Biomedical subjects

Gillian M Ring

Publications and source records attributed to Gillian M Ring.

2 recordsLinked to original sources

PubMed2004

Purification and assay of recombinant ADAR proteins expressed in the yeast Pichia pastoris or in Escherichia coli.

ADARs are found in Metazoans but are not present in yeasts. We have found that the methanol-utilizing yeast Pichia pastoris can be used to efficiently express enzymatically active epitope-tagged ADARs. We describe plasmid construction and protein expression procedures for producing Drosophila ADAR in this system.ADAR expression in Pichia pastoris uses the methanol-inducible alcohol oxidase AOX1 promoter for induction. A Zeocin resistance gene on the plasmid is used to select high copy number tandem integrations of the plasmid constructs. Preparation of extracts by grinding cultures in liquid nitrogen and purification protocols using 6 x HIS and FLAG epitope tags are described. Procedures for preparing radiolabeled dsRNA and for assaying the non-specific RNA editing activity of ADARs are described.ADARs produced in Escherichia coli are not enzymatically active. We describe expression of the ADAR dsRNA binding domains in E. coli using current versions of the T7 promoter based Studier vectors as well as the purification of the domains.

Adenosine Deaminase↗

PubMed2003

An ADAR that edits transcripts encoding ion channel subunits functions as a dimer.

In this report, we establish that Drosophila ADAR (adenosine deaminase acting on RNA) forms a dimer on double-stranded (ds) RNA, a process essential for editing activity. The minimum region required for dimerization is the N-terminus and dsRNA-binding domain 1 (dsRBD1). Single point mutations within dsRBD1 abolish RNA-binding activity and dimer formation. These mutations and glycerol gradient analysis indicate that binding to dsRNA is important for dimerization. However, dimerization can be uncoupled from dsRNA-binding activity, as a deletion of the N-terminus (amino acids 1-46) yields a monomeric ADAR that retains the ability to bind dsRNA but is inactive in an editing assay, demonstrating that ADAR is only active as a dimer. Different isoforms of ADAR with different editing activities can form heterodimers and this can have a significant effect on editing in vitro as well as in vivo. We propose a model for ADAR dimerization whereby ADAR monomers first contact dsRNA; however, it is only when the second monomer binds and a dimer is formed that deamination occurs.

Adenosine Deaminase↗