Expression of E. coli tag gene encoding 3-methyladenine glycosylase I in NIH-3T3 murine fibroblasts.
NIH-3T3 fibroblasts were co-transfected with pSV2neo and pSG5tag by the calcium-phosphate precipitation method. The stable integration of the tag gene sequence and its transcription was verified by Southern and Northern blot analysis. 3-Methyladenine glycosylase activity in pSG5tag transfected 3T3 cells was approximately 400 times higher than in cells transfected with the control plasmid pSG5 or in the parental cells and was inhibited by 3-methyladenine. Bacterial tag gene can thus be expressed in mammalian cells and the encoded enzyme is functionally active. These transfected cells could serve as an important tool to investigate the importance of the repair of N3-adenine as a mechanism of protection against the mutagenicity and cytotoxicity of alkylating agents.
Taverna
P
P
Cancer Chemotherapy Laboratory, Centro Daniela e Catullo Borgomainerio, Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy.
Garattini
E
E
Citti
L
L
Damia
G
G
D'Incalci
M
M
D016475:3T3 Cells / Q000378:metabolism; D000818:Animals; D002455:Cell Division; D045647:DNA Glycosylases; D004260:DNA Repair; D004926:Escherichia coli / Q000235:genetics*; D051379:Mice; D009699:N-Glycosyl Hydrolases / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000096:biosynthesis; D014162:Transfection
D012333:RNA, Messenger; D011994:Recombinant Proteins; C021565:3-methyladenine-DNA glycosylase; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases