Beta-Glucuronidase

 b-Glucuronidase (E.C. 3.2.1.31; GUS) is the most widely used reporter gene in plant molecular biology (1). The gusA gene cloned from Escherichia coli encodes a 68-kDa b-glucuronidase that forms a stable homotetramer and catalyzes the hydrolysis of a large number of glucuronides, in which D-glucuronic acid is conjugated through a b-O-glycosidic linkage to any aglycone. b-Glucuronidase has emerged as the reporter gene of choice to be used in plants, because there is very little or no endogenous glucuronidase activity across the phyla. This minimizes the detection of background activity and allows very small quantities of GUS to be detected. GUS activity may be followed in cell lysates or in situ. The major use of GUS has been to study gene expression patterns in transgenic plants by expressing GUS under the control of regulatory sequences of interest. A second use has been to monitor the intracellular fate of chimeric proteins by generating GUS fusion genes. For example, GUS has been fused to gene leader sequences that target the fusion gene to different organelles and allow intracellular trafficking of proteins to be studied (2.(

GUS activity may be detected using a variety of substrates. As in the case of beta-galactosidase and X-gal, GUS expression is most commonly detected using histochemical substrates, such as X-GlcU (5-bromo-4-chloro-3-indolyl b-D-glucuronide), which gives a dark blue precipitate upon hydrolysis. Alternative substrates include 5-bromo-6-chloro-3-indolyl b-D-glucuronide, 6-chloro-3-indolyl b-D-glucuronide, and indoxyl b-D-glucuronide that give magenta, pink, and blue precipitates, respectively (3). The variety of substrates also makes GUS a valuable component of dual (or multiple) reporter gene systems; for example, GUS and lacZ expression could be detected in the same tissues by using substrates that give different colored histochemical precipitates. In addition to histochemical substrates, a number of fluorescent and chemiluminescent substrates have been

developed that are analogous to the substrates for b-galactosidase, such as CFDG-GlcU (Molecular Probes Inc.) and 4-methylumbelliferyl b-D-glucuronide (MUGlcU) (4).

In the case of lacZ, problems may be encountered in loading substrates into cells; to load FDG, for example, cells must undergo a moderate osmotic shock that can affect cell viability. In the case of GUS, however, a second gene, gusB, may be expressed that encodes a permease that actively takes up and transports glucuronide substrates into the cell. In addition to substrates that allow reporter gene visualization, a number of other bioactive molecules can be conjugated to glucuronides, which could then be released by hydrolysis in GUS-expressing cells. Thus, combined use of gusA and gusB increases the use of the reporter gene, from merely indicating gene expression to controlling a specific cell manipulation (1).

 References

1.R. A. Jefferson (1989) Nature 342, 837–838. 

2.R. A. Jefferson, T. A. Kavanagh, and M. W. Bevan (1987) EMBO J. 6, 3901–3907. 

3.G. A. Hull and M. Devic (1995) Methods. Mol. Biol. 49, 125–141. 

4. C. E. Olesen, J. J. Fortin, J. C. Voyta, and I. Browstein (1997) Methods Mol. Biol. 63, 61–70.

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مواضيع ذات صلة

Micro-RNA and Long Noncoding RNA

Histone Organization

Noncoding DNA

DNA Synthesis Occurs During the S Phase of the Cell Cycle

Formation of Replication Bubbles

Initiation & Elongation of DNA Synthesis

The Exact Function of Much of the Mammalian Genome is Not Well Understood

DNA is organized into Chromosomes

Some Regions of Chromatin are “Active” & Others are “Inactive”

The Nucleosome Contains Histone & DNA

Histones Are the Most Abundant Chromatin Proteins

Specific Nucleases Digest Nucleic Acids