Synthesis of chloramphenicol acetyltransferase coded by bacterial gene carried on P1CM bacteriophage in extracts of human blood platelets

Abstract

Extracts of human blood platelets, which do not contain endogenous DNA, were found to take part in the de novo synthesis of the bacterial enzyme chloramphenicol acetyltransferase (CAT) (EC 2.3.1.28) in a cell-free system. The synthesis was demonstrated in the presence of DNA from bacteriophage P1CM, a membrane fraction from Escherichia colt K175 endowed with transcription activity (P1(S)), or purified RNA polymerase (EC 2.7.7.6), amino acids, nucleotides, phosphoenolpyruvate, and K+ and Mg2+ ions. Addition of external cyclic AMP (0.1 mM) was found to augment the synthesis of CAT, whereas addition of either actinomycin D (10 μg/ml) or rifampicin (8 μg/ml) completely inhibited it, indicating its dependence on DNA-linked RNA synthesis.The formation of CAT in the above cell-free system was not sensitive to dihydrostreptomycin (100 μg/ml) and chloramphenicol (10 μg/ml) which inhibited the formation of the enzyme in the cell-free system of E. coli K175. On the other hand, it was inhibited by cycloheximide (28 μg/ml) which was not inhibitory for the bacterial cell-free system.The activity of CAT formed in the presence of either the platelet or the bacterial cell-free extracts was inhibited by an antiserum against the CAT of E. coli K12 D1R1, produced in rabbits, indicating the antigenic identity of the CAT proteins produced in these extracts. By means of this rabbit antiserum, precipitation of [3H]leucine-labeled protein was obtained from the platelet-extract system. No such 3H-labeled protein precipitation was obtained if either the platelet extract or the bacterial membranous fraction (or RNA polymerase) was excluded.