Chloramphenicol acetyl

Azuma, T., Motoyama, N., Fields, L.E., Loh, D.Y. (1993). Mutations of the chloramphenicol acetyl transferase transgene driven by the immunoglobulin promoter and intron enhancer. Int. Immunol. 5, 121-130. 

The nuclear envelope acts as a permeability barrier for a variety of macromolecules, and DNA is no exception. In 1980, Mario Capecchi demonstrated that when a pBR322-derived plasmid expressing thymidine kinase (TK) was microinjected into the nuclei of TK-deficient mouse fibroblasts, between 50% and 100% of the injected cells showed TK activity at 24 hours post-injection (Capecchi, 1980). By contrast, in over 1000 cytoplasmically injected cells, no gene expression was detected in any cell during the same time frame. Similar results were obtained in another microinjection study using rat TK-deficient cells and a plasmid expressing chloramphenicol acetyl transferase (CAT) driven by a herpes TK promoter. When 1000-2000 copies of the plasmid were injected into the cytoplasm, less than 3% of the activity was seen as compared to cells injected in the nucleus with the same number of plasmids (Graessman et al., 1989). Zabner et al. 

CAT folding reporter antibiotic resistance correctly folded proteins impart antibiotic resistance to organism via a chloramphenicol acetyl-transferease fusion tag Maxwell, 1999... 

Fig. 6 Transfection activity of polyamine cholesterol cationic derivatives with linear (a-c) and T-shaped (d—f) structures. The activity of their mixtures with the helper lipid DOPE was measured in vivo as chloramphenicol acetyl transferase (CAT) expression in mouse lung [36]...

Chloramphenicol acetyl transferase (CAT). This bacterial enzyme was the first reporter protein used for studying the transcriptional activity of eukaryotic regulatory sequences (Gorman et al., 1982). CAT inactivates chloramphenicol, an inhibitor of prokaryotic protein synthesis, by converting it to the mono- or di-acetylated species. Measurement of CAT activity requires a 14C-radiolabeled chloramphenicol or acetyl-CoA and, therefore, an additional step is neccessary to separate the radio-labeled reactant from the product. Novel detection methods based on fluorescent substrates or ELISA assays, which do not use radiolabeled reagents, have been described more recently (Bullock and Gorman, 2000). 

Chloramphenicol Binds to 23S rRNA in the 50S ribosomal subunit and inhibits translation Chloramphenicol acetyl transferase acetylates the antibiotic... 

Chloramphenicol (9) is liable to breakdown by chloramphenicol acetyl-transferases [185]. Fluoro derivatives (57, 58) resist enzymatic attack but little has been heard of these, apparently because of their toxicity [319], Aminoglycoside antibiotics (AGACs) may be chemically modified by AMEs. Some derivatives (e.g. amikacin, 43) are more recalcitrant than others, e.g. kanamycin (42) (see Figure 4.2). Other enzyme-resistant AGACs of low toxicity are needed. 

Chloramphenicol Acetyl Transferase, Secreted Placental Alkaline Phosphatase, P-Galactosidase... 

Nakaya [224] reported the presence of chloramphenicol acetyl transferase in a strain of Ps. aeruginosa. The enzyme activity was reported to be low and no chloramphenicol esters were detected. R factor mediated resistance to chloramphenicol in pseudomonas strains has been reported by Witchitz and Chabbert [225], However, Ingram, Richmond and Sykes [205] failed to transfer the R factor mediated chloramphenicol resistance gene to Ps. aeruginosa from a strain of Klebsiella aerogenes. 

Mechanisms of resistance to fusidic acid include alterations in elongation factor G (whose inhibition at the ribosome level mediates the effect of fusidic acid), altered drug permeability, binding by chloramphenicol acetyl-transferase type 1, and enhanced efflux (12). 

A second domain is necessary for binding to subunits Ej and Eg, while the third major 250-residue domain contains the catalytic acyltransferase center.3 3 309 center closely resembles that of chloramphenicol acetyl-transferase (Qiapter I2).3i0/3ii jj g jjpgyj30i.309.3i2... 

CAT assay. An enzyme assay. CAT stands for chloramphenicol acetyl transferase, a bacterial enzyme which inactivates chloramphenicol by acety-lating it. CAT assays are often performed to test the function of a promoter. The gene coding for CAT is linked onto a promoter (transcription control region) from another gene, and the construct is transfected into cultured cells. Largely supplanted by the reporter gene luciferase. 

However, by far the most important strategy involves the production of enzymes that destroy or deactivate the antibiotics. Bacterial enzymes that inactivate chloramphenicol (e.g., chloramphenicol acetyl transferase) and the aminoglycosides (usually enzymes that acetylate or phosphorylate the aminoglycosides) are quite common, but it is the penicillinases or, as they are now called, the beta-lactamases, that have had the most impact on antibacterial chemotherapy. The beta-lactam antibiotics, including the penicillins, cephalosporins and the newer variants called penems, carbapen-ems, cephamycins and monobactams, all possess the four-membered ring... 

The improved PEP/pyruvate system developed by Kim and Swartz [7] yielded 350 pg mL of chloramphenicol acetyl-transferase (CAT) in the first hour of an E. coli batch reaction. The same reaction system yielded 750 pg mL CAT in 3 hours when periodically fed with amino acids, PEP, and magnesium acetate [6]. This is remarkable efficiency for a system lacking a dialysis membrane, and is a feature that weU suits robotic handling and high-throughput screening (HTS) strategies. 

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