Quantification and comparison of chloramphenicol acetyltransferase activity in transformed plant protoplasts using high-performance liquid chromatography- and radioisotope-based assays.
Rice and petunia leaf and cell suspension protoplasts were transformed by electroporation in the presence of pDW2. This plasmid contains a chloramphenicol acetyltransferase (CAT) coding region under the control of a promoter constructed from sequences of the cauliflower mosaic virus genome. We have compared two different approaches to measuring CAT activity in this system, namely high-performance liquid chromatography (HPLC) and a radioisotope-based method. Our results show that both techniques have a similar detection limit of 10 mU CAT and (with an activity greater than 10 mU CAT) the standard error for measuring known amounts of CAT activity was less than +/- 12% for both assays. The HPLC technique, however, has a greater linear response range of 10-600 mU CAT than the radioisotope method, which has a range of 10-400 mU CAT. The HPLC assay also requires a shorter assay time. As a result of this work we believe that HPLC is a viable alternative to the radioisotope-based assay described.
Davis
A S
AS
Department of Botany, University of Nottingham, University Park, United Kingdom.
Davey
M R
MR
Clothier
R C
RC
Cocking
E C
EC
D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000235:genetics; D012275:Oryza / Q000201:enzymology; D010944:Plants / Q000201:enzymology*; D011523:Protoplasts / Q000201:enzymology*; D011868:Radioisotopes; D014170:Transformation, Genetic
D011868:Radioisotopes; D015500:Chloramphenicol O-Acetyltransferase