Purification and assay of ADAR activity.
ADAR editing enzymes are found in all multicellular animals and are conserved in sequence and protein organization. The number of ADAR genes differs between animals, ranging from three in mammals to one in Drosophila. ADAR is also alternatively spliced to generate isoforms that can differ significantly in enzymatic activity. Therefore, to study the enzyme in vitro, it is essential to have an easy and reliable method of expressing and purifying recombinant ADAR protein. To add to the complexity of RNA editing, the number of transcripts that are edited by ADARs differs in different organisms. In humans there is extensive editing of Alu sequences, whereas in invertebrates transcripts expressed in the central nervous system are edited and this editing increases during development. It is possible to quantify site-specific RNA editing by sequencing of clones derived from RT-PCR products. However, for routine assaying of an edited position within a particular transcript, this is both expensive and time consuming. Therefore, a nonradioactive method based on poison primer extension assay is an ideal alternative.
MRC Human Genetics Unit, Western General Hospital, Edinburgh, United Kingdom.
Liam P
LP
Keegan
Joshua J
JJ
Rosenthal
Loretta M
LM
Roberson
Mary A
MA
O'Connell
D000243:Adenosine Deaminase / Q000737:chemistry* / Q000302:isolation & purification*; D001483:Base Sequence; D001671:Biochemistry / Q000379:methods; D002138:Calibration; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010843:Pichia / Q000378:metabolism; D017393:RNA Editing; D011994:Recombinant Proteins / Q000737:chemistry
D017931:DNA Primers; D011994:Recombinant Proteins; D000243:Adenosine Deaminase