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)

Trypanosomes compartmentalize most of their glycolytic enzymes in a peroxisome-like microbody, the glycosome. The specificity of glycosomal targeting was examined by expression of chloramphenicol acetyltransferase fusion proteins in trypanosomes and monkey cells. Compartmentalization was assessed by cell fractionation, differential detergent permeabilization, and immunofluorescence. The targeting signal of trypanosome phosphoglycerate kinase resides in the COOH-terminal hexapeptide, NRWSSL; a basic amino acid is not required. The minimal targeting signal is, as for mammalian cells, a COOH-terminal tripeptide related to -SKL. However, the acceptable degeneracy of the signal for glycosomal targeting in trypanosomes is considerably greater than that for peroxisomal targeting in mammals, with particularly relaxed requirements in the penultimate position.
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PMID:Glycosome assembly in trypanosomes: variations in the acceptable degeneracy of a COOH-terminal microbody targeting signal. 144 92

Typical of other housekeeping genes, the promoter for the human hypoxanthine phosphoribosyl transferase-encoding gene (HPRT) is G + C-rich, lacks a TATA box and has multiple transcription start points. To test the hypothesis that these features may result in relaxed control over the direction of transcription, we examined the effect of orientation on the ability of the HPRT promoter to control expression of the following reporter genes in transfected cells: luc (firefly luciferase), cat (bacterial chloramphenicol acetyltransferase) and neo (neomycin resistance). A 376-bp fragment containing the HPRT promoter efficiently expressed the luc gene irrespective of orientation, and the 5' ends of luciferase RNA produced in cells transfected with inverted promoter constructs mapped to within the HPRT promoter, indicating that the HPRT promoter has bidirectional activity. However, in the presence of two divergently-flanking reporter genes expression from the inverted HPRT promoter was only 10-20% compared to the noninverted orientation. Furthermore, the inverted HPRT promoter expressed cat less well than luc, and was unable to express neo sufficiently well to produce any colonies under appropriate selection conditions. Attempts to detect endogenous divergent HPRT transcripts were unsuccessful. The promoter of another housekeeping gene, encoding 3-phosphoglycerate kinase (PGK), expressed moderate levels of cat (40%) but not luc (less than 5%) in the inverted orientation. By comparison, two TATA-box containing promoters functioned extremely poorly when inverted. This study indicates that two plasmid-borne housekeeping promoters have at least a limited potential for bidirectional activity, but the functional significance of this is unclear if the corresponding endogenous housekeeping promoters express divergent transcripts at similarly low levels. The poor activity of the HPRT and PGK promoters in the inverted orientation suggests that there is a mechanism which influences the direction of transcription from these promoters.
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PMID:Limited bidirectional activity of two housekeeping gene promoters: human HPRT and PGK. 234 94

The Cre-loxP recombination system of bacteriophage P1 is frequently utilized in genetic manipulation in embryonic stem (ES) cells. The level of Cre expression is critical to induce loxP site-specific recombination in ES cells. To compare the efficiency of recombination, we constructed four Cre expression vectors driven by different promoters: cytomegarovirus/chicken beta-actin (CAG) promoter, human polypeptide chain elongation factor 1alpha (hEF-1alpha) promoter, mouse phosphoglycerate kinase-1 (mPGK) promoter, and polyoma enhancer/herpes simplex virus thymidine kinase (MC1) promoter. We introduced these Cre expression vectors by electroporation into three ES cell lines carrying a single copy of CAG-loxP-chloramphenicol acetyltransferase (CAT) gene-loxP-beta-galactosidase (beta-gal) gene construct. Since the Cre-mediated recombination leads to excision of the CAT gene, the efficiency of recombination can be monitored as beta-gal expression. No selection system was used in the experiments. The maximum recombination frequency was obtained when the CAG promoter was used, followed by the hEF-1alpha promoter, the mPGK promoter and the MC1 promoter in order. These results indicate that the efficiency of recombination in transient expression system correlates with the promoter activity of Cre expression vector. Thus, it is important to choose the promoter for effective recombination by Cre.
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PMID:Efficiency of recombination by Cre transient expression in embryonic stem cells: comparison of various promoters. 944 13

A 40-bp DNA, consisting of seven tandem GATA repeats, is located near the HS5 site in the 5' boundary area of the locus control region (LCR) of human beta-globin gene. This (GATA)(7) motif, named 5a, exhibits silencer activity in erythroid cells. In transfected, recombinant plasmids containing the chloramphenicol acetyltransferase (CAT) reporter gene, 5a repressed the activity of the cis-linked housekeeping phosphoglycerate kinase (pgk) promoter; 5a also repressed the activity of the cis-linked HS2 enhancer regardless of whether the CAT gene was driven by the pgk or the epsilon-globin promoter. Repression by 5a was most severe when 5a was spliced upstream of HS2 at a distance of less than 200 bases from the HS2 enhancer core. The silencer activity of 5a was independent of whether the component GATA motifs were in head to tail orientation as in the wild type 5a or in head to head or tail to tail orientation as in a mutant 5a. Band shift experiments show that the GATA-1 protein binds to both 5a and the mutant 5a and forms a large protein complex. Together, the results suggest that GATA-1 bound at 5a is a strong, proximal repressor of HS2 enhancer activity.
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PMID:A (GATA)(7) motif located in the 5' boundary area of the human beta-globin locus control region exhibits silencer activity in erythroid cells. 1093 58

In Trypanosoma brucei, the PGKB and PGKC genes-encoding phosphoglycerate kinase are co-transcribed as part of a polycistronic RNA. PGKB mRNA and the cytosolic PGKB protein are much more abundant in the procyclic life-cycle stage than in bloodstream forms, whereas PGKC mRNA and glycosomal PGKC protein are specific to bloodstream forms. We here show that a sequence between nucleotides 558 and 779 in the 3'-untranslated region of the PGKC mRNA causes low expression of the chloramphenicol acetyltransferase (CAT) reporter gene in procyclic trypanosomes. In procyclics, depletion of the RRP45 component of the exosome (3'-->5' exonuclease complex) or the 5'-->3' exonuclease XRNA increased the abundance of CAT-PGKC mRNA as a consequence of effects on the degradation of precursor and/or mature mRNAs. In bloodstream forms, inhibition of both trans splicing and transcription resulted in immediate exponential decay of PGKC mRNA with a half-life of 46 min. Inhibition of transcription alone gave non-exponential kinetics and inhibition of splicing alone resulted in a longer apparent half-life. We also found that production of mRNAs using T7 polymerase can affect the apparent half-life, and that large amounts of CAT enzyme may be toxic in trypanosomes.
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PMID:Regulated expression of glycosomal phosphoglycerate kinase in Trypanosoma brucei. 1718 72