating that either c-jun or a protein antigenically related to c-jun is a component of the factor. These results suggest that the TNF-responsive element is not activated by AP-1 or CREB in U937 cells and that a novel DNA binding factor is important for constitutive and inducible TNF gene expression.
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PMID:Identification of a tumor necrosis factor-responsive element in the tumor necrosis factor alpha gene. 182 93

We have isolated non-globin cDNA clones specific for erythroid differentiation from K562 human erythroleukemia cells and have identified those that may regulate globin gene transcription. A cDNA library was constructed from K562 cells induced by hemin for production of embryonic and fetal hemoglobins and screened against cDNA from uninduced K562 cells. Full-length clones specific for induced K562 cells were ligated into a eukaryotic expression vector and transfected into HeLa cells to allow for production of the corresponding coded polypeptide. The ability to increase epsilon- or gamma-globin promoter activity was identified using cotransfection with a second vector containing a globin gene promoter fused to a reporter gene. Six of the induced K562-specific clones exhibited the ability to increase the levels of the reporter genes, bacterial chloramphenicol acetyltransferase and human growth hormone. Sequencing analyses of these clones indicated that five were homologous to ferritin heavy and light chains and one had no homology with known DNA or protein sequences. The ferritin light chain cDNA had the greatest effect on globin gene promoter activation, increasing the gamma-globin promoter activity by 6-8-fold. The activation of the globin gene promoter in the absence of globin gene translation suggests that ferritin (or iron) may have a direct role in globin gene transcription. The subtractive library cloning strategy has enabled us to isolate cDNA clones that activate specific gene promoter without the requirement of direct DNA binding. This approach may allow further identification of the genes encoding proteins that are involved in the control of erythropoiesis.
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PMID:Activation of globin gene expression by cDNAs from induced K562 cells. Evidence for involvement of ferritin in globin gene expression. 184 May 94

A 600-base-pair (bp) enhancer region upstream from the major IE94 gene of simian cytomegalovirus (SCMV) produces very strong basal expression of associated gene products. This domain consists of multiple sets of interspersed repetitive elements, including 11 copies of a conserved 16-bp palindromic sequence with the consensus CCATTGACGTCAATGG. These series I repeats contain an 8-bp core TGACGTCA that resembles the cyclic AMP (cAMP) response element (CRE) of cellular genes. In transient chloramphenicol acetyltransferase assays in K562 human erythroleukemia cells, a set of deleted variants of the IE94 promoter all responded up to 15-fold to induction by cAMP. However, successive removal of most of the SCMV 16-bp motifs reduced basal expression over 20-fold. The cAMP stimulation was also manifested at the steady-state RNA level after SCMV infection of K562 cells and was detectable within 1.5 h after treatment of DNA-transfected cells. Addition of a single 30-bp oligonucleotide encompassing the 16-bp palindrome conveyed up to 10-fold cAMP responsiveness onto a heterologous weak promoter but had no effect on basal expression. In contrast, two or more adjacent copies produced 20- to 40-fold increases in basal expression and provided greater than 200-fold activation in the presence of cAMP. Similar effects were obtained when the oligonucleotides were placed in a downstream location relative to the reporter gene. Studies with mutant oligonucleotides revealed that both the core CRE and the flanking sequence portions of the 16-bp elements were essential for enhancer function. Both components were also important for maximum cAMP responsiveness. Band shift assays with fractionated nuclear extracts from Raji lymphocytes revealed multiple competable complexes with cellular DNA-binding factors that recognized the series I elements. Three distinct CREB-like factors were detected that required only the core 8-bp elements for binding. We conclude that the 16-bp series I repeats provide a major contribution to the constitutive enhancer properties of the IE94 promoter and also act as functional CREs. The cAMP response properties appear likely to play a key role in reactivation of the virus from a latent state in appropriately differentiating cell types.
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PMID:The palindromic series I repeats in the simian cytomegalovirus major immediate-early promoter behave as both strong basal enhancers and cyclic AMP response elements. 215 15

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

We have studied the 5'-flanking sequences required for the transcriptional regulation of human epsilon-globin gene expression. A series of deletion mutants of the human epsilon-globin gene 5'-flanking sequences were constructed and linked to the bacterial chloramphenicol acetyltransferase gene. Expression of these constructs was tested in HeLa cells and the human erythroleukemia K-562 cells. By measuring chloramphenicol acetyltransferase activities and mRNA levels we found that the sequence between -177 and -392 base pairs (bp) relative to the mRNA initiation site exerts a negative effect on epsilon-globin promoter activity. This effect is more pronounced in HeLa cells compared with K-562 cells. To further characterize the negative control region we cloned the DNA sequence between -177 and -392 bp either 5' or 3' of the epsilon-globin promoter and in either orientation. Our data indicate that this negative control region inhibits the epsilon-globin promoter activity in a position- and orientation-independent manner, thus suggesting that it is a silencer. In addition, the silencer also inhibits the expression from the Herpesvirus thymidine kinase promoter. Sequence comparison reveals that there are three short regions within the silencer that share extensive homology with those found in other negative control DNA elements. Our results therefore indicate that an upstream silencer element is present in the epsilon-globin gene and that it may play an important role in the control of epsilon-globin gene expression during development.
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PMID:Identification of a transcriptional silencer in the 5'-flanking region of the human epsilon-globin gene. 274 86

Hemin-induced differentiation of the human erythroleukemia cell line K562 results in the expression and accumulation of erythroid-specific gene products such as embryonic and fetal hemoglobins and the elevated synthesis of the major heat shock protein HSP70. This activity was suggested to represent activation of a heat shock gene during erythroid maturation independent of stress induction. In this study, we demonstrate that hemin induces the transcription of two members of the human HSP70 gene family, HSP70 and GRP78 (BiP). However, the induction of HSP70 by hemin showed characteristics consistent with the molecular events associated with a heat shock or stress response. The increase in HSP70 gene transcription was accompanied by induction of the stress-induced form of the heat shock transcription factor. Moreover, a heat shock element was required for the hemin responsiveness of chimeric heat shock promoter-chloramphenicol acetyltransferase genes transiently expressed in transfected K562 cells.
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PMID:Hemin-induced transcriptional activation of the HSP70 gene during erythroid maturation in K562 cells is due to a heat shock factor-mediated stress response. 279 86

Transcriptional properties of the mouse globin genes and their promoters were examined in COS cells using transient expression assays. Transfected intact mouse alpha 1-globin genes generate transcripts, whereas beta maj and beta min globin transcripts are detected only if the genes are associated with a strong exogenous (SV40) enhancer sequence. Under these conditions the ratio of accumulated beta maj and beta min mRNA sequences approximates the 4:1 ratio observed in reticulocytes and murine erythroleukemia cells (MELC) induced to differentiate by hexamethylene bisacetamide (HMBA). As determined using hybrid genes consisting of globin gene 5' regions fused to the chloramphenicol acetyltransferase (CAT) structural gene, this 4:1 ratio appears dependent upon the relative activities of the two 5' promoter regions.
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PMID:Regulation of murine alpha-, beta major-, and beta minor-globin gene expression. 282 66

Studies of recombinants between murine leukemia viruses (MuLVs) that cause thymic or erythroid leukemias have shown that enhancer sequences in the long-terminal repeats (LTRs) can determine the target tissues for pathogenesis. It has been inferred that the enhancers may specifically target viral expression into the cells that then become neoplastic. However, the neoplasms in those studies formed after latencies and contained ultimate viruses (called MCFs) that differed from the injected viruses in their enhancer sequences and envelope (env) genes. Transcriptional activities of LTRs from these proximal and ultimate viruses have not been thoroughly analyzed in different hematopoietic lineages. We present evidence that the enhancer of Friend spleen focus-forming virus (SFFV), an ultimate erythroleukemogenic retrovirus, contains an unstable 42-nucleotide direct repeat. Other ultimate erythroleukemogenic MuLVs (Friend MCFs) contain an enhancer nearly identical to that of SFFV both in its sequence and in its specific instability. The instability occurs in sequences that contain inverted repeats and we propose that it occurs by a simple reverse transcriptase hop mechanism. We constructed plasmids that contain the two forms of the SFFV LTR linked to the bacterial chloramphenicol acetyltransferase (CAT) gene, and we compared these in transient transfection assays with LTR-CAT plasmids constructed from Friend and Moloney MuLVs. The assays employed erythroleukemia cells, thymic lymphoma cells, and fibroblasts. The tropisms of expression correlated only weakly with tissue specificities of pathogenesis and each LTR was active in all cells. The SFFV 42-nucleotide duplication reduced expression in erythroid cells and increased expression in fibroblasts. We conclude that retroviral enhancers do not stringently direct gene expression into specific cell lineages, but on the contrary they are leaky and contain replicative instabilities that also may facilitate viral entrenchment throughout the host. These results have important implications for understanding murine retroviral evolution and the multi-step process of leukemogenesis.
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PMID:An enhancer sequence instability that diversifies the cell repertoire for expression of a murine leukemia virus. 283 56

Recombination studies have established that retroviral long terminal repeats (LTRs) are important genetic determinants of the viral capacity to induce hematopoietic tumors and to specify the type of cell making up the tumor. Plasmids containing LTRs of several murine leukemia viruses linked to the chloramphenicol acetyltransferase gene were tested in transient assays to measure relative rates of transcriptional activity in different types of hematopoietic cells. LTRs of the thymomagenic viruses SL3-3, Moloney leukemia virus, and a Moloney mink cell focus-forming virus all expressed to higher levels than other LTRs in T-lymphocyte cell lines. Conversely, the LTRs of Friend leukemia virus and a polycythemic spleen focus-forming virus expressed to higher levels than other LTRs in erythroleukemia cells. The LTR of nonleukemogenic Akv virus induced a relatively low level of activity compared with the others in all cells tested. Thus the relative level of LTR-driven expression in various types of cells corresponds to the type of tumor caused by the intact virus in vivo. These results provide direct evidence that the tissue specificity of the transcriptional activity of LTRs plays a critical role in determining the target cell for retroviral oncogenesis.
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PMID:Correlation of leukemogenic potential of murine retroviruses with transcriptional tissue preference of the viral long terminal repeats. 302

The efficiency of DNA transfer into human hematopoietic cells by electroporation was investigated and compared to conventional transfection procedures. Important parameters of electroporation were optimized in human erythroleukemia cells using the chloramphenicol acetyltransferase (acetyl-CoA; chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28) gene linked to the cytomegaloviral enhancer-promoter. In addition, selected chemicals with different modes of action were studied for their ability to aid DNA entry and gene expression in this system, and several were found to enhance gene transfection by electroporation in a significant manner. Using these chemical stimulators, many but not all human and mouse suspension cultures tested were successfully electroporated by the Baekon 2000 instrument. From these studies, it appears that electroporation can be enhanced by chemical additives. Because of its efficiency, reproductivity, and convenience electroporation is an attractive method of gene transfer in human hematopoietic cells.
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PMID:Short-term efficient expression of transfected DNA in human hematopoietic cells by electroporation: definition of parameters and use of chemical stimulators. 328 63


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