Transfection of the Escherichia coli ada gene coding for O6-alkylguanine-DNA
alkyltransferase results in expression of ada in mammalian cells and transmission of nitrosourea resistance to cells lacking
alkyltransferase activity. We have used a replication-incompetent retrovirus to transfer into mammalian cells a chimeric gene consisting of 548 bp of the promoter-regulatory region of the gene for P-enolpyruvate carboxykinase (
GTP) (EC 4.1.1.32) (PEPCK) linked to ada. The PEPCK promoter was used because it is inducible and highly expressed in liver and kidney cells both in vitro and in vivo. After
retrovirus infection of the rat kidney cell line, NRK, intact proviral
DNA was integrated into the genome of cloned cells. Individual NRK clones produced up to 200 units/mg
protein of bacterial
alkyltransferase activity compared to 65 units/mg
protein of mammalian
alkyltransferase in the parent cell line. Transcription of ada
mRNA originating from the PEPCK promoter was induced with Bt2cAMP or
dexamethasone and the combination caused a 4-fold increase in ada
mRNA while total
alkyltransferase activity was induced up to 2-fold. NRK clones expressing ada had up to 2.0-fold increased resistance to 1,3-bis(2- chloroethyl)-1- nitrosourea. Thus, retroviral gene transfer of the PEPCKada chimeric gene allows efficient and inducible expression of ada with a resulting increase in
alkyltransferase activity and nitrosourea drug resistance. This retrovirus may be used to study the role of
alkyltransferase in repair of mutagenic DNA lesions in mammalian cells in vivo.