N-Acetoxy-N-acetyl-2-aminofluorene-induced mutatuion spectrum in a human hprt cDNA shuttle vector integrated into mammalian cells

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Abstract
The spectrum of mutations induced by N-acetoxy-N-acetyl-2-aminofluorene (N-AcO-AAF) was examined by the pZipHprtNeo shuttle vector in mammalian cells. The vector carries a cDNA of the human hypoxanthine phosphoribosyl transferase (hprt) gene, which is stably integrated into chromosomal DNA of a mouse cell line, VH12. After treatment of the cell with N-AcO-AAF, 48 independent 6-thioguanine-resistant clones were obtained and altered sequences of the mutated cDNA hprt genes were determined. Frameshifts and deletions were the predominant mutational events (68%) induced by N-AcO-AAF and the remainder were base substitutions (32%) of various types. Analysis of sequence alterations at all the sites of mutation revealed that: (i) > 65% of mutations occurred at G:C sites, suggesting C8G adducts are responsible premutagenk lesions for these mutations; and (ii) short sequence repeats were frequently found at the sites of frameshift and deletion, and slippage-misalignment is the suggested mechanism for the induction of mutations at these sites. Implied significance of slippage - misalignment as a fundamental mechanism for mutagenesis is discussed.

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