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Abstract
BackgroundWith the advent of sequence-based approaches in the mutagenesis studies, it is now possible to directly evaluate the genome-wide pattern of experimentally induced DNA sequence changes for a diverse array of organisms. To gain a more comprehensive understanding of the mutational bias inherent in mouse ENU mutagenesis, this study describes a detailed evaluation of the induced mutational pattern obtained from a sequence-based screen of ENU-mutagenized mice.ResultsBased on a large-scale screening data, we derive the sequence-based estimates of the nucleotide-specific pattern and frequency of ENU-induced base replacement mutation in the mouse germline, which are then combined with the pattern of codon usage in the mouse coding sequences to infer the spectrum of amino acid changes obtained by ENU mutagenesis. We detect a statistically significant difference between the mutational patterns in phenotype- versus sequence-based screens, which presumably reflects differential phenotypic effects caused by different amino acid replacements. We also demonstrate that the mutations exhibit strong strand asymmetry, and that this imbalance is generated by transcription, most likely as a by-product of transcription-coupled DNA repair in the germline.ConclusionThe results clearly illustrate the biased nature of ENU-induced mutations. We expect that a precise understanding of the mutational pattern and frequency of induced nucleotide changes would be of practical importance when designing sequence-based screening strategies to generate mutant mouse strains harboring amino acid variants at specific loci. More generally, by enhancing the collection of experimentally induced mutations in unambiguously defined genomic regions, sequence-based mutagenesis studies will further illuminate the molecular basis of mutagenic and repair mechanisms that preferentially produce a certain class of mutational changes over others.
Highlights
With the advent of sequence-based approaches in the mutagenesis studies, it is possible to directly evaluate the genome-wide pattern of experimentally induced DNA sequence changes for a diverse array of organisms
Site-specific pattern and frequency of induced mutations Whereas our mutagenesis screen includes nongenic portion of the mouse genome [13], the present analysis focuses on the sequence changes identified within the protein-coding genes so that the detected mutations are classified as base changes on the nontranscribed strand
The overall frequency of base replacement mutation is obtained as 7.18 × 10-7 per nucleotide site per generation. (Inclusion of mutations detected in intergenic sequences yields a slightly higher rate of 7.49 × 10-7 [13].) Based on a preliminary experiment utilizing Taq polymerase-induced errors as positive controls, we determined that under the experimental conditions used in our mutation screening [13], the rate of mutation discovery by our temperature gradient capillary electrophoresis (TGCE) system was ~50%; the absolute rate of ENUinduced heritable mutation is estimated roughly as 1.4 × 10-6 per nucleotide site, which is approximately two orders of magnitude higher than the spontaneous mutation rate in humans (~2 × 10-8 per site per generation [15,16,17])
Summary
With the advent of sequence-based approaches in the mutagenesis studies, it is possible to directly evaluate the genome-wide pattern of experimentally induced DNA sequence changes for a diverse array of organisms. To gain a more comprehensive understanding of the mutational bias inherent in mouse ENU mutagenesis, this study describes a detailed evaluation of the induced mutational pattern obtained from a sequence-based screen of ENU-mutagenized mice. N-ethyl-N-nitrosourea (ENU) is known to generate a spectrum of alleles, mainly by introducing single base replacement changes (i.e. transitions and transversions), and has been the mutagen of choice for enhancing the genetic resource for biomedical applications [1,2]. ENU-induced nucleotide changes are known to exhibit a strong bias towards particular lesions [4,5], and possibilities remain that this property should restrict the general applicability of ENU mutagenesis
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