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 purpose of this investigation was to identify and characterize the regulatory elements involved in the transcriptional activation of the beta gamma (leaky-late or gamma 1) genes of herpes simplex virus type 1 (HSV-1) by using the major capsid protein (VP5 or ICP5) gene as model. Gel mobility shift assays with nuclear extracts from uninfected and infected HeLa cells enabled us to identify two major protein-DNA complexes involving the VP5 promoter. The mobilities of these two complexes remained unaltered, and no unique complexes were observed when infected cell nuclear extracts were used. DNase I and orthophenanthroline-Cu+ footprint analyses revealed that the two complexes involve a single binding site, GGCCATCTTGAA, located between -64 and -75 bp relative to the VP5 cap site. To determine the function of this leaky-late binding site (LBS) in VP5 gene activation, we tested the effect of mutations in this region by using transient expression of a cis-linked chloramphenicol acetyltransferase gene. Deletion of the above sequence resulted in a seven- to eightfold reduction in the level of transactivation of the chloramphenicol acetyltransferase gene by superinfection with HSV-1 or by cotransfection of HSV-1 immediate-early genes. From these results, we conclude that the LBS sequence and a cellular factor(s) are involved in the transactivation of the VP5 gene. A search of published gene sequences revealed that sequences related to the LBS exist in a number of other HSV-1, cytomegalovirus, retrovirus, and cellular promoters. Sequence homologies of binding sites and results of unpublished competition binding studies suggest that this leaky-late binding factor may be related to, or the same as, a ubiquitous cellular transcriptional factor called YY1 or common factor-1 (also known as NF-E1, delta, and UCRBP).
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PMID:Transactivation of the major capsid protein gene of herpes simplex virus type 1 requires a cellular transcription factor. 131 6

The chicken ovalbumin gene is regulated at the level of transcription by four classes of steroid hormones. A steroid-dependent regulatory element (SDRE) found from -900 to -732 is required for this steroid-mediated induction. To define more precisely sequences of the SDRE required for steroidal induction, a series of exonuclease III deletions were made in the 3' end of the SDRE. Fusion genes containing the mutant ovalbumin 5'-flanking sequences linked to the chloramphenicol acetyltransferase structural gene (CAT) were transfected into steroid-responsive primary oviduct cells. These functional studies defined a region of the SDRE from -793 to -759 that is essential for induction by steroids. Analysis of protein interactions in this 34-base pair region by copper-phenanthroline footprinting and methylation interference assays defined nucleotides required for protein binding. Footprinting showed protection of residues extending from -784 to -765, an area that included nucleotides that, when methylated, interfered with protein binding. In addition, this footprinted region contained 10 nucleotides that were identical to sequences contained in the beta-interferon gene regulatory element. An oligomer synthesized to this region of homology produced two DNA-protein complexes with oviduct nuclear proteins. Although this region of the interferon gene regulatory element binds the transcription factor NF-kappa B, an oligomer from the immunoglobulin kappa light chain gene known to bind NF-kappa B did not compete with the SDRE oligomer for binding to oviduct nuclear proteins. Surprisingly, this same NF-kappa B oligomer was able to restore steroid responsiveness to an SDRE mutant, while an oligomer from the immunoglobulin heavy chain gene inserted in the same position did not affect induction by steroids. These data suggest that a protein binding to sequences in the SDRE that are similar to an NF-kappa B-binding site participates in the steroid-mediated increase in transcription of the chicken ovalbumin gene.
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PMID:A protein with a binding specificity similar to NF-kappa B binds to a steroid-dependent regulatory element in the ovalbumin gene. 203 95

To define the import pathway for apoiso-1-cytochrome c in vivo, the coding region for bacterial chloramphenicol acetyltransferase (CAT) or yeast copper metallothionein (CuMT) was fused to the carboxy terminus of the apoiso-1-cytochrome c (iso-1) coding region. When the resulting iso-1/CAT and iso-1/CuMT fusion proteins were individually expressed in Saccharomyces cerevisiae, they were specifically targeted to the mitochondria and protected from trypsin digestion. Although iso-1/CAT was accessible to heme modification, it remained membrane associated because of the folded conformation of the CAT domain. A small deletion disrupting CAT structure resulted in the translocation of the resulting fusion protein, iso-1/CAT delta, to the intermembrane space, where it functioned efficiently in respiratory electron transfer. Similarly, iso-1/CuMT was heme modified and nearly identical to iso-1 in its ability to support respiratory growth, indicating that the CuMT domain was compatible with translocation to the IMS. Inclusion of copper in the growth medium, which converts the loosely structured apo-CuMT to a tightly folded holo-CuMT, inhibited both heme attachment and respiratory growth without affecting mitochondrial targeting. Thus, by altering the folded conformation of the reporter moiety of these fusion proteins, it was possible to differentiate between those molecules arrested at the mitochondrial targeting step of the cytochrome c import pathway and those translocated to the intermembrane space. By replacing the heme-binding cysteine residues with serines, this system was used to demonstrate that the import requirement for heme attachment operated at the level of membrane translocation and not on mitochondrial targeting in vivo.
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PMID:In vivo expression and mitochondrial targeting of yeast apoiso-1-cytochrome c fusion proteins. 217 84

Three nuclear factors, the Ah receptor, XF1, and XF2, bind sequence specifically to the Ah response elements or xenobiotic response elements (XREs) of the cytochrome P450IA1 (P450c) gene. The interactions of these factors with the Ah response element XRE1 were compared by three independent methods, methylation interference footprinting, orthophenanthroline-Cu+ footprinting, and mobility shift competition experiments, using a series of synthetic oligonucleotides with systematic alterations in the XRE core sequence. These studies established the following (i) all three factors interact sequence specifically with the core sequence of XRE1; (ii) the pattern of contacts made with this sequence by the Ah receptor are different from those made by XF1 and XF2; and (iii) although XF1 and XF2 can be distinguished by the mobility shift assay, the sequence specificities of their interactions with XRE1 are indistinguishable. Further characterization revealed the following additional differences among these three factors: (i) XF1 and XF2 could be extracted from nuclei under conditions quite different from those required for extraction of the Ah receptor; (ii) XF1 and XF2 were present in the nuclei of untreated cells and did not respond to polycyclic compounds, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and beta-napthoflavone, while nuclear Ah receptor was undetectable in untreated cells and rapidly increased in response to TCDD; (iii) inhibition of protein synthesis did not affect the TCDD-induced appearance of the Ah receptor but substantially decreased the constitutive activities of XF1 and XF2, suggesting that the Ah receptor must be present in untreated cells in an inactive form that can be rapidly activated by polycyclic compounds, while the constitutive expression of XF1 and XF2 depends on the continued synthesis of a relatively unstable protein; (iv) the receptor-deficient and nuclear translocation-defective mutants of the hepatoma cell line Hepa1, which are known to lack nuclear Ah receptor, expressed normal levels of XF1 and XF2, suggesting that the former factor is genetically distinct from the latter two; and (v) a divalent metal ion, probably Zn2+, is known to be an essential cofactor for the Ah receptor but was not required for the DNA-binding activities of XF1 and XF2. Together, these findings indicate that the Ah receptor is distinct from XF1 and XF2, while the latter two activities may be related. Because the DNA-binding domains of these three factors overlap substantially, their binding to XREs is probably mutually exclusive, which suggests that the interplay of these factors at Ah response elements may be important to the regulation of CYP1A1 gene transcription. The results of preliminary transfection experiments with constructs harboring XREs upstream of the chloramphenicol acetyltransferase gene driven by a minimal simian virus 40 promoter are presented that are consistent with this hypothesis.
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PMID:Multiple DNA-binding factors interact with overlapping specificities at the aryl hydrocarbon response element of the cytochrome P450IA1 gene. 217 7

Rat cell lines were established in which the bacterial chloramphenicol acetyltransferase (CAT) gene under control of the human immunodeficiency virus (HIV) long terminal repeat (LTR) was stably integrated. The cell lines showed a repressed phenotype for CAT expression, but could be induced for it by inhibition of protein synthesis, as well as by heat-shock and chemical inducers of the cellular stress response, such as sodium arsenite, 8-hydroxyquinoline and the heavy metals cadmium and copper. A decameric sequence present in the NF-kB binding sites in the HIV LTR (GGGACTTTCC) resembles the cellular heat-shock core sequence and may therefore be involved in the heat-shock response.
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PMID:Heat-shock induction of the human immunodeficiency virus long terminal repeat. 318 32

The human metallothionein (MT)-IG gene (hMT-IG) is tandemly linked in a head-to-head fashion with the hMT-IF gene. The hMT-IG gene encodes a MT-I polypeptide and has a tripartite structure. The 5'-flanking region of the hMT-IG gene has a TATAA box, four GC motifs, and at least four metal responsive elements. The 3'-untranslated region has a variation of the polyadenylation signal, AATTAA, and the 3'-flanking region a YGTGTTYY RNA processing signal. This gene is expressed in hepatoma-derived cell lines (Hep G2 and Hep3B2) in response to the heavy metals (cadmium, copper, and zinc) but not to the glucocorticoid analogue dexamethasone. In contrast, the lymphoblastoid cell line (Wi-L2) does not express the hMT-IG gene. These results suggest that the hMT-IG gene is regulated differentially and in a cell type-specific manner. Transient expression studies of the chloramphenicol acetyltransferase (CAT) gene under the transcriptional control of either the hMT-IG or hMT-IF promoter in Hep G2 cells has demonstrated that both promoters contain all the necessary cis-acting elements to elicit a similar pattern of heavy metal inducibility. However, the hMT-IG promoter in all instances is five times more active than the hMT-IF promoter. The differences in promoter activity of these genes could possibly be due to inherent differences in their basal level regulatory sequences. The expression of MT-IGcat in transfected Wi-L2 cells demonstrates that the hMT-IG promoter is not cell type-specific.
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PMID:Structure and expression of the human metallothionein-IG gene. Differential promoter activity of two linked metallothionein-I genes in response to heavy metals. 340 43

Occipital horn syndrome (OHS), an X-linked connective tissue disorder, has recently been shown to result from mutations in the Menkes disease gene (MNK), which encodes a copper-transporting ATPase. By Southern analysis we detected a small deletion in a region 5' to the MNK gene in one patient with OHS. Genomic clones from an unaffected individual were isolated and sequenced, revealing three tandem 98 bp repeats situated upstream of the reported transcription start site, and analysis of the patient's DNA showed a deletion of one of the repeats. The deletion is likely to be responsible for the disease in this patient, as it was not observed in 110 unaffected individuals analyzed, and no other mutation in the patient was detected by RT-PCR and chemical cleavage mismatch analysis or by cDNA sequence analysis. The deletion is associated with a dramatic decrease in expression of a chloramphenicol acetyltransferase reporter gene, implicating the repeat sequences in regulation of MNK expression, although a quantitative analysis of MNK mRNA from a cell line derived from the patient shows no detectable reduction. Other experiments revealed no effect on the site of transcription initiation, termination or on splicing.
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PMID:A repeated element in the regulatory region of the MNK gene and its deletion in a patient with occipital horn syndrome. 892 1

A panel of HepG2-derived cell lines (CAT-Tox [L] assay, Xenometrix), harboring stress genes consisting of a sequence for chloramphenicol acetyltransferase (CAT) under the transcriptional regulation from mammalian promoters and response elements, was exposed for 18-24 hr to aqueous suspensions of urban dusts (SRM-1648, SRM-1649, EHC-93) or PM2.5 particles (particulate matter < 2.5 micron). Expression of CAT protein was measured by enzyme-linked immunosorbent assay. Induction of the CAT genes was verified with benzo[a]pyrene (CYP1A1, cytochrome P450 1A1 promoter; GSTYa, glutathione transferase subunit Ya promoter; XRE, xenobiotic response element), cadmium sulfate, and copper sulfate (HMTIIa, metallothionein IIa promoter; HSP70, heat shock protein 70 promoter). The urban dust suspensions were active on CYP1A1, GSTYa, and XRE cell lines. SRM-1648 and SRM-1649 were twice as potent as EHC-93 per unit mass in inducing the xenobiotic-dependent responses, which correlated with contents in polycyclic aromatic hydrocarbons. These three reference particles, as well as six PM2.5 preparations collected on hi-vol filters in the Great Lakes basin, were also found to induce HMTIIa and HSP70, the magnitude of the responses correlating closely with the amount of soluble copper in the particulate preparations. The results indicate that bioavailable chemical species in the unfractionated particles can directly and quantitatively induce xenobiotic, metal, and stress-dependent responses in a target cell model, resulting in patterns of gene induction consistent with the chemical compositions of the environmental materials. We propose that cell culture models could be helpful for toxicodynamic inferences in adjunct to environmental monitoring and exposure assessments.
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PMID:Regulation of promoter-CAT stress genes in HepG2 cells by suspensions of particles from ambient air. 932 24

Two regulatory genes (lcoR and lcoS) were identified from a plasmid-borne lactococcal copper resistance determinant and characterized by transcriptional fusion to the promoterless chloramphenicol acetyltransferase gene (cat). RT-PCR analysis indicates that lcoR and lcoS are organized within an operon, controlling the transcription of cat in a copper-inducible manner. The amino acid sequences deduced from lcoR and lcoS show homology to the response and sensor proteins of known two-component regulatory systems. Deletion within either lcoS or both genes inactivated the copper-dependent activity, suggesting the presence of no trans-acting lcoR and lcoS homologs in the lactococcal host chromosome. The transcription start site involved in copper induction was mapped by primer extension.
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PMID:A plasmid-encoded two-component regulatory system involved in copper-inducible transcription in Lactococcus lactis. 1009 23

The Cu/Zn superoxide dismutase (SODI) catalyzes the dismutation of superoxide radicals produced in the course of biological oxidations. When placed under the control of the rat SOD1 gene promoter and transfected into human HepG2 hepatoma cells, the activity of a chloramphenicol acetyltransferase reporter gene was found to increase three- to four-fold in the presence of heavy metals (cadmium, zinc and copper). Functional analysis of mutant derivatives of the SOD1 gene promoter and the use of a heterologous promoter system confirmed that the induction of the SOD1 gene by metal ions requires a metal-responsive element (MRE) located between positions -273 and -267 (GCGCGCA). It was also shown by gel mobility shift assays that an MRE binding protein is induced by the exposure of the human liver cell line HepG2 to heavy metals. These results suggest that the MRE participates in the induction of the SOD1 gene by heavy metals.
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PMID:Heavy metal-mediated activation of the rat Cu/Zn superoxide dismutase gene via a metal-responsive element. 1051 27


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