Spontaneous and mutagen-mediated amplification of a neo gene integrated at different genomic sites in rat 2 fibroblasts.
A promoterless neo gene was stably transfected in rodent fibroblasts to act as a reporter gene for rearrangements resulting in its expression at the different genomic integration sites. Nine clones were isolated which had integrated a varying copy number of neo at one or more genomic sites but still displayed a Neo- phenotype (G418-sensitive). These clones were analyzed for their ability to become Neo+ (G418-resistant) either spontaneously or after mutagen treatment. They were all able to generate G418-resistant subclones spontaneously at frequencies ranging from 2 x 10(-8) to 6 x 10(-5). The acquired G418-resistance was always associated with amplification and enhanced transcription of neo. No correlation was observed between the frequency of occurrence of G418-resistance and the number of copies or integration sites of neo. When treated with the mutagens mitomycin C or methylmethane sulfonate, only one clone, RH15, produced G418-resistant subclones in a dose-related fashion. In this mutagen-inducible clone, DNA lesions of a different nature (monoadducts or cross-links) were equally efficient in the induction of G418-resistance. Amplification and enhanced transcription of the neo gene were observed in both the spontaneous and mutagen-induced G418-resistant subclones of RH15 cell line. These findings indicate that the exogenous neo gene integrated at different genomic sites was acting as a reporter gene for amplification. Interestingly, while all nine integration sites were observed to amplify spontaneously, only one could be induced to amplify by mutagens. This suggests that different genomic regions display differing susceptibilities to mutagen-mediated amplification. This may be important in view of the major role played by mutagen-mediated gene amplification in carcinogenesis.
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Dipartimento di Biochimica e Biotecnologie Mediche, II Facoltà di Medicina e Chirurgia, Università degli Studi di Napoli, Italy.
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Mallardo
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De Lorenzo
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D000818:Animals; D015139:Blotting, Southern; D004351:Drug Resistance / Q000235:genetics; D005347:Fibroblasts; D005784:Gene Amplification / Q000187:drug effects / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D005839:Gentamicins / Q000494:pharmacology; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D016685:Mitomycin / Q000633:toxicity; D051381:Rats; D014162:Transfection
D005839:Gentamicins; D016685:Mitomycin; C010680:antibiotic G 418; D008741:Methyl Methanesulfonate