The GUS reporter system (''GUS'':
β-glucuronidase) is a
reporter gene
In molecular biology, a reporter gene (often simply reporter) is a gene that researchers attach to a regulatory sequence of another gene of interest in bacteria, cell culture, animals or plants. Such genes are called reporters because the charac ...
system, particularly useful in plant
molecular biology
Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and phys ...
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.
Purpose
The purpose of this technique is to analyze the activity of a gene transcription
promoter (in terms of
expression
Expression may refer to:
Linguistics
* Expression (linguistics), a word, phrase, or sentence
* Fixed expression, a form of words with a specific meaning
* Idiom, a type of fixed expression
* Metaphorical expression, a particular word, phrase, o ...
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
''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus '' Esc ...
, which codes for the enzyme,
β-glucuronidase; this enzyme, when incubated with specific colorless or non-fluorescent
substrates, can convert them into stable
color
Color (American English) or colour (British English) is the visual perceptual property deriving from the spectrum of light interacting with the photoreceptor cells of the eyes. Color categories and physical specifications of color are associ ...
ed or
fluorescent
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, ...
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 A fusion gene is a hybrid gene formed from two previously independent genes. It can occur as a result of translocation, interstitial deletion, or chromosomal inversion. Fusion genes have been found to be prevalent in all main types of human neopl ...
. The insertion of the uidA gene will cause production of GUS, which can then be detected using various glucuronides as substrates.
Substrates
There are different possible
glucuronide
A glucuronide, also known as glucuronoside, is any substance produced by linking glucuronic acid to another substance via a glycosidic bond. The glucuronides belong to the glycosides.
Glucuronidation, the conversion of chemical compounds to gl ...
s that can be used as substrates for the β-glucuronidase, depending on the type of detection needed (
histochemical
Histology,
also known as microscopic anatomy or microanatomy, is the branch of biology which studies the microscopic anatomy of biological tissues. Histology is the microscopic counterpart to gross anatomy, which looks at larger structures vis ...
,
spectrophotometrical,
fluorimetrical). The most common substrate for GUS histochemical staining is
5-bromo-4-chloro-3-indolyl glucuronide
X-Gluc is a chemical compound with the molecular formula C14H13BrClNO7. It is used as a reagent to detect β-glucuronidase, an enzyme produced by the '' E. coli'' bacterium. It is used to detect ''E. coli'' contamination in food, water and the ur ...
(
X-Gluc). X-Gluc is
hydrolyzed
Hydrolysis (; ) is any chemical reaction in which a molecule of water breaks one or more chemical bonds. The term is used broadly for substitution, elimination, and solvation reactions in which water is the nucleophile.
Biological hydrolysis ...
by GUS into the product 5,5'-dibromo-4,4'-dichloro-indigo (diX-indigo). DiX-indigo will appear blue, and can be seen using light microscopy.
This process is analogous to hydrolysis of
X-gal by
Beta-galactosidase
β-Galactosidase (EC 3.2.1.23, lactase, beta-gal or β-gal; systematic name β-D-galactoside galactohydrolase), is a glycoside hydrolase enzyme that catalyzes hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides.
β ...
to produce blue cells as is commonly practiced in bacterial reporter gene assays.
For other types of detection, common substrates ar
p-nitrophenyl β-D-glucuronidefor the spectrophotometric assay an
4-methylumbelliferyl-beta-D-glucuronide(MUG) for the fluorimetric assay.
History
The system was originally developed by
Richard Anthony Jefferson during his
Ph.D. at the
University of Colorado at Boulder
The University of Colorado Boulder (CU Boulder, CU, or Colorado) is a public research university in Boulder, Colorado. Founded in 1876, five months before Colorado became a state, it is the flagship university of the University of Colorado sy ...
.
[''Cambia'' Organization Website]
biography of Richard A. Jefferson
He adapted the technique for the use with plants as he worked in the ''Plant Breeding Institute'' of
Cambridge
Cambridge ( ) is a university city and the county town in Cambridgeshire, England. It is located on the River Cam approximately north of London. As of the 2021 United Kingdom census, the population of Cambridge was 145,700. Cambridge bec ...
, between 1985 and 1987.
Since then thousands of labs have used the system, making it one of the most widely used tools in plant molecular biology, as underlined by thousands of citations in scientific literature.
Target organisms
An organism is suitable for a GUS assay if it lacks naturally occurring β-glucuronidase activity or if the activity is very low (''background'' activity). For this reason, the assay is not useful in most
vertebrates
Vertebrates () comprise all animal taxa within the subphylum Vertebrata () (chordates with backbones), including all mammals, birds, reptiles, amphibians, and fish. Vertebrates represent the overwhelming majority of the phylum Chordata, with ...
and many
molluscs
Mollusca is the second-largest phylum of invertebrate animals after the Arthropoda, the members of which are known as molluscs or mollusks (). Around 85,000 extant species of molluscs are recognized. The number of fossil species is estim ...
.
Since there is no detectable GUS activity in
higher plants
Vascular plants (), also called tracheophytes () or collectively Tracheophyta (), form a large group of land plants ( accepted known species) that have lignified tissues (the xylem) for conducting water and minerals throughout the plant. They al ...
,
mosses
Mosses are small, non-vascular flowerless plants in the taxonomic division Bryophyta (, ) ''sensu stricto''. Bryophyta (''sensu lato'', Schimp. 1879) may also refer to the parent group bryophytes, which comprise liverworts, mosses, and hornw ...
,
algae
Algae (; singular alga ) is an informal term for a large and diverse group of photosynthetic eukaryotic organisms. It is a polyphyletic grouping that includes species from multiple distinct clades. Included organisms range from unicellular micr ...
,
fern
A fern (Polypodiopsida or Polypodiophyta ) is a member of a group of vascular plants (plants with xylem and phloem) that reproduce via spores and have neither seeds nor flowers. The polypodiophytes include all living pteridophytes exce ...
s,
fungi
A fungus ( : fungi or funguses) is any member of the group of eukaryotic organisms that includes microorganisms such as yeasts and molds, as well as the more familiar mushrooms. These organisms are classified as a kingdom, separately fr ...
and most
bacteria
Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria were am ...
,
the assay is ideally suited for gene expression studies in these organisms, and considered the reporter gene of choice for in plant science.
Benefits and limitations
The GUS assay does not require the presence of any cofactors or ions for function. Beta-glucuronidase can function through a wide range of pH values, and is fairly resistant to thermal inactivation. However, GUS is susceptible to inhibition from certain heavy metal ions, such as Cu
2+ and Zn
2+.
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.
[{{Cite journal, last1=Citovsky, first1=V., last2=Zupan, first2=J., last3=Warnick, first3=D., last4=Zambryski, first4=P., date=1992-06-26, title=Nuclear localization of Agrobacterium VirE2 protein in plant cells, url=https://www.science.org/doi/10.1126/science.1615325, journal=Science, language=en, volume=256, issue=5065, pages=1802–1805, doi=10.1126/science.1615325, issn=0036-8075, pmid=1615325, bibcode=1992Sci...256.1802C] GUS assays can be carried out in the presence of
potassium ferricyanide
Potassium ferricyanide is the chemical compound with the formula K3 e(CN)6 This bright red salt contains the octahedrally coordinated 3−.html" ;"title="e(CN)6sup>3−">e(CN)6sup>3− ion. It is soluble in water and its solution shows some g ...
to prevent the stain from diffusing.
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
Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence, and is usually distinguished from a photoprotein. The name was first used by Raphaël Dubois who invented the words '' luciferin'' and ''luciferase'' ...
,
GFP,
beta-galactosidase
β-Galactosidase (EC 3.2.1.23, lactase, beta-gal or β-gal; systematic name β-D-galactoside galactohydrolase), is a glycoside hydrolase enzyme that catalyzes hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides.
β ...
,
chloramphenicol acetyltransferase
Chloramphenicol acetyltransferase (or CAT) is a bacterial enzyme () that detoxifies the antibiotic chloramphenicol and is responsible for chloramphenicol resistance in bacteria. This enzyme covalently attaches an acetyl group from acetyl-CoA to ...
(CAT),
alkaline phosphatase
The enzyme alkaline phosphatase (EC 3.1.3.1, alkaline phosphomonoesterase; phosphomonoesterase; glycerophosphatase; alkaline phosphohydrolase; alkaline phenyl phosphatase; orthophosphoric-monoester phosphohydrolase (alkaline optimum), systematic ...
. 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.
Other uses
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.
Sources
Molecular biology