Characterization of Arabidopsis thaliana mismatch specific endonucleases: application to mutation discovery by TILLING in pea

Plant J. 2007 Sep;51(6):1116-25. doi: 10.1111/j.1365-313X.2007.03201.x. Epub 2007 Jul 25.

Abstract

Scanning DNA sequences for mutations and polymorphisms has become one of the most challenging, often expensive and time-consuming obstacles in many molecular genetic applications, including reverse genetic and clinical diagnostic applications. Enzymatic mutation detection methods are based on the cleavage of heteroduplex DNA at the mismatch sites. These methods are often limited by the availability of a mismatch-specific endonuclease, their sensitivity in detecting one allele in a pool of DNA and their costs. Here, we present detailed biochemical analysis of five Arabidopsis putative mismatch-specific endonucleases. One of them, ENDO1, is presented as the first endonuclease that recognizes and cleaves all types of mismatches with high efficiency. We report on a very simple protocol for the expression and purification of ENDO1. The ENDO1 system could be exploited in a wide range of mutation diagnostic tools. In particular, we report the use of ENDO1 for discovery of point mutations in the gibberellin 3beta-hydrolase gene of Pisum sativum. Twenty-one independent mutants were isolated, five of these were characterized and two new mutations affecting internodes length were identified. To further evaluate the quality of the mutant population we screened for mutations in four other genes and identified 5-21 new alleles per target. Based on the frequency of the obtained alleles we concluded that the pea population described here would be suitable for use in a large reverse-genetics project.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Arabidopsis / enzymology*
  • Arabidopsis / genetics
  • Arabidopsis Proteins / classification
  • Arabidopsis Proteins / metabolism
  • Arabidopsis Proteins / physiology*
  • Deoxyribonucleases / metabolism
  • Deoxyribonucleases / physiology*
  • Endonucleases / classification
  • Endonucleases / metabolism
  • Endonucleases / physiology*
  • Mixed Function Oxygenases / genetics*
  • Molecular Sequence Data
  • Peas / genetics*
  • Phylogeny
  • Plant Proteins / genetics*
  • Point Mutation
  • Sequence Alignment
  • Sequence Analysis, DNA / methods

Substances

  • Arabidopsis Proteins
  • BFN1 protein, Arabidopsis
  • Plant Proteins
  • Mixed Function Oxygenases
  • gibberellin 3beta-hydroxylase
  • Deoxyribonucleases
  • Endonucleases