Rapid generation of hypomorphic mutations

Laura L. Arthur, Joyce J. Chung, Preetam Jankirama, Kathryn M. Keefer, Igor Kolotilin, Slavica Pavlovic-Djuranovic, Douglas L. Chalker, Vojislava Grbic, Rachel Green, Rima Menassa, Heather L. True, James B. Skeath, Sergej Djuranovic

Research output: Contribution to journalArticlepeer-review

10 Scopus citations

Abstract

Hypomorphic mutations are a valuable tool for both genetic analysis of gene function and for synthetic biology applications. However, current methods to generate hypomorphic mutations are limited to a specific organism, change gene expression unpredictably, or depend on changes in spatial-temporal expression of the targeted gene. Here we present a simple and predictable method to generate hypomorphic mutations in model organisms by targeting translation elongation. Adding consecutive adenosine nucleotides, so-called polyA tracks, to the gene coding sequence of interest will decrease translation elongation efficiency, and in all tested cell cultures and model organisms, this decreases mRNA stability and protein expression. We show that protein expression is adjustable independent of promoter strength and can be further modulated by changing sequence features of the polyA tracks. These characteristics make this method highly predictable and tractable for generation of programmable allelic series with a range of expression levels.

Original languageEnglish (US)
Article number14112
JournalNature communications
Volume8
DOIs
StatePublished - Jan 20 2017

ASJC Scopus subject areas

  • General Chemistry
  • General Biochemistry, Genetics and Molecular Biology
  • General Physics and Astronomy

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