Mutational analysis of N-ethyl-N-nitrosourea (ENU) in the fission yeast Schizosaccharomyces pombe.

Abstract

Forward genetics has boosted our knowledge on genic function in a multitude of biological models and it has significantly contributed to the understanding of genetic bases of development, ageing and human diseases. With the advent of the next generation sequencing and use of powerful bioinformatic tools, this traditional genetic strategy has acquired a new impulse. At present, whole genome sequencing assisted by in silico analysis allows the rapid and efficient identification of gene variants that are responsible for a particular phenotype. In this experimental pipeline, it is crucial to start by provoking a large number of random changes in the genome of the model organisms to be screened. A range of chemical mutagens are used to this end because most of them display particular reactivity properties and act differently over DNA. Here we use N-ethyl-N-nitrosourea (ENU) as a mutagen for the first time to our knowledge in the fission yeast Schizosaccharomyces pombe. By comparison to the extensively used Ethyl methanesulfonate (EMS) in a phenotype-based study, we conclude that ENU is a very potent and easy-to-use mutagen. Judging from DNA sequence analysis of the identified mutants, ENU induces base changes rather than indels and the mutational spectrum in the fission yeast seems similar to that found in mice but different to that described in other single-celled organisms such as budding yeast and E. coli. Using ENU in S. pombe, we have gathered a collection of 49 auxotrophic mutants including two deleterious alleles of ATIC human ortholog. Defective alleles of this gene are causative of AICA-Ribosiduria, a severe genetic disease. We have also identified 5 aminoglycoside-resistance inactivating mutations in APH genes. All these mutations reported here may be of interest in the metabolism and antibiotic resistance research fields.