GUS reporter system

  The GUS reporter system (GUS: beta-glucuronidase) is a reporter gene system, particularly useful in plant molecular biology. ^[1] Several kinds of GUS reporter gene assay are actually available, depending of the substrate used: the term GUS staining refers to the most common of these, an histochemical technique.

Additional recommended knowledge

Weighing the right way

Guide to balance cleaning: 8 simple steps

What is the Correct Way to Check Repeatability in Balances?

Purpose

The purpose of this technique is to analyze the activity of a promoter (in terms of expression of a gene under that promoter) either in a quantitative way or through visualization of its activity in different tissues. The technique is based on beta-glucuronidase, an enzyme from the bacterium Escherichia coli;^[2] this enzyme, when incubated with some specific colorless or not fluorescent substrates, can transform them into coloured or fluorescent products. ^[3]

Substrates

There are actually different possible glucuronides that can be used as substrates for the beta-glucuronidase, depending on the type of detection needed (histochemical, spectrophotometrical, fluorimetrical). The most common substrate for GUS hystochemical staining is 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc): the product of the reaction is in this case a clear blue color. Other common substrates are p-nitrophenyl β-D-glucuronide for the spectrophotometrical assay and 4-methylumbelliferyl-beta-D-glucuronide (MUG) for the fluorimetrical assay.^[4]

History

The system was originally developed by Richard Anthony Jefferson during his Ph.D. at the University of Colorado at Boulder. ^[5] He adapted the technique for the use with plants as he worked in the Plant Breeding Institute of Cambridge, between 1985 and 1987 ^[1]. Since then thousands of labs made use of the system, making probably the most widely used tool in plant molecular biology, as underlined by over 6000 citations in scientific literature. ^[5]

Target organisms

  An organism is suitable for a GUS assay if it has no own beta-glucuronidase or the own activity is very low (background activity). For this reason the assay is not useful in almost all vertebrates and many molluscs.^[4] Since there is no detectable GUS activity in higher plants, mosses, algae, ferns, fungi and most bacteria, ^[4] these organisms are perfectly suited for the assay and this is the reason why the assay is widespread in plant science.

Other reporter systems

The GUS system is not the only available gene reporter system for the analysis of promoter activity. Other competing systems are based on e.g. luciferase, GFP, beta-galactosidase, chloramphenicol acetyltransferase (CAT), alkaline phosphatase. The use of one or the other system is mainly dependent on the organism of interest.

Other uses

  The GUS assay, as well as other reporter gene systems, can be use for other kinds of studies other than the classical promoter activity assay. Reporter systems have been used for the determination of the efficiency of gene delivery systems, the intracellular localization of a gene product, the detection of protein-protein or protein-DNA interactions, the efficiency of translation initiation signals and the success of molecular cloning efforts.

Intellectual Property

The use of the uidA gene for the GUS assay is covered by U.S. Patent 5,268,463 , U.S. Patent 5,432,081  and U.S. Patent 5,599,670 .

Sources

1. ^ ^{a b} GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. R.A. Jefferson, T. A. Kavanagh, and M. W. Bevan EMBO J. 1987 December 20; 6(13): 3901–3907 [1]
2. ^ Cloning and endonuclease restriction analysis of uidA and uidR genes in Escherichia coli K-12: determination of transcription direction for the uidA gene. C. Blanco, P. Ritzenthaler, and M. Mata-Gilsinger J Bacteriol 1982 February; 149(2): 587–594. [2]
3. ^ beta-Glucuronidase from Escherichia coli as a gene-fusion marker. R.A. Jefferson, S.M. Burgess, and D. Hirsh PNAS 1986 November; 83(22): 8447–8451 [3]
4. ^ ^{a b c} U.S. Patent 5,268,463 
5. ^ ^{a b} Cambia Organization Website: biography of Richard A. Jefferson