Evaluation of chromosome painting to assess the induction and persistence of chromosome aberrations in bone marrow cells of mice treated with benzene |
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Authors: | Stronati Laura Farris Alessandra Pacchierotti Francesca |
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Institution: | Department of Pathophysiology, Center of Environmental Genomics, Zhejiang University School of Medicine, 353 Yan’an Road, Hangzhou 310031, China |
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Abstract: | Intact pZ189 DNA was allowed to replicate in FL-FEN-1− cell line that was established in this laboratory in which the expression of FEN-1 gene was blocked by dexamethasone-inducible expression of antisense RNA to FEN-1. E. coli MBM7070 was transfected with the replicated plasmid, and those with mutations in the supF gene were identified. The frequency of mutants that did not contain recognizable changes in the electrophoretic mobility of the plasmid DNA was scored. The frequency of such mutants was 19.1 × 10−4 (34/17781), significantly higher than those of 2.9 × 10−4 (4/13668) and 3.0 × 10−4 (3/9857) in the corresponding controls, respectively. Sequence analysis of the supF genes of these mutants showed that all (37/37) the base substitutions occurred at C:G base pairs; 68% (23/37) of the base substitutions were base transversions, while 32% (12/37) were transitions. Approximately 76% (23/37) of these base substitutions occurred frequently at nine positions; two of these sites contain triple pyrimidine (T or C) repeat upstream to the mutated base; four of these sites consist of 5′-TTN1N2 and mutations occurred at N1 site sequence; another two sites have the characteristics of triple A flanked at both 5′ and 3′ side by TCT, with the base substitution occurring at C in the context sequence. These data suggested that these sites are the hot spot of mutagenesis in plasmid replicated in FEN-1-deficient cells. Besides the mutator phenotype of the FEN-1-deficient cell, it was also demonstrated that FEN-1-deficient cell exhibited an increased N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) sensitive phenotype. |
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Keywords: | FEN-1 MNNG Mutator phenotype Sequence specificity Spontaneous mutagenesis |
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