L. Watrin et D. Prieur, PUROMYCIN-RESISTANT MUTANTS OF THE HYPERTHERMOPHILIC ARCHAEON PYROCOCCUS-ABYSSI, Journal of marine biotechnology, 6(2), 1998, pp. 124-128
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.