PUROMYCIN-RESISTANT MUTANTS OF THE HYPERTHERMOPHILIC ARCHAEON PYROCOCCUS-ABYSSI

Mutant strains of Pyrococcus abyssi strain GE5 were selected by platin g cells on solid media with progressively increasing levels of puromyc in. Spontaneous and UV-treated resistant mutants were isolated on soli d medium at 90 mu g of puromycin per milliliter and with low reversion frequency (about 10(-8)). Acetyl-puromycin resulting from acetylation of puromycin in the resistance mechanism was shown to be inhibitory a t higher concentrations compared with puromycin, both in vivo and in v itro for P. abyssi mutants. The permeability of the mutant membrane to puromycin and acetyl-puromycin was also proved by checking the uptake of both compounds. Puromycin was shown to inhibit poly(U)-dependent p olyphenylalanine synthesis in P. abyssi mutants extracts, much more th at acetylpuromycin. Finally, the higher puromycin acetyltransferase ac tivity was observed with P. abyssi mutant (400 mU/mg of protein). Thus , the pac-type gene of these mutants, which is responsible for acetyla tion of puromycin and which conferred resistance to puromycin, seems t o be a potential selective marker in gene transfer experiments with P. abyssi.