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)

Fifty clinical isolates of chloramphenicol-resistant staphylococci from diverse sources were screened for the presence of chloramphenicol acetyltransferase (CAT) and were found to contain the inducible chloramphenicol-inactivating enzyme in each case. Polyacrylamide disc gel electrophoresis revealed four distinct types of CAT of which three were purified to a state of homogeneity. Each purified CAT preparation was shown to exist as a tetrameric protein with a native molecular weight of 80,000 and an identical subunit size of 20,000. All four staphylococcal types of CAT exhibited identical catalytic and immunological properties, but they possessed variable sensitivity to heat denaturation and to inhibition by mercuric ion. Each of the three purified variants of staphylococcal CAT was capable of undergoing reversible denaturation in 6 m guanidine hydrochloride. In vitro hybridization was successful between pairs of each of the three purified staphylococcal enzymes. Only one heteromeric (hybrid) species was observed in addition to the parental types rather than the three predicted from the known quaternary structure of staphylococcal CAT.
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PMID:Mechanism of chloramphenicol resistance in staphylococci: characterization and hybridization of variants of chloramphenicol acetyltransferase. 479 May 93

DNA shuffling is widely used for optimizing complex properties contained within DNA and proteins. Demonstrated here is the amplification of a gene library by PCR using uridine triphosphate (dUTP) as a fragmentation defining exchange nucleotide with thymidine, together with the three other nucleotides. The incorporated uracil bases were excised using uracil-DNA-glycosylase and the DNA backbone subsequently cleaved with piperidine. These end-point reactions required no adjustments. Polyacrylamide urea gels demonstrated adjustable fragmentation size over a wide range. The oligonucleotide pool was reassembled by internal primer extension to full length with a proofreading polymerase to improve yield over Taq. We present a computer program that accurately predicts the fragmentation pattern and yields all possible fragment sequences with their respective likelihood of occurrence, taking the guesswork out of the fragmentation. The technique has been demonstrated by shuffling chloramphenicol acetyltransferase gene libraries. A 33% dUTP PCR resulted in shuffled clones with an average parental fragment size of 86 bases even without employment of a fragment size separation, and revealed a low mutation rate (0.1%). NExT DNA fragmentation is rational, easily executed and reproducible, making it superior to other techniques. Additionally, NExT could feasibly be applied to several other nucleotide analogs.
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PMID:Nucleotide exchange and excision technology (NExT) DNA shuffling: a robust method for DNA fragmentation and directed evolution. 1606 32