Nonsense-mediated mRNA decay in barley mutants allows the cloning of mutated genes by a microarray approach

We have previously described a microarray approach to identify and clone genes from mutants of higher organisms. In the method cDNA of two mutants with similar phenotype are competitively hybridized to DNA clones arrayed on a glass slide. Clones corresponding to an mRNA that is not expressed in one of the strains due to a mutation will be specifically highlighted in the hybridization, which provides a possibility to identify and eventually clone the mutated gene. The approach is dependent on mutations that affect the amount of mRNA. Nonsense mutations. which prematurely terminate translation, can be such mutations as a surveillance system known as nonsense-mediated decay (NMD) has been developed by organisms to reduce the abundance of mRNA with nonsense codons. In the present study, we have analysed the barley (Hordeum vulgare L.) magnesium chelatase mutants xantha-f(26), xantha-f(27) and xantha-f(40) in order to investigate the presence of NMD in barley, as well as the importance of the position of the stop codon for NMD. Both nonsense-mutants xantha-f(27) and xantha-f(40). but not the missense mutant xantha-f(26), showed NMD. This was not expected for xantha-f(27) as its mutation is in the last exon of the gene. We conclude the NMD expands the number of mutants that can be used for gene cloning by our described microarray approach. (C) 2004 Elsevier SAS. All rights reserved.