Chloramphenicol acetyltransferase CAT

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. 

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). 

Strategically similar, but structurally distinct, are the chloramphenicol acetyltransferases (CATs). Chloramphenicol binds to the large ribosomal subunit at the nexus of the peptidyltraus-fer ceuter aud the peptide exit muuel (28, 29). CATs are trimeric euzymes with active sites at the iuterface of the monomers... 

APH). Chloramphenicol is attacked by chloramphenicol acetyltransferases (CAT). Acetyltransferases attack susceptible amino groups and require acetyl coenzyme A, while AAD or APH enzymes attack susceptible hydroxyl groups and require ATP (or another nucleotide triphosphate). 

Figure 20.5 Transfection efficiency of pc DNA3.1 plasmid encoding chloramphenicol acetyltransferase (CAT) mediated by chitosan (CS) and alkylated chitosan (ACS). Source From Ref. [68].

This chapter covers j8-galactosidase (jSGal or LacZ) and luciferase (Lux/Luc) gene systems as typical reporter systems. Note that alkaline phosphatase (PhoA) can also be used as an excellent reporter system and has been described in Chapter 5. As selection markers, the ampicillin resistance system (Bla or ApO will be described. The chloramphenicol acetyltransferase (CAT) gene system will also be discussed as a typical marker/reporter with dual functions. 

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