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

Abstract

19 Forward genetics has boosted our knowledge on genic function in a multitude of 20 biological models and it has significantly contributed to the understanding of genetic 21 bases of development, ageing and human diseases. With the advent of the next 22 generation sequencing and use of powerful bioinformatic tools, this traditional genetic 23 strategy has acquired a new impulse. At present, whole genome sequencing assisted by 24 in silico analysis allows the rapid and efficient identification of gene variants that are 25 responsible for a particular phenotype. In this experimental pipeline, it is crucial to start 26 by provoking a large number of random changes in the genome of the model organisms 27 to be screened. A range of chemical mutagens are used to this end because most of them 28 display particular reactivity properties and act differently over DNA. Here we use N-29 ethyl-N-nitrosourea (ENU) as a mutagen for the first time to our knowledge in the 30 fission yeast Schizosaccharomyces pombe. By comparison to the extensively used Ethyl 31 methanesulfonate (EMS) in a phenotype-based study, we conclude that ENU is a very 32 potent and easy-to-use mutagen. Judging from DNA sequence analysis of the identified 33 mutants, ENU induces base changes rather than indels and the mutational spectrum in 34 the fission yeast seems similar to that found in mice but different to that described in 35 other single-celled organisms such as budding yeast and E. coli. Using ENU in S.36 pombe, we have gathered a collection of 49 auxotrophic mutants including two 37 deleterious alleles of ATIC human ortholog. Defective alleles of this gene are causative 38 of AICA-Ribosiduria, a severe genetic disease. We have also identified 5 39 aminoglycoside-resistance inactivating mutations in APH genes. All these mutations 40 reported here may be of interest in the metabolism and antibiotic resistance research 41 fields.3 42 Introduction 43 The search of a particular phenotype of interest after natural or induced random 44 mutation in biological models, and the consequent identification of the underlying 45 genetic variant, is known as forward genetics. This strategy provides an extremely 46 powerful tool to characterise individual gene functions as well as complex genetic 47 pathways interplay, when two or more mutations coexist into the same cell. Human 48 genetic diseases are usually provoked by hypomorphic mutations (partial loss of 49 function) that allow embryo development and birth but become deleterious at later 50 stages. Therefore, by inducing DNA changes into model organisms, genetic bases of 51 human diseases can be very closely reproduced to be studied; even in single-celled 52 organisms such as yeast [1]. 53A number of physical and chemical mutagens have been used to boost the 54 number of random changes over the natural spontaneous mutation rate. In addition to 55 single base substitutions, most of these agents also provoke small insertions and 56 deletions (generically known as indels) [2]. Indels usually lead to frame shif...