Non-radioactive detection of ?-glucuronidase and chloramphenicol acetyltransferase activities in co-transformed protoplasts by HPLC

Abstract

The use of transient gene expression assays for the study of natural or engineered plant promoters is affected by a considerable degree of inter-experiment variability. As a means of obtaining interpretable data from a limited number of experiments, we worked out conditions for the simultaneous determi nation of the activity of two reporter genes, a "sample" and a "reference", ona single extract of co-transformed protoplasts. ß-glucuronidase (GUS) and chloramphenicol acetyl transferase (CAT) genes, both under the control of the CaMV 35S promoter, were transferred into tobacco (Nicotiana tabacum L.) protoplasts on two independent plasmids. The parallel expression of the two reporter genes in several independent co-transformation experiments was verified. Conditions for the use of a single protoplast extraction buffer and for the simultaneous assay of both reporter gene activities were set up. A HPLC method for the non-radioactive determination of both enzyme activities on a single aliquot of the reaction mixture was developed. The resulting procedure was tested using the GUS gene as "reference" and the CAT gene, under the control of either wild type or upstream-deleted (-90) CaMV 35S promoter, as "sample". The protocol is simple and allows the fast analysis of plant promoters in the presence of a true internal standard under conditions in which assay manipulations are reduced to a minimum and both reporter gene activities are subjected to the same experimental treatments.

Quantitative analysis by flow cytometry of abscisic acid-inducible gene expression in transiently transformed rice protoplasts

“…2, 3), as reported for other species (42)(43)(44)(45). This result highlights an advantage of FCM over biochemical methods, namely, the ability to quantify gene expression on a per-cell basis in a small (Ͻ6%; Fig.…”

“…Previous studies on DNA uptake in plant protoplasts showed that only a subset of cells is competent to take up DNA, and the amount of uptake is limited (42)(43)(44)(45). Therefore, the effect of the amount of input DNA on ABAinducible gene expression was determined.…”

Collective Resistance in Microbial Communities by Intracellular Antibiotic Deactivation

“…To confirm that Cm R cells actually deactivate Cm in the growth medium, we analyzed culture supernatant (S) by high-performance liquid chromatography (HPLC) [44]. As shown in Fig 2C, within 4 h of growth, Cm R cells entirely converted an initial Cm concentration of 5 μg ml −1 , as evidenced by the disappearance of the corresponding Cm peak at wavelength 278 nm.…”

“…As shown in Fig 2C, within 4 h of growth, Cm R cells entirely converted an initial Cm concentration of 5 μg ml −1 , as evidenced by the disappearance of the corresponding Cm peak at wavelength 278 nm. New peaks (at later elution times) appeared and gradually increased in HPLC profiles of S collected after 1, 2, and 4 h of cultivation; these peaks were previously shown to correspond to acetylated Cm derivates (1- and 3-acetylchloramphenicol) [44]. …”

“…Four wells were sampled and pooled per time point (combined volume of 1.2 ml), centrifuged to remove cells, and filtered through a 0.2 μm filter. HPLC analysis was carried out using an Agilent 1260 Infinity system (Agilent Technologies) with ultraviolet (UV) detection at 278 nm (maximum absorbance of Cm) [44]. An Aeris Peptide XB-C18 column (Phenomenex) with 3.6 μm particle and a size of 250 × 4.60 mm was used.…”