GUS reporter system

Molecular biology technique

The GUS reporter system (GUS: β glucuronidase) is a reporter gene system, particularly useful in plant molecular biology and microbiology. Several kinds of GUS reporter gene assay are available, depending on the substrate used. The term GUS staining refers to the most common of these, a histochemical technique.

The purpose of this technique is to analyze the activity of a gene transcription promoter (in terms of expression of a so called reporter gene under the regulatory control of that promoter) either in a quantitative manner, involving some measure of activity, or qualitatively (on versus off) through visualization of its activity in different cells, tissues, or organs. The technique utilizes the uidA gene of Escherichia coli, which codes for the enzyme, β glucuronidase; this enzyme, when incubated with specific colorless or non fluorescent substrates, can convert them into stable colored or fluorescent products. The presence of the GUS induced color indicates where the gene has been actively expressed. In this way, strong promoter activity produces much staining and weak promoter activity produces less staining. The uidA gene can also be fused to a gene of interest, creating a gene fusion. The insertion of the uidA gene will cause production of GUS, which can then be detected using various glucuronides as substrates. This process is analogous to hydrolysis of X gal by Beta galactosidase

The system was originally developed by Richard Anthony Jefferson during his Ph. D. at the University of Colorado at Boulder. He adapted the technique for the use with plants as he worked in the Plant Breeding Institute of Cambridge, between 1985 and 1987. However, GUS is susceptible to inhibition from certain heavy metal ions, such as Cu2+ and Zn2+. Additionally, the interpretation of the assay is limited by the movement of diX indigo throughout the cell. DiX indigo, can associate with lipids to diffuse far from the site of enzyme activity, which shows a lack of cytosolic localization and irregularity of substrate penetration. This can potentially lead to an incorrect interpretation of GUS protein localization. Despite a lack of cellular localization, nuclear localization of GUS has been well observed. GUS assays can be carried out in the presence of potassium ferricyanide to prevent the stain from diffusing.

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 and the imaging and microscopy technologies available to the laboratories conducting the research.

The GUS assay, as well as other reporter gene systems, can be used 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.