Induction of human high K-m 5 '-nucleotidase in cultured 293 cells

Human 293 cells were stably transfected with a plasmid introducing a recept or for the ecdysone analog muristerone. The cells were further stably trans fected with muristerone-inducible expression vectors carrying either the cD NA far the human high K-M 5'-nucleotidase or the coding sequence of the nuc leotidase linked to the 5'-end of the sequence for the green fluorescent pr otein. Upon induction, both types of transfectants overproduced nucleotidas e activity in a time- and dose-dependent manner. Western blots gave values close to the expected subunit molecular masses of 65 and 92 kDa, respective ly, excluding processing of the induced proteins, Cells induced to overexpr ess the nucleotidase showed a decreased growth rate and contained smaller p ools of each of the four common ribonucleoside triphosphates. They showed n o increased resistance to the toxicity of 2-chlorodeoxyadenosine. (C) 1999 Academic Press.