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 U3 regions of the long terminal repeats (LTRs) of simian immunodeficiency viruses SIVmac251 and SIVmac239 were analyzed for basal transcriptional activity and for interaction with cellular factors in the T-cell line HUT-78 and the monocyte/macrophage cell line U937. A number of 5' deletions and mutations were made in the U3 regions of the two LTRs, and these constructs were placed upstream of a plasmid containing the bacterial chloramphenicol acetyltransferase reporter gene. The nucleotide sequences between -225 and +18 were sufficient to maintain full transcriptional activity of both LTRs in HUT-78 and U937 cells. Nucleotide sequence analysis revealed several differences between SIVmac251 and SIVmac239 within this region. Analysis of deletion mutants revealed that an additional removal of bases, from -124 to -225, had little effect on the transcriptional activity of the clone 239 LTR, whereas this deletion resulted in a significant reduction of activity in the clone 251 LTR. DNase protection assays using nuclear extracts from HUT-78 and U937 cells showed that bases within this region bound cellular factors. In addition, the NF-kappa B site was protected in DNase assays with HUT-78 cells and 12-O-tetradecanoylphorbol-13-acetate-treated U937 cells. An additional DNase footprint was detected in SIVmac239, at -52 to -38, just upstream of the TATA box. This site overlaps the 3' half of the 3'-most Sp-1 site and is downstream of 11 bases that are found in SIVmac239 but not SIVmac251. Thus, differences in the sequences in the U3 region of the LTRs of SIVmac251 and SIVmac239 have been identified which appear to alter the transcriptional activity of these promoters as well as changing the interaction of cellular proteins with sequences in the LTRs.
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PMID:Comparison of the transcriptional activity of the long terminal repeats of simian immunodeficiency viruses SIVmac251 and SIVmac239 in T-cell lines and macrophage cell lines. 198 14

To gain insight into the normal controls mediating expression of the c-Ki-ras protooncogene, we have identified DNA sequence elements within its promoter that are essential for transcriptional activity. Transient expression assays using the bacterial chloramphenicol acetyltransferase gene were used initially to localize regions directing primary promoter function. Stepwise deletion of 5' promoter sequences resulted in a gradual decrease in the ability to drive transcription of the reporter gene, suggesting that this promoter is composed of multiple cis-acting elements. Gel mobility-shift and DNase protection studies involving a 166-base-pair DNA fragment allowed the identification of protein-binding sites corresponding to these multiple regulatory elements. One element demonstrating particular transcriptional influence exists as a homopurine/homopyrimidine-rich region that in vitro exhibits S1 nuclease sensitivity and binds at least one nuclear protein. Data from competition binding experiments suggest that this nuclear factor may be influential in the regulation of other essential growth-control genes as well.
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PMID:An S1 nuclease-sensitive homopurine/homopyrimidine domain in the c-Ki-ras promoter interacts with a nuclear factor. 218 46

Uteroglobin is expressed in various tissues of the rabbit under complex hormonal control. In the endometrium the uteroglobin gene is transcribed only in epithelial cells after administration of ovarian hormones. In this paper we demonstrate that within the promoter region of the rabbit uteroglobin gene, there is a functional estrogen-responsive element (ERE) located between -265 and -252. Hybrid constructions containing sequences of the uteroglobin promoter up to -299, linked to the chloramphenicol acetyltransferase gene of E. coli respond to estrogens in gene transfer experiments, whereas a deletion that removes half of the ERE does not. A synthetic oligonucleotide corresponding to the putative ERE is able to confer estrogen inducibility to an otherwise unresponsive promoter. Binding experiments with purified estrogen receptor from calf uterus reveal a DNase-I footprint over the ERE. Within this protected region six guanine residues that have been shown to be contacted by the receptor in other EREs are protected against methylation by dimethylsulfate in the presence of the estrogen receptor. We compare this ERE with the vitellogenin A2 ERE from Xenopus and find that the relative affinity of the uteroglobin ERE is slightly lower than that of the vitellogenin ERE. Thus, this uteroglobin ERE could be involved in physiological regulation of uteroglobin expression in the genital tract.
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PMID:The uteroglobin promoter contains a noncanonical estrogen responsive element. 228 Jul 77

Expression of the skeletal troponin I (sTnI) gene is regulated transcriptionally in a muscle-specific fashion. We show here that the region of the sTnI gene between -160 and +61 (relative to the transcription initiation site) is able to direct expression of the bacterial chloramphenicol acetyltransferase (CAT) gene is muscle cultures at a level approximately 100 times higher than in fibroblast cultures. RNA analysis demonstrated that transcription of the CAT gene was initiated at the same site as transcription of the endogenous sTnI gene and that CAT activity levels were approximately proportional to CAT mRNA levels. Deletion analysis demonstrated that the region between nucleotides -160 and -40 contained sequences essential for full promoter activity. Surprisingly, 3' deletion analysis indicated that the first exon (-6 to +61) of the sTnI gene was also required for full activity of the sTnI promoter in skeletal muscle cells. Chimeric promoter experiments, in which segments of the sTnI and the herpes simplex virus thymidine kinase promoter were interchanged, indicated that reconstitution of a muscle-specific promoter required inclusion of both the upstream and exon I regions of the sTnI gene. Exon I, and the region immediately upstream, showed DNase protection over sequence motifs related to those found in other genes, including the tar region of human immunodeficiency virus type 1. These results demonstrate that expression of the sTnI promoter in embryonic skeletal muscle cells requires complex interaction between two separate promoter regions, one of which resides within the first 61 transcribed nucleotides of the gene.
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PMID:Muscle-specific activity of the skeletal troponin I promoter requires interaction between upstream regulatory sequences and elements contained within the first transcribed exon. 235 14

We have previously identified a series of five DNase-I hypersensitive (HS) sites within and around the rat phosphoenolpyruvate carboxykinase (PEPCK) gene. The far upstream region has now been sequenced, and the tissue-specific HS site has been mapped more precisely at 4,800 base pairs upstream of the transcription start site of the PEPCK gene. DNA fragments that include the HS site were cloned upstream of various promoters to test whether these regions modulate transcription of the chloramphenicol acetyltransferase reporter gene. Chloramphenicol acetyltransferase activity was enhanced when the DNA fragment encompassing the upstream HS site was linked to various lengths of the PEPCK promoter or to the heterologous simian virus 40 promoter. This upstream region in conjunction with the proximal promoter, which may contain a tissue-specific element, conferred maximum activation in H4IIE hepatoma cells, which express the endogenous PEPCK gene. When these experiments were performed in XC cells, in which the gene is not expressed, transcriptional activation by the upstream element was still significant. Evidence of a specific protein-DNA interaction, using DNA mobility shift and DNase I footprinting assays, was obtained only when using H4IIE cell nuclear extracts. Competition assay showed that the interacting factor may be similar or identical to the liver-specific factor HNF3. We suggest that this protein factor binds to DNA within the HS site and interacts with the proximal promoter region to control tissue-specific high-level expression of the PEPCK gene.
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PMID:Interaction of a liver-specific factor with an enhancer 4.8 kilobases upstream of the phosphoenolpyruvate carboxykinase gene. 235 22

We analyzed control sequences of the human papovavirus JC virus (JCV) to define the cis-acting elements that regulate specific expression of the viral early region genes in glial cells. Nuclear run-on transcription, S1 analysis, and chloramphenicol acetyltransferase enzyme activity in a transient transfection assay established that the cell type-specific expression of JCV early genes is determined at the transcriptional level. Using DNase footprinting analysis of nuclear proteins prepared from glial and nonglial cells, we located four regions within the JCV control sequences that specifically interacted with the proteins. In glial cells, all four domains contributed to the specific expression of a heterologous promoter, whereas in nonglial cells, two protein-binding regions showed no effect on basal transcriptional activity and the other two domains significantly downregulated transcription of the promoter. We conclude that cell type-specific transcription of the JCV early promoter is under both positive and negative regulation in eucaryotic cells.
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PMID:Cell type-specific expression of JC virus early promoter is determined by positive and negative regulation. 253 50

The transcriptional enhancer sequences of the papillomaviruses are regulated by trans-acting factors encoded by the viral E2 open reading frame. We have performed detailed functional and physical analyses of the enhancer of the human papillomavirus type 11 (HPV-11). Using the chloramphenicol acetyltransferase (CAT) assay in transiently transfected monkey CV-1 cells, the enhancer region has been localized to a 270-bp tract immediately preceding the E6 open reading frame, and it consists of two functional components. The first is a constitutive enhancer containing sequences homologous to the GT-, Sph-, and P-motifs found in the SV40 and polyomavirus enhancers; others resemble the recognition sequence for CTF (NF-1), a factor which stimulates transcription of certain eukaryotic genes and replication of adenovirus DNA. The second component is an inducible enhancer with a consensus sequence ACCN6GGT responsive to the E2 protein encoded by papillomaviruses. Tandem copies of portions of the constitutive enhancer function as an E2-independent enhancer, whereas multiple copies of HPV-11 DNA restriction fragments or synthetic oligonucleotides containing the E2-responsive sequence (E2-RS) act as an enhancer in the presence of the E2 protein encoded by HPV-1, HPV-11, or bovine papillomavirus type 1 (BPV-1). The inducible activity is lost when mutations are introduced into the E2-RS or when a mutant palindromic sequence is substituted. We have also expressed the E2 proteins of HPV-1, HPV-11, and BPV-1 in Escherichia coli and studied their physical interactions with the E2-responsive sequence in vitro. Filter-binding analyses with crude Escherichia coli lysates show that the E2 proteins bind to the E2-RS, but not to mutated motifs, with an affinity proportional to the copy number. These E2 proteins have been purified to near-homogeneity by sequence-specific DNA affinity chromatography using the synthetic E2-RS as a ligand. The purified proteins protect a DNA segment containing the E2-RS and several flanking nucleotides in pancreatic DNase I footprinting analyses. Based on these results, we conclude that E2 proteins activate the enhancer by binding directly to the E2-RS and interacting with other transcriptional factors and that the sequence ACCN6GGT is both necessary and sufficient for the E2 protein binding in vitro and for activation of RNA transcription in vivo.
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PMID:Functional mapping of the human papillomavirus type 11 transcriptional enhancer and its interaction with the trans-acting E2 proteins. 283 26

The alpha chain of the interleukin-2 receptor (IL-2R alpha) is a key regulator of lymphocyte proliferation. To analyze the mechanisms controlling its expression in normal cells, we used the 5'-flanking region (base pairs -2539/+93) of the mouse gene to drive chloramphenicol acetyltransferase expression in four transgenic mouse lines. Constitutive transgene activity was restricted to lymphoid organs. In mature T lymphocytes, transgene and endogenous IL-2R alpha gene expression was stimulated by concanavalin A and up-regulated by IL-2 with very similar kinetics. In thymic T cell precursors, IL-1 and IL-2 cooperatively induced transgene and IL-2R alpha gene expression. These results show that regulation of the endogenous IL-2R alpha gene occurs mainly at the transcriptional level. They demonstrate that cis-acting elements in the 5'-flanking region present in the transgene confer correct tissue specificity and inducible expression in mature T cells and their precursors in response to antigen, IL-1, and IL-2. In a complementary approach, we screened the 5' end of the endogenous IL-2R alpha gene for DNase-I hypersensitive sites. We found three lymphocyte specific DNase-I hypersensitive sites. Two, at -0.05 and -5.3 kilobase pairs, are present in resting T cells. A third site appears at -1.35 kilobase pairs in activated T cells. It co-localizes with IL-2-responsive elements identified by transient transfection experiments.
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PMID:Mouse interleukin-2 receptor alpha gene expression. Delimitation of cis-acting regulatory elements in transgenic mice and by mapping of DNase-I hypersensitive sites. 773 12

Cell type-specific expression of the catecholamine synthetic enzyme, tyrosine hydroxylase (TH), appears to be mediated in part by cis-acting elements located at the 3' end of the human gene. Further delineation of this region indicated sequences corresponding to a CACGTG motif significantly stimulated transcription of a heterologous promoter in various cell types. Mutation of this site led to a complete loss of activity. DNase footprinting, gel retardation, and UV cross-linking experiments indicated that a 74-kDa cellular factor(s) bound specifically to the CACGTG motif in the pheochromocytoma cell line PC12. The size of this protein and its pattern of expression are compatible with those of the CACGTG binding protein TFE3. Transgenic animals were created using a 261-bp human TH 3' fragment encompassing the CACGTG motif in front of a thymidine kinase promoter/chloramphenicol acetyltransferase reporter gene. In three lines of mice this fragment was sufficient to direct a pattern of mRNA expression in peripheral neuroendocrine tissues that mimicked TH mRNA distribution. However, these sequences were not sufficient for CNS-specific patterns of expression. Thus, multiple cell type-specific enhancers may regulate TH gene expression in the CNS and periphery.
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PMID:The 3' flanking region of the human tyrosine hydroxylase gene directs reporter gene expression in peripheral neuroendocrine tissues. 779 Aug 65

A thyroid transcription factor-1 (TTF-1)-binding element in the rat TSH receptor (TSHR) promoter, between -189 and -175 basepairs (bp), is important for both thyroid-specific expression and thyroid-specific TSH/cAMP autoregulation of the TSHR. The identification of an up-stream TTF-1-binding site and its relationship to the function of the down-stream TTF-1 element are the subjects of this report. Sequence analysis identifies a potential TTF-1 site at -878 bp; deoxyribonuclease-I footprinting shows that the -881 to -866 bp region is protected by recombinant TTF-1 protein and by nuclear extracts from FRTL-5 thyroid cells that contain TTF-1, but not by extracts from nonfunctioning FRT thyroid or Buffalo rat liver (BRL) cells, which have no TTF-1, or by Pax-8. FRTL-5, but not FRT or BRL cell nuclear extracts, form a specific protein-DNA complex with this region in gel mobility shift analyses; its formation is prevented by TTF-1-binding elements from the thyroglobulin promoter. The upstream TTF-1 site acts as an enhancer when coupled to a heterologous simian virus-40 promoter-chloramphenicol acetyltransferase (CAT) chimera and transfected into FRTL-5 thyroid cells. There is a greater increase, 3-vs. 2-fold (P < 0.05), when TSHR promoter-CAT chimeras, which contain the up-stream TTF-1 element, pTRCAT5'-907 or pTRCAT5'-886, as opposed to those in which it is deleted, pTRCAT5'-907 delta USTTF-1, are transfected into FRTL-5 cells or cotransfected with a TTF-1 expression vector into BRL cells, which have no endogenous TTF-1. The TTF-1-dependent activity of pTRCAT5'-907 delta USTTF-1 is the same as that of the minimal promoter, -220 to -39 bp, containing only the down-stream TTF-1 site in both cells. Transfection of chimeric TSHR-CAT plasmids with the down- and/or up-stream TTF-1 site deleted reveals that the down-stream TTF-1 element functions in the absence of the up-stream element, but function of the up-stream site requires the down-stream TTF-1 element. Like the down-stream TSHR TTF-1-binding site, the up-stream TTF-1 site is different from TTF-1 sites in the thyroglobulin and thyroid peroxidase promoter, in that it does not interact with Pax-8.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Characterization of an up-stream thyroid transcription factor-1-binding site in the thyrotropin receptor promoter. 782 40


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