Summary
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.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Abbreviations
- CaMV:
-
cauliflower mosaic virus
- CAT:
-
chloramphenicol acetyl transferase
- EDTA:
-
ethylenediaminetetraacetic acid
- GUS:
-
ß-glucuronidase
- HPLC:
-
high performance liquid chromatography
- MES:
-
2-morpholinoethanesulphonic acid
- MS:
-
medium after Murashige and Skoog (1962)
- MUG:
-
4-methyl umbelliferyl glucuronide
- MU:
-
methylumbelliferone
- NOS:
-
nopaline synthase
- PEG:
-
polyethylene glycol
- TRIS:
-
tris-hydroxymethyl aminomethane
- UV:
-
ultraviolet
References
Bradford MM (1976) Anal Biochem 72: 248–254
Ebert PR, Har SB, An G (1987) Proc Natl Acad Sci USA 84: 5745–5749
Fromm ME, Taylor LP, Walbot V (1986) Nature 319: 791–793
Gorman C, Moffet L, Howard B (1982) Mol Cell Biol 5: 1044–1051
Jefferson RA, Kavanagh TA, Bevan MW (1987) EMBO J 6: 3901–3907
Junker B, Zimny J, Luhrs R, Lorz H (1987) Plant Cell Rep 6: 329–332
Labarca C, Paigen K (1980) Anal. Biochem 102: 344–347
Lepetit M, Ehling M, Gigot C, Hahne G (1991) Plant Cell Rep 10: 401–405
Mass C, Werr W (1989) Plant Cell Rep 8: 148–151
Murashige T, Skoog F (1962) Physiol Plant 15: 473–497
Nagy JI, Maliga P (1976) Pflanzenphysiol 78: 453–455
Ow DW, Jacobs JD, Howell SH (1987) Proc Natl Acad Sci USA 84: 4870–4874
Paszkowski J, Shillito RD, Saul M, Mandak V, Hohn T, Hohn B, Potrykus I (1984) EMBO J 3: 2717–2722
Power JB, Chapman JV (1985) In: Plant Cell Culture: a Practical Approach, Isolation, Culture and Genetic Manipulation of Plant Protoplasts — (Dixon RA ed) IRL press pp. 37–65
Sala F, Marchesi ML, Castiglione S, Paszkowski J, Saul M, Potrykus I, Negrutiu I (1989) In: Biotechnology in agriculture and forestry, Volume 9, Plant protoplasts and genetic engineering II (Bajaj YPS, ed) Berlin Heidelberg: Springer-Verlag, pp. 217–227
Sambrook J, Fritsch EF, Maniathis T (1989) In: Molecular Cloning. A laboratory manual, 2nd edition. Cold Spring Harbor Laboratory Press
Scott R, Draper J, Jefferson R, Dury G, Jacob L (1988) In: Plant genetic transformation and gene expression (Draper J, Scott R, Armitage P, eds). Blackwell Scientific Publications, pp 263–339
Young SL, Jackson AE, Puett D, Melner MH (1985) DNA 6: 469–475
Author information
Authors and Affiliations
Additional information
Communicated by I. Potrykus
Rights and permissions
About this article
Cite this article
Branca, C., Ricci, A., Torelli, A. et al. Non-radioactive detection of β-glucuronidase and chloramphenicol acetyltransferase activities in co-transformed protoplasts by HPLC. Plant Cell Reports 12, 361–365 (1993). https://doi.org/10.1007/BF00234693
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF00234693