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)

In the human genome, the erythroid-specific hypersensitive site HS2 enhancer regulates the transcription of the downstream beta-like globin genes 10-50 kilobases away. The mechanism of HS2 enhancer function is not known. The present study employs RNA protection assays to analyze the transcriptional status of the HS2 enhancer in transfected recombinant chloramphenicol acetyltransferase (CAT) plasmids. In erythroid K562 cells in which the HS2 enhancer is active, the HS2 sequence directs the synthesis of long enhancer transcripts that are initiated apparently from within the enhancer and elongated through the intervening DNA into the cis-linked CAT gene. In nonerythroid HL-60 cells in which the HS2 enhancer is inactive, long enhancer transcripts are not detectable. Splitting the HS2 enhancer between two tandem Ap1 sites abolishes the synthesis of a group of long enhancer transcripts and results in loss of enhancer function and transcriptional silencing of the cis-linked CAT gene. In directing the synthesis of RNA through the intervening DNA and the gene by a tracking and transcription mechanism, the HS2 enhancer may (i) open up the chromatin structure of a gene domain and (ii) deliver enhancer binding proteins to the promoter sequence where they may stimulate the transcription of the gene at the cap site.
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PMID:Transcription of the hypersensitive site HS2 enhancer in erythroid cells. 145 1

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

The c-fos proto-oncogene seems to play an important role during differentiation and activation of cells from the hematopoietic lineage. Therefore, it is of interest to investigate the mechanism underlying its transcriptional activation in these cells. To delineate the sequences and factors involved in c-fos transcriptional activation during the course of myeloid cell differentiation, we have used the K 562 chronic leukemic cell line as a model. K 562 cells were transfected with chloramphenicol transacetylase (CAT) reporter constructs, including various regions of the human c-fos promoter, and induced to differentiate by two distinct agents: 12-O-tetradecanoyl phorbol-13-acetate (TPA), which activates a differentiation program along the megakaryoblastic pathway; and hemin, which induces erythroid differentiation. We show here that TPA treatment of K 562 cells induces fos CAT reporter constructs activation, whereas treatment with hemin does not. Furthermore, predifferentiation of the cells with hemin blocks a subsequent induction by TPA, in correlation with the inhibition by hemin of megakaryoblastic differentiation markers appearance. Both the induction by TPA and the inhibition by hemin are mediated by a dyad symmetry element (DSE) located in the upstream regulatory region, between -318 and -296. These results suggest that the protein complex binding to the DSE regulatory element is the target for c-fos activation by TPA and inhibition by hemin in K 562 cells. However, no modulation of protein affinity for the DSE sequence was detected by gel shift assay during the course of induction or inhibition, suggesting that the structural change responsible for the transcriptional modulation is too unstable or too subtle to be detected by this method.
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PMID:The dyad symmetry element is the molecular target for c-fos induction and inhibition during K 562 differentiation along mutually exclusive lineages. 189 33

B19 parvovirus is absolutely tropic for human erythroid progenitor cells. Among the untested mechanisms underlying this tropism is the possibility of cell-specific positive regulation of the promoter in permissive cells. Using the bacterial chloramphenicol acetyltransferase and firefly luciferase reporter genes, we detected strong activity from the B19 P6 promoter in transfected nonpermissive cells. Very high-level expression was seen in a T lymphoblastoid cell line, CEM. No transcriptional enhancement occurred in an erythropoietin-dependent semipermissive cell line. A putative second B19 promoter at map unit 44 (P44) was nonfunctional and unable to confer tissue specificity. Thus, tropism is unlikely to be regulated at the level of transcriptional initiation from either the P6 or P44 promoter.
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PMID:Indiscriminate activity from the B19 parvovirus p6 promoter in nonpermissive cells. 202 72

We have used a competition assay to investigate the influence of erythroid-specific cellular factors on transcription from the human epsilon-globin major cap site promoter and the minor promoter located 200 base pairs (bp) upstream from the epsilon-globin cap site. In the human erythroid cell line K562, competition of the epsilon-globin major cap site promoter linked to the chloramphenicol acetyltransferase (CAT) gene (epsilon P-CAT) with the same promoter fragment linked to a neomycin resistance gene (epsilon P-NEO) leads to a reduction in CAT activity. This indicates the specific presence of K562 cells of factor(s) which interact with the 200-bp promoter fragment (isolated from the gene body or flanking sequences) to activate transcription from the epsilon-globin major cap site. Competition of the epsilon-globin major promoter (as epsilon P-CAT) with the upstream minor epsilon-globin promoter (as epsilon P2-NEO) also leads to a reduction in CAT activity, indicating that both promoters share erythroid-specific trans-acting factors. The reverse competition (epsilon P2-CAT with epsilon P-NEO) leads to an increase in CAT activity, suggesting that the existence of erythroid-specific factor(s) which repress transcription from the 200-bp-upstream epsilon-globin promoter.
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PMID:Interaction of epsilon-globin cis-acting control elements with erythroid-specific regulatory macromolecules. 223 25

We report the isolation and complete sequence of the gene encoding the rabbit erythroid-cell-specific 15-lipoxygenase (RBC 15-LOX), containing 14 exons spanning 8.0 kb. The transcription start point was mapped by S1 nuclease-protection experiments and comparison with the sequence of the RBC 15-LOX mRNA, as defined previously by primer extension experiments. The promoter contains a TATA-like motif, but no CCAAT motif in the canonical position, and lies within a 'CpG-rich island'. Functional analysis of the immediate 5'-flanking DNA by transfection experiments shows that a 150 nucleotide (nt) 5' fragment linked to the chloramphenicol acetyltransferase gene acts as a functional promoter in both erythroid and nonerythroid cell lines and responds in an erythroid-specific manner to the enhancer from the Friend murine leukaemia virus long terminal repeat, whereas a 40-nt fragment is inactive. Intron 7 contains eight copies of a 54-nt repeat containing a region with homology to the simian virus 40/immunoglobulin gene enhancers.
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PMID:The promoter structure and complete sequence of the gene encoding the rabbit erythroid cell-specific 15-lipoxygenase. 261 16

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

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

An enhancer specific to erythroid cells was identified previously in the 3' flanking sequence of the chicken adult beta-globin gene and shown to act on the beta-globin promoter. This enhancer lies between the adult beta-globin gene and the embryonic epsilon-globin gene, about equidistant from the two promoters. To determine whether this enhancer acts also on the epsilon-globin promoter, we constructed plasmids containing the enhancer and either the beta- or the epsilon-globin promoter fused to the bacterial chloramphenicol acetyltransferase gene. Primary chicken erythrocytes of both primitive and definitive lineages were transfected with these plasmids. We show that the enhancer is able to stimulate expression from the epsilon-globin promoter as well as the beta-globin promoter. Levels of expression change with the developmental stage of the cell in a way that is partially consistent with the observed developmental regulation of the beta- and epsilon-globin genes in vivo. There appear to be no other enhancer elements either 5' of the epsilon-globin gene or within 6 kilobase pairs of its 3' end. Thus, the enhancer between the beta- and epsilon-globin genes apparently serves to regulate both genes.
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PMID:Bidirectional control of the chicken beta- and epsilon-globin genes by a shared enhancer. 335 80

We describe a method for studying transient gene expression in primary avian erythroid cells that involves controlled osmotic shock, followed by DNA transfection using DEAE-dextran. Cells treated in this way reproducibly express high levels of chloramphenicol acetyltransferase (CAT) when transfected with a plasmid having the cat gene coupled to an appropriate viral promoter. An observed correlation between levels of CAT expression and extent of hemoglobin release during controlled shock makes it possible to choose optimum conditions for expression in erythroid cells at various stages of embryonic development. Using these techniques, we have investigated the effect on CAT expression of fusing to the cat gene various portions of the chicken adult beta-globin (beta A) gene. We show that in 9-day or 12-day embryonic erythrocytes, the promoter activity of the 5' flanking region of the beta A gene (in the absence of any viral promoters) is strongly stimulated by a downstream sequence, located in the region 110-588 base pairs on the 3' side of the poly(A) signal, that acts as an enhancer. Its activity is reduced in 5-day embryonic cells and absent in primary chicken fibroblasts and mouse L cells, suggesting that this transient expression system will be useful in studying developmentally regulated globin gene expression.
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PMID:Regulated gene expression in transfected primary chicken erythrocytes. 345 75


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