Single RNA species injected in Xenopus oocyte directs the synthesis of active choline acetyltransferase.
In vitro synthesized complementary RNA (cRNA) transcribed from a non-full length Drosophila choline acetyltransferase (ChAT) cDNA clone will direct the synthesis of enzymatically active and immunologically recognizable protein when injected into Xenopus oocytes. The levels of ChAT activity expressed in injected oocytes are proportional, over 4 orders of magnitude difference, to the concentration of injected 'sense' orientation cRNA. GpppG capping of the in vitro synthesized cRNA is not necessary for expression of active ChAT but inclusion of the capping compound during in vitro transcription results in higher levels of enzyme expression at lower levels of cRNA injection. In addition the capped cRNA results in increasing ChAT expression by the oocytes up to 7 days after injection while uncapped cRNA results in maximum enzyme activity after a single day and decreasing levels of activity during subsequent days. A single immunologically detectable protein is produced by oocytes injected with 'sense' cRNA which has a molecular size of 75 kDa and is indistinguishable from the major form of ChAT present in Drosophila. Oocytes making enzymatically active ChAT also accumulate significant levels of acetylcholine. We conclude from these results that our our non-full length Drosophila ChAT cDNA clone contains all the necessary coding information to make a functional protein which appears to have the same size and activity as native Drosophila ChAT.
McCaman
R E
RE
Division of Neuroscience, Beckman Research Institute of the City of Hope, Duarte, CA 91010.
Carbini
L
L
Maines
V
V
Salvaterra
P M
PM
D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D002794:Choline / Q000378:metabolism; D002795:Choline O-Acetyltransferase / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000235:genetics; D004330:Drosophila / Q000201:enzymology / Q000235:genetics; D005260:Female; D007700:Kinetics; D009865:Oocytes / Q000201:enzymology*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics*; D014158:Transcription, Genetic; D014981:Xenopus
D012333:RNA, Messenger; D004247:DNA; D002795:Choline O-Acetyltransferase; D002794:Choline; D000109:Acetylcholine