Chloramphenicol acetyltransferase gene

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

Sleigh, M. J. (1986) A nonchromatographic assay for expression of the chloramphenicol acetyltransferase gene in eukaryotic cells. Anal. Biochem. 156(1), 251-256. 

Different tissues have been shown to display different DNA pharmacokinetics. Genes and gene products have been observed as long as 19 months after direct injection without indication of plasmid integration or replication (175). Direct injection of the chloramphenicol acetyltransferase gene into the thyroid resulted in elimination of DNA from the gland with a half-life of 10 hours. The enzyme activity was maximal for 24 hours and eliminated through first-order kinetics with an apparent half-life of 40 hours (176). Similar results were recorded for synovial fluid intra-articular administration of plasmid DNA (177). 

Marcus-Sekura, C J., Woerner, A. M., Shinozuka, K, Zon, G, and Quinnan, G V (1987) Comparative inhibition of chloramphenicol acetyltransferase gene expression by antisense oligonucleotide analogues having alkyl phospho-triester, methylphosphonate and phosphorothioate linkage Nucl Acids Res 15,5749-5752. 

CAT, chloramphenicol acetyltransferase gene LUC, luciferase gene SeAP, secreted alkaline phosphatase gene. 

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. 

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). 

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). 

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. 

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. 

Chloramphenicol acetyltransferase is used as a genetic marker system to study transformation and gene expression in plant protoplasts, cells, and tissues. 

Fig. 16. Map of the plasmid used for expression of flavocytochrome 62 in Escherichia coli. The flavocytochrome 62 coding region (CYB2) is located between a strong E. coli promoter (p) and terminator (t). Selection is based on ampicillin resistance conferred by the /3-lactamase gene (bla). coRI, BglU, and HindW. cleavage sites are indicated ori, origin of replication cat, chloramphenicol acetyltransferase coding sequence rbs, ribosome-binding site.

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