The protein content of each sample is then determined and the rates are standardized and expressed as pmol 4-MU/µg protein/min (see Note 5). Genetics Home Reference has merged with MedlinePlus. Prepare sufficient wells to assay each sample in duplicate (see Note 7). Deposit a drop of embedding solution (Reichert-Jung) onto the sample holder and place small pieces of fresh or frozen plant tissue in the droplet. Finally, the sample is fixed in either Solution A at room temperature or Solution B at 4°C for 4 h and rinsed twice with Na-phosphate buffer.

The addition of potassium ferricyanide and ferrocyanide, at a final concentration of 0.1–0.5 mM, to the X-Gluc solution prevents the diffusion of the indoxyls by accelerating the dimerization. Measure the absolute fluorescence values of the calibration wells using the Read Endpoint option. Alternative methods of visualization of GUS activity are discussed in Notes 10 and 11. [11][17], Repeating unit of the heparan sulfate substrate of β-glucuronidase, Surface depiction of active site pocket of β-glucuronidase with catalytic residues shown[1], Mechanism of β-glucuronidase hydrolysis of a sugar substrate with high energy transition states showing oxocarbenium ion character depicted[15], Potential stabilization of the nucleophilic residue Glu540 by Tyr504 in β-glucuronidase[16], Predicted activity of the conserved Asn450 residue in stabilization of the β-glucuronidase sugar substrate[11][17], Potential salt bridge between Glu352 and Arg216 in human beta-glucuronidase[1][18], Deficiencies in β-glucuronidase result in the autosomal recessive inherited metabolic disease known as Sly syndrome or Mucopolysaccharidosis VII.

The Deltasoft program displays kinetic plots of fl.u/min for each well. CAS  To give a protocol for fixation, embedding and sectioning that would be suitable for all plant tissues is impossible. Deltasoft can then directly interpolate the concentration of unknown samples from the standard curve. [11] Beta-glucuronidase contains several notable structural formations, including a type of beta barrel known as a jelly roll barrel and a TIM barrel. deficiency presenting as hydrops fetalis or as early mucopolysaccharidosis VII. GUS, is short for glucuronidase, an enzyme in the bacterium E. coli. MedlinePlus links to health information from the National Institutes of Health and other federal government agencies.

However UniProtKB may contain entries with identical sequences in case Prechill the cryostat chamber to between −20°C and −35°C. The reactions proceed through a transition state with oxocarbenium ion characteristics. However, it is recommended to prepare fresh X-Gluc solution for each assay. The only way fluorescence occurs is by GUS enzyme activity. Metrics details. PubMed  This enzyme is located in lysosomes, compartments within cells that digest and recycle different types of molecules. Each GUS assay is performed in duplicate and the mean and variation of replica samples are automatically calculated using the Deltasoft statistics program. For the staining of seeds, it is necessary to dissect the fruit. In this case, Deltasoft can directly convert the rates of activity from fl.u/mm into U GUS/min. We propose two different fixation solutions—Solution A, for most tissues (to be tried first); and Solution B, as a starting protocol for more recalcitrant tissues such as mature seeds. Taxes to be calculated in checkout.

), Springer Nature is developing a new tool to find and evaluate Protocols. You are facing a dilemma: On the one hand, good fixation is required to maintain a good structure of the sample, and, on the other hand, some components of the fixative solutions, especially the glutaraldehyde, have an inhibitory effect on β-glucuronidase activity. USA 80: 4803–4807. Cell. When the GUS reporter gene is under the control of a strong promoter, the staining can “leak” into neighboring cells and the addition of potassium ferricyanide and ferrocyanide is necessary to achieve a true localization of the sites of β-glucuronidase activity. Martin, T., Schmidt, R., Altmann, T., and Frommer, W. (1992) Nondestructive assay systems for detection of beta-glucuronidase activity in higher plants. Mozo, T. and Hooykaas, P. J. J.

The checksum is computed as the sequence 64-bit Cyclic Redundancy Check value (CRC64) β-glucuronidase fromEscherichia coli as a gene-fusion marker.Proc. To break down these large molecules, individual sugars are removed one at a time from one end of the molecule. Although less sensitive than the fluorometric assay, the spectrophotometric assay is simple and cheap (16).

Learn more. Researchers believe that the accumulated GAGs may also interfere with the functions of other proteins inside the lysosomes and disrupt many normal functions of cells. Plant Mol Biol Rep 5, 387–405 (1987). This treatment makes the slides sticky so that the sections will adhere strongly. Aliquot approx 2–5 mg (50–200 seeds) of Arabidropsis seeds into a 1.5-mL Eppendorf tube and grind thoroughly in a 150 µL GUS extraction buffer using a motorized micropestle. Upon integration into UniProtKB, each entry is assigned a unique accession number, which is called 'Primary (citable) accession number'.



This subsection of the 'Entry information' section shows the date of integration of the entry into UniProtKB, the date of the last sequence update and the date of the last annotation modification ('Last modified'). PubMed  Monitoring β-glucuronidase activity through the use of a GUS assay allows determination of the spatial and temporal expression of the gene in question.

(35). 2: 987–995. Richard A. Jefferson. PubMed  US$ 39.95. Wenn dieses DNA-Konstrukt (bspw. After grinding, add 150 µL of GUS extraction buffer and mix by vortexing.

Van den, Broeck, G., Timko, M., Kausch, A. P., Cashmore, A. R., Van Montagu, M., and Herrera-Estrella, L. (1985). Stoeber, F. (1961).



This subsection of the Names and taxonomy section indicates the name(s) of the gene(s) that code for the protein sequence(s) described in the entry. Bevan, M. W. (1984). This process is experimental and the keywords may be updated as the learning algorithm improves. The shortage (deficiency) of β-glucuronidase leads to the accumulation of dermatan sulfate, heparan sulfate, and chondroitin sulfate within lysosomes in virtually all tissues and organs. coli. Also, the problem of substrate penetration is alleviated. Initially, these mechanisms, because of this oxocarbenium characteristic of the transition state, were suggested to be SN1 reactions proceeding through a discrete oxocarbenium ion intermediate. (Possible sources of background activity are discussed in Note 1.). The fluorometric and histochemical assays described here are all carried out at neutral pH, which is the optimum pH for bacterial β-glucuronidase, and we have not been troubled by endogenous activity. To allow a better penetration of the fixatives, the object can be cut into small pieces of 5 mm in length. In case of problems, it is possible to add nonionic detergents to the X-Gluc solution (e.g., 0.05% Triton X100 or 0.1 % Tween 20) to decrease surface tension By analyzing many samples, even in difficult conditions of staining, a good idea of the general pattern of GUS expression in the tissue should be obtained.

(34) have developed a method for ultrastructural detection of GUS activity. Monitoring β-glucuronidase activity through the use of a GUS assay allows determination of the spatial and temporal expression of the gene in question. Natl. (17) reported that an addition of 53 amino acids is tolerated with no significant drop in activity, and that β-glucuronidase fusions are still active (albeit at a reduced level) when a 126 amino acid extension is added. At the same time, place 5 µL of extract in a 1.6-mL disposable cuvet.

β-glucuronidase is involved in the breakdown of large molecules called glycosaminoglycans (GAGs).
In general, it is better to stain before the fixation. These lifetimes are too short to assign to a reaction intermediate.

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