Using Group II Introns for Attenuating the In Vitro and In Vivo Expression of a Homing Endonuclease

PLoS One. 2016 Feb 24;11(2):e0150097. doi: 10.1371/journal.pone.0150097. eCollection 2016.

Abstract

In Chaetomium thermophilum (DSM 1495) within the mitochondrial DNA (mtDNA) small ribosomal subunit (rns) gene a group IIA1 intron interrupts an open reading frame (ORF) encoded within a group I intron (mS1247). This arrangement offers the opportunity to examine if the nested group II intron could be utilized as a regulatory element for the expression of the homing endonuclease (HEase). Constructs were generated where the codon-optimized ORF was interrupted with either the native group IIA1 intron or a group IIB type intron. This study showed that the expression of the HEase (in vivo) in Escherichia coli can be regulated by manipulating the splicing efficiency of the HEase ORF-embedded group II introns. Exogenous magnesium chloride (MgCl2) stimulated the expression of a functional HEase but the addition of cobalt chloride (CoCl2) to growth media antagonized the expression of HEase activity. Ultimately the ability to attenuate HEase activity might be useful in precision genome engineering, minimizing off target activities, or where pathways have to be altered during a specific growth phase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chaetomium / enzymology*
  • Chaetomium / genetics
  • Cobalt / pharmacology
  • Deoxyribonucleases, Type II Site-Specific / biosynthesis*
  • Deoxyribonucleases, Type II Site-Specific / genetics
  • Fungal Proteins / biosynthesis*
  • Fungal Proteins / genetics
  • Gene Expression Regulation, Enzymologic / drug effects
  • Gene Expression Regulation, Enzymologic / physiology*
  • Gene Expression Regulation, Fungal / drug effects
  • Gene Expression Regulation, Fungal / physiology*
  • Introns*
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / genetics

Substances

  • Fungal Proteins
  • Recombinant Proteins
  • Cobalt
  • Deoxyribonucleases, Type II Site-Specific
  • cobaltous chloride

Grants and funding

This research is supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada to GH. The authors also gratefully acknowledge support for TKG from a University of Manitoba Faculty of Science Graduate Award and the University of Manitoba Faculty of Graduate Studies GETS program.