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)

Sequence variations of the 5'-upstream region of latent membrane protein 1 (LMP-1) in two Epstein-Barr virus (EBV) strains have been reported before (Chen et al., 1992). To investigate the effect of these variations on gene expression, we constructed a series of deletion plasmids encompassing positions -950 to +20 of the LMP-1 promoter region and tested for the ability to drive chloramphenicol acetyltransferase (CAT) gene expression in C33A cells. Results showed that the promoter activities of constructs from NPC strain were 3-fold lower than the corresponding constructs from the B95-8 strain. In addition, the region between -54 and +20 contained the basic, constitutive promoter activity for both strains. Sequence analysis of this region indicated that an activating transcription factor (ATF) binding site, TGACGTAG, which is present in B95-8 strain was changed to TCTCGTAG in NPC strain. A chimeric plasmid study suggested that these sequence variations in the ATF binding site may contribute to the 3-fold increase of CAT activity observed for B95-8 strain. Furthermore, the activity of the promoter constructs was not activated by EBV-encoded nuclear antigen 2 (EBNA-2) in C33A cells. However, the promoter activities were upregulated in B-lymphocyte cells such as CG3 and CA46 cells. The biological significance of this difference in promoter activity of LMP-1 gene between two strains and the involvement of the cellular factors were discussed.
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PMID:Characterization of 5'-upstream sequence of the latent membrane protein 1 (LMP-1) gene of an Epstein-Barr virus identified in nasopharyngeal carcinoma tissues. 748 24

A very small plasmid vector system is described for construction and high-level production of C-terminal chloramphenicol acetyltransferase (CAT) fusion proteins in Escherichia coli. The only functional elements of the plasmid are a minimal region of the ColE1 origin of DNA replication and the Tn9 cat gene, both under control of a tac promoter. Since C-terminal fusion to CAT does not interfere with chloramphenicol (Cm) resistance, plasmids are maintained under Cm selection. Because of its small size (1392 bp), the system is especially convenient for building and expression of synthetic genes and gene fragments. This concept was utilized to generate a fusion with a synthetic gene encoding the multiple-epitope fragment from the rubella virus E1 membrane protein. Affinity-purified fusion proteins were obtained in mg amounts from 100-ml batches of culture fluid, and incorporated as a specific antigen in a rubella immunoglobulin G enzyme-linked immunosorbent assay.
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PMID:An Escherichia coli plasmid vector system for high-level production and purification of heterologous peptides fused to active chloramphenicol acetyltransferase. 768 30

The effects of defined mutations in the lipopolysaccharide (LPS) and the outer membrane protein OmpA of the recipient cell on mating-pair formation in liquid media by the transfer systems of the F-like plasmids pOX38 (F), ColB2 and R100-1 were investigated. Transfer of all three plasmids was affected differently by mutations in the rfa (LPS) locus of the recipient cell, the F plasmid being most sensitive to mutations that affected rfaP gene expression which is responsible for the addition of pyrophosphorylethanolamine (PPEA) to heptose I of the inner core of the LPS. ColB2 transfer was more strongly affected by mutations in the heptose II-heptose III region of the LPS (rfaF) whereas R100-1 was not strongly affected by any of the rfa mutations tested. ompA but not rfa mutations further decreased the mating efficiency of an F plasmid carrying a mutation in the mating-pair stabilization protein TraN. An F derivative with a chloramphenicol acetyltransferase (CAT) cassette interrupting the traA pilin gene was constructed and pilin genes from F-like plasmids (F, ColB2, R100-1) were used to complement this mutation. Unexpectedly, the results suggested that the differences in the pilin sequences were not responsible for recognizing specific groups in the LPS, OmpA or the TraT surface exclusion protein. Other corroborating evidence is presented suggesting the presence of an adhesin at the F pilus tip.
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PMID:The role of the pilus in recipient cell recognition during bacterial conjugation mediated by F-like plasmids. 785 27

The Na,K-ATPase is an integral plasma membrane protein consisting of alpha and beta subunits, each of which has discrete isoforms expressed in a tissue-specific manner. Of the three functional alpha isoform genes, the one encoding the alpha 3 isoform is the most tissue-restricted in its expression, being found primarily in the brain. To identify regions of the alpha 3 isoform gene that are involved in directing expression in the brain, a 1.6 kb 5'-flanking sequence was attached to a reporter gene, chloramphenicol acetyltransferase (CAT). The alpha 3-CAT chimeric gene construct was microinjected into fertilized mouse eggs, and transgenic mice were produced. Analysis of adult transgenic mice from different lines revealed that the transgene is expressed primarily in the brain. To further delineate regions that are needed for conferring expression in this tissue, systematic deletions of the 5'-flanking sequence of the alpha 3-CAT fusion constructs were made and analyzed, again using transgenic mice. The results from these analyses indicate that DNA sequences required for mediating brain-specific expression of the alpha 3 isoform gene are present within 210 bp upstream of the transcription initiation site. alpha 3-CAT promoter constructs containing scanning mutations in this region were also assayed in transgenic mice. These studies have identified both a functional neural-restrictive silencer element as well as a positively acting cis element.
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PMID:The presence of both negative and positive elements in the 5'-flanking sequence of the rat Na,K-ATPase alpha 3 subunit gene are required for brain expression in transgenic mice. 798 27

The MIP (major intrinsic protein) gene, a member of an ancient family of membrane channel genes, encodes the predominant fiber cell membrane protein of the ocular lens. Its specific expression in the lens fibers is temporally and spatially regulated during development. To study the regulation of expression of MIP and delineate the regulatory elements underlying its tissue specificity and ontogenic profile, we have cloned 2840 bp of the human MIP 5'-flanking sequence. The human MIP 5'-flanking sequence contains three complete Alu repetitive elements in tandem at position between nt -1699 and -2684 (nt -1699/-2684). These Alu elements appear to have had a complex evolutionary history with insertions at different times. We have fused DNA fragments containing MIP 5'-flanking sequences to the bacterial cat reporter gene encoding chloramphenicol acetyltransferase and assayed them in primary cultures of chicken lens cells. We have mapped two negative regulatory regions in the human MIP 5'-flanking sequences -1564/-1696 and -948/-1000. We demonstrated that the human MIP 5'-flanking sequence -253/+42 contains a functional promoter in lens cells but is inactive in kidney epithelial cells or mouse fibroblasts, suggesting that this sequence contains regulatory elements responsible for the lens-specific expression of MIP.
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PMID:Isolation and characterization of the 5'-flanking sequence of the human ocular lens MIP gene. 856

Use of beta-lactamase in gene fusions to study membrane protein topology permits exploitation of its biological activity to select for positive (external) hybrids on ampicillin agar plates. When the enzyme is attached to cytoplasmic loops of a membrane protein, it is not secreted and is therefore unable to confer ampicillin resistance. In this study, we examine the use of the cytoplasmic enzyme chloramphenicol acetyltransferase (Cat) as a complement to the use of periplasmic beta-lactamase, in gene fusion studies. This enzyme is responsible for chloramphenicol resistance in Escherichia coli. We show that Cat confers substantial antibiotic resistance when fused to cytoplasmic loops of lactose permease. As expected, periplasmically exposed Cat is enzymatically active in vitro but unable to confer significant chloramphenicol resistance, presumably because of the absence of acetylcoenzyme A in the periplasm. Therefore, Cat may serve as a topogenic sensor in gene fusion studies. The new Cat fusion approach is discussed with regard to its potential use for selecting E. coli mutants which are defective in the assembly of membrane proteins.
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PMID:Biogenesis and topology of integral membrane proteins: characterization of lactose permease-chloramphenicol acetyltransferase hybrids. 871 79

The triose phosphate 3-phosphoglycerate phosphate translocator (TPT) is a chloroplast envelope inner membrane protein whose transit peptide has structural properties typical of a mitochondrial presequence. To study the TPT transit peptide in more detail, we constructed two chimeric genes encompassing the TPT transit peptide and either 5 or 23 amino-terminal residues of the mature TPT, both linked to the reporter chloramphenicol acetyltransferase (cat) gene. The precursors were synthesized in vitro and translocated to and processed in purified plant mitochondria. However, this import was not specific since both precursors were also imported into isolated chloroplasts. To extend this analysis in vivo, the chimeric genes were introduced into tobacco by genetic transformation. Analysis of CAT distribution in subcellular fractions of transgenic plants did not confirm the data obtained in vitro. With the construct retaining only 5 residues of the mature TPT, CAT was found in the cytosolic fraction. Extension of the TPT transit peptide to 23 residues of the mature TPT allowed specific import and processing of CAT into chloroplasts. These results indicate that, despite its unusual structure, the TPT transit peptide is able to target a passenger protein specifically into chloroplasts, provided that NH2-terminal residues of the mature TPT are still present. The discrepancy between the in vitro and in vivo data suggests that the translocation machinery is more stringent in the latter case and that sorting of proteins might not be addressed adequately by in vitro experiments.
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PMID:Different in vitro and in vivo targeting properties of the transit peptide of a chloroplast envelope inner membrane protein. 918 51

Episomal vectors, described for efficient and regulated expression of heterologous proteins in mammalian cells, have the advantage that they persist in multiple copies in the cell without integrating into the chromosome. To efficiently express heterologous proteins we used such a vector based on elements of the Epstein-Barr virus (EBV), namely the sequences coding for Epstein-Barr nuclear antigen 1 and the viral origin of replication. Because constitutive expression is often deleterious to the cell, we combined the interferon-inducible Mx promoter with this EBV-derived vector. This resulted in an efficient and strictly regulated expression of the reporter gene chloramphenicol acetyltransferase (CAT) and of the neurotransmitter receptor h5-HT(1B), reaching levels of 16 ng CAT/mg cytoplasmic protein and 1300 fmol receptor/mg membrane protein, respectively. For both proteins, the expression levels were influenced by the orientation of the expression cassette. The higher expression in the favored orientation did not result from a higher copy number of these episomes. Northern analysis revealed a transcriptional read-through from the thymidine kinase promoter on the episomal vector, which interfered with the transcription of the heterologous gene in the less favored orientation.
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PMID:Orientation-dependent gene expression with Epstein-Barr virus-derived vectors. 1467 61

The spc operon of Escherichia coli encodes 11 ribosomal proteins and SecY. The secY gene and downstream rpmJ encoding a ribosomal protein, L36, are located distal to the promoter of the spc operon. It has been suggested that the stability of SecY mRNA depends on rpmJ unless a rho-independent terminator is inserted immediately downstream of secY. Moreover, it has been suggested that RpmJ is dispensable for E. coli. We constructed rpmJ null strains, AY101 (DeltarpmJ::tetA) and AY201 (DeltarpmJ::cat), by replacing rpmJ with tetA, which encodes a membrane protein responsible for tetracycline-resistance, and cat, which encodes a cytoplasmic chloramphenicol acetyltransferase, respectively. Depletion of RpmJ did not inhibit protein synthesis, whereas the growth of AY101 was defective at high temperatures. The level of SecY mRNA decreased significantly in both disruptants even though the rho-independent terminator was inserted immediately downstream of secY. Some periplasmic proteins were missing in the disruptants with a concomitant increase in the amount of phage shock protein in the inner membrane. These phenotypes caused by the rpmJ null mutation were corrected by a plasmid carrying secY, but not by one carrying rpmJ.
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PMID:Disruption of rpmJ encoding ribosomal protein L36 decreases the expression of secY upstream of the spc operon and inhibits protein translocation in Escherichia coli. 1611 91

Previous studies have demonstrated that Helicobacter pylori (Hp) delays its entry into macrophages and persists inside megasomes, which are poorly acidified and accumulate early endosome autoantigen 1. Herein, we explored the role of Hp urease in bacterial survival in murine peritoneal macrophages and J774 cells. Plasmid-free mutagenesis was used to replace ureA and ureB with chloramphenicol acetyltransferase in Hp Strains 11637 and 11916. ureAB null Hp lacked detectable urease activity and did not express UreA or UreB as judged by immunoblotting. Deletion of ureAB had no effect on Hp binding to macrophages or the rate or extent of phagocytosis. However, intracellular survival of mutant organisms was impaired significantly. Immunofluorescence microscopy demonstrated that (in contrast to parental organisms) mutant Hp resided in single phagosomes, which were acidic and accumulated the lysosome marker lysosome-associated membrane protein-1 but not early endosome autoantigen 1. A similar phenotype was observed for spontaneous urease mutants derived from Hp Strain 60190. Treatment of macrophages with bafilomycin A1, NH4Cl, or chloroquine prevented acidification of phagosomes containing mutant Hp. However, only ammonium chloride enhanced bacterial viability significantly. Rescue of ureAB null organisms was also achieved by surface adsorption of active urease. Altogether, our data indicate a role for urease and urease-derived ammonia in megasome formation and Hp survival.
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PMID:Role of urease in megasome formation and Helicobacter pylori survival in macrophages. 1654 3


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