Chloramphenicol acetyltransferases

The major mechanism of resistance to chloramphenicol is mediated by the chloramphenicol acetyltransferases (CAT enzymes) which transfer one or two acetyl groups to one molecule of chloramphenicol. While the CAT enzymes share a common mechanism, different molecular classes can be discriminated. The corresponding genes are frequently located on integron-like structures and are widely distributed among Gramnegative and - positive bacteria. 

Plasmid- ortransposon-encoded chloramphenicol acetyltransferases (CATs) are responsible for resistance by inactivating the antibiotic. CATs convert chloramphenicol to an acetoxy derivative which fails to bind to the ribosomal target. Several CATs have been characterized and found to differ in properties such as elecfrophorefic mobilify and cafalyfic acfivify. 

Shaw WV, AGW Leslie (1991) Chloramphenicol acetyltransferase. Annu Rev Biophys Chem 20 363-386. 

They used different generations of intact dendrimers to transfect plasmid DNA in a variety of cells (Table 18.2) using luciferase, CAT (chloramphenicol acetyltransferase) and /i-galactosidasc reporter genes to quantify transfection efficiency. 

There are other reporter gene systems, such as j8-galactosidase (a bacterial enzyme), chloramphenicol acetyltransferase (a bacterial enzyme), and aequorin (a jellyfish protein). 

Chloramphenicol is able to inhibit the peptidyl transferase reaction and so bacterial protein synthesis by binding reversibly to the 50s ribosomal subunit. Resistance can occur due to the plasmid-mediated enzyme chloramphenicol acetyltransferase which inactivates the drug by acetylation. Such resistance is often a part of plasmid-mediated multidrug resistance. Resistance can also occur by an altered bacterial permeability. However in most instances resistance to chloramphenicol only develops slowly and remains partial. 

Low-level resistance to chloramphenicol may emerge from large populations of chloramphenicol-susceptible cells by selection of mutants that are less permeable to the drug. Clinically significant resistance is due to production of chloramphenicol acetyltransferase, a plasmid-encoded enzyme that inactivates the drug. 

Of the many mechanisms of bacterial resistance to chloramphenicol and thiamphenicol, the plasmid-mediated transmissible resistance conferred by the presence in resistant bacteria of chloramphenicol-acetyltransferases (CAT) is the most important. 

DNA injection directly into mouse diaphragm has also resulted in luciferase expression and there appeared to be no damage to the diaphragm due to the DNA injections (Davis and Jasmin, 1993). In a related study, /3-galactosidasc ( /3-gal)-encoding pDNA injected into the articular space of rabbit knee joints resulted in /3-gal expression in the joints (Yovandich etal., 1995). In the same study, chloramphenicol acetyltransferase (CAT) encoding pDNA injected into rat knee joints also led to reporter gene expression, with peak expression 48 hours after injection and with no detectable activity 15 days later. 

The targeting of antibodies to the periplasm requires the use of signal peptides. The pelB leader of the pectate lyase gene of Erwinia carotovora (56) is commonly used. The gill leader (9), the phoA leader of the E. coli alkaline phosphatase, and the ompA leader of E. coli outer membrane protein OmpA have also been used, being common to many protein expression vectors (57,58). Further examples are the heat-stable enterotoxin II (stll) signal sequence (47) and the bacterial chloramphenicol acetyltransferase (cat) leader (59). 

IA Murray, JA Gil, DA Hopwood, WV Shaw. Nucleotide sequence of the chloramphenicol acetyltransferase gene of Streptomyces acrimycini. Gene 85 283-291, 1989. 

Tratschin, J. D., West, M. H., Sandbank, T. and Carter, B. J. (1984). A human parvovirus, adeno-associated virus, as a eucaryotic vector Transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase. Mol. Cell. Biol. 4, 2072-2081. 

Bullock C, Gorman C (2000), Fusions to chloramphenicol acetyltransferase as a reporter, Methods Enzymol. 326 202-221. 

Gorman CM, Moffat LF, Howard BFI (1982), Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells, Mol. Cell. Biol. 2 1044-1051. 

As pointed out above, nt1 receptors have been discovered in lymphoma cells selected for resistance to the cytolytic glucocorticoid effect. Since receptors from which the M domain had been eliminated by cDNA manipulation still function to some extent in transfection studies it was important to find out whether nt1 receptors would also be able to mediate some hormonal response. This was in fact observed when nt lymphoma variants were transfected with a DNA construct consisting of the LTR region of the mouse mammary tumour virus coupled to the gene for chloramphenicol acetyltransferase (U. Gehring and H. Losert, unpublished experiments). Hormonal induction of enzyme activity was consistently observed but was low, as one might expect. 

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