Marker Gene Monthly Newsletter   

August, 2007

Volume 6, Number 8

© Copyright MGT, Inc., 2007.  Published by Marker Gene Technologies, Inc., The University of Oregon Riverfront Research Park, 1850 Millrace Drive, Eugene, Oregon 97403-1992 USA.  All rights reserved.  For information on the use or copying of the material contained in this document, please contact us at techservice@markergene.com.  Please see below for subscription information and updates.  This newsletter is labeled as an ADVERTISEMENT in accordance with the CAN-SPAM act of 2003, S.877 Public Law: 108-187.

Alkylguanine Transferase (AGT) Fusion.

A new marker gene system has been developed in the laboratories of Dr. Kai Johnsson and colleagues at the Institute of Chemical Sciences and Engineering, Swiss Federal Institute of Technology in Lausanne, Switzerland using the enzyme O⁶-alkylguanine-DNA alkyltransferase (AGT) and allied fusion proteins.  The AGT enzyme is usually responsible for a DNA repair mechanism in mammalian cells, whereby alkylated (methylated) guanine residues are dealkylated.  Upon activity, the resulting alkyl group is irreversibly attached to the enzyme, inactivating it.  The wide specificity of this enzyme has been used to allow reaction with a variety of substituted O⁶-benzylguanine (BG) derivatives with substituents at the 4-position of the benzyl ring.  Recently, many fluorescent analogs of BG have been prepared (containing fluorescein, rhodamine, CY3, CY5, etc.) and when these compounds are applied to live bacterial, yeast or mammalian cells, they are found to specifically label cells that have been transfected with a vector containing the AGT gene.  These new probes and systems are now available commercially from Covalys Biosciences AG.

The AGT gene, which can be fused to either the N or C terminus of the gene of interest, codes for a small, monomeric protein (207 aa), which can even be truncated to 177 residues without significantly affecting the activity against BG derivatives.  The relatively narrow substrate specificities of E. coli and yeast AGTs, which do not readily react with BG, allow for direct labeling of AGT fusion proteins in these hosts. However, labeling in mammalian cells requires the use of AGT-deficient cell lines.  For more information about this new marker gene, please see our website or visit the references or links below. 

• George, N., Pick, H., Vogel, N., Johnsson, N., Johnsson K., (2004) “Specific labeling of cell surface proteins with chemically diverse compounds.” J. Am. Chem. Soc. 126: 8896-8897.
• Keppler, A., Pick, H., Arrivoli, C., Vogel, H., Johnsson K. (2004) “Labeling of fusion proteins with synthetic fluorophores in live cells.” Proc. Natl. Acad. Sci. USA 101: 9955-9959.
• http://www.covalys.com/technologies/snap-tag.html
• Keppler, A., Gendreizig, S. Gronemeyer, T. Pick, H. Vogel, H. and Johnsson K. (2003) “A general method for the covalent labeling of fusion proteins with small molecules in vivo.” Nat. Biotechnol. 21: 86-89.
• Keppler, A., Kindermann, M., Gendreizig, S., Pick, H., Vogel, H., Johnsson K. (2004) “Labeling of fusion proteins of O6-alkylguanine-DNA alkyltransferase with small molecules in vivo and in vitro.” Methods 32: 437-444.
•   Juillerat, A., Heinis, C., Sielaff, I., Barnikow, J., Jaccard, H., Kunz, B., Terskikh, A., Johnsson K. (2005) “Engineering substrate specificity of O6-alkylguanine-DNA alkyltransferase for specific protein labeling in living cells” ChemBioChem 6: 1263-1269.
• Keppler, A., Arrivoli, C., Sironi, L., Ellenberg J., (2006)  “Fluorophores for live cell imaging of AGT fusion proteins across the visible spectrum” BioTechniques 41(2): pp 167-175.
  

Alkaline Phosphatase Quantitation.

There are a variety of intracellular phosphatases that utilize an assortment of phosphate and polyphosphate esters derived from membrane structural components, phosphoproteins, nucleotides and energy reservoirs as substrates.  Simple phosphatases, such as alkaline and acid phosphatase, catalyze the hydrolysis of phosphomonoesters with release of inorganic phosphate.  Alkaline Phosphatase plays an important role in many biochemical regulatory pathways, including a possible role in cell differentiation.  In mammalian cells, alkaline phosphatase (AP) exists as a dimer, where the N-terminus of one monomer embraces the other, stretching toward the active site. Alkaline Phosphatase activity can be monitored using the fluorescent substrate, Fluorescein diphosphate, tetraammonium salt (FDP) (Product No. M1222-002), contained in our new MarkerGene^TM Fluorescent Alkaline Phosphatase Assay Kit (M1222).  Alkaline Phosphatase catalyzes a two-step hydrolysis of FDP, releasing the highly fluorescent compound, fluorescein (Abs 488nm, EM 512nm), and activity measurements are easily obtained either in vitro, in cell lysate preparations, or in vivo. The kit contains enough substrate for up to 100 assays and control experiments, and also contains an inhibitor cocktail, reference standards and a detailed protocol for use. Please visit our website or see the references below for more information and applications.

• Ali A.T., Penny C.B., Paiker J.E., van Niekerk C., Smit A., Ferris W.F., Crowther N.J., (2005). “Alkaline Phosphatase is involved in the control of adipogenesis in the murine preadipocyte cell line, 3T3-L1.” Clin. Chim. Acta 354(1-2):  101-9  
• Rider D.A., Young S.P., (2003) “Measuring the specific activity of the CD45 protein tyrosine phosphatase.” J. Immunol. Methods 277: 127-134.
• Pastula C., Johnson I., Beechem J.M., Patton W.F., (2003). “Development of fluorescence-based selective assays for serine/threonine and tyrosine phosphatases.” Comb. Chem. High Throughput Screen. 6: 341-346
•   Wang Q., Scheigetz J., Roy B., Ramachandran C., Gresser M.J., (2002).  “Novel caged fluorescein diphosphates as photoactivatable substrates for protein tyrosine phosphatases.”  Biochim. Biophys. Acta 1601(1): 19-28. 
• Waddleton D, Ramachandran C, Wang Q. (2000). “Development of a method for evaluating protein tyrosine phosphatase CD45 inhibitors using jurkat cell membrane.” Anal. Biochem. 285: 58-63.
• Huang Z., Wang Q., Ly H.D., Gorvindarajan A., Scheigetz J., Zamboni R., Desmarais S., Ramachandran C., (1999). “3,6-Fluorescein diphosphate: a sensitive fluorogenic and chromogenic substrate for protein tyrosine phosphatases.”  J. Biomol. Screen. 4(6): 327-334.
• Wang Q., Scheigetz J., Gilbert M., Snider J., Ramachandran C., (1999) “Fluorescein monophosphates as fluorogenic substrates for protein tyrosine phosphatases.” Biochim. Biophys. Acta 1431: 14-23.
• Rotman B., Zderic J.A., Edelstein M., (1963). “Fluorogenic substrates for beta-D-galactosidases and phosphatases derived from fluorescein (3,6-dihydroxyfluoran) and its monomethyl ether.” Proc. Natl. Acad. Sci. USA 50: 1.

Direct Fluorescent Labeling for Determination of Sugars.

m-Dansylaminophenylboronic acid (M0329) is a popular fluorescent labeling reagent for direct labeling of cis-diols (including sugars) when reacted at high pH values.  Labeling can occur on cell surfaces or by simply mixing the carbohydrate compound with the reagent in vitro for total carbohydrate determination in glycoproteins and for HPLC analysis of sugars. The fluorescence emission of the dansyl fluorophore has a very large Stoke's shift (EX 337nm, EM 517nm ) but the emission can be environmentally sensitive and is blue shifted under some circumstances.  M0329 has found utility for labeling and measuring glycoproteins, carbohydrates, some aminosugars or other compounds that contain cis-diol functions, including the active site of some proteins.

The procedure for labeling sugars or glycoproteins involves mixing M0329 with the sugar or protein at an equimolar or with a 2-3X molar excess of the reagent in a high pH buffer (e.g. 0.1 M sodium phosphate buffer pH 9.0, or equivalent).  The dansylaminophenylboronic acid is soluble in this buffer at low concentrations, but if multiple samples are being analyzed, it is suggested to first dissolve the labeling reagent in a water miscible solvent (like DMSO) at a high concentration, and then add a small sample of this reagent solution to the saccharide or analyte.  The product can then be analyzed by purification using common techniques (HPLC, TLC, gel chromatography, etc.) where the fluorescently labeled products will fluoresce at 517 nm when excited at 337 nm.  Note that the fluorescence is somewhat environment sensitive, and the emission wavelength will shift to about 490 nm when it is bound to proteins.  For more information about these assays, please visit our website or see the references below.

• Svatos, A., Antonchick, A., Schneider, B., (2004) “Determination of brassinosteroids in the sub-femtomolar range using dansyl-3-aminophenylboronate derivatization and electrospray mass spectrometry.”    Rapid Commun. Mass Spec. 18(7):  816-821.
• Luis, G.P., Granda, M., Badia, R., Dıaz-Garcıa, M.E., (1998) "Selective Fluorescent Chemosensor for Fructose." Analyst 123: 155.
• Dryjanski M, Pratt RF. (1995) "Steady-state kinetics of the binding of beta-lactams and penicilloates to the second binding site of the Enterobacter cloacae P99 beta-lactamase." Biochemistry 34: 3561-3568.
•   Dubus A, Normark S, Kania M, Page MG. (1995) "Role of asparagine 152 in catalysis of beta-lactam hydrolysis by Escherichia coli AmpC beta-lactamase studied by site-directed mutagenesis." Biochemistry 34: 7757-7764.
•   Watanabe K, Mizuta M. (1995) "Fluorometric detection of glycosphingolipids on thin-layer chromatographic plates." J Lipid Res 36: 1848-55.
• Gamoh K, et al. (1990)  "Determination of Traces of Natural Brassinosteroids as Dansylaminophenylboronates by Liquid Chromatography with Fluorometric Detection." Anal. Chim. Acta 228: 101.
• Pazhanisamy S, Pratt RF. (1989) "Beta-lactamase-catalyzed aminolysis of depsipeptides: peptide inhibition and a new kinetic mechanism." Biochemistry 28: 6875-6882.
• Pazhanisamy S, Pratt RF. (1989)  "Beta-lactamase-catalyzed aminolysis of depsipeptides: proof of the nonexistence of a specific D-phenylalanine/enzyme complex by double-label isotope trapping." Biochemistry 28: 6870-6875.
• O'Connor CJ, Yaghi BM. (1989) "N-(5-Dimethylaminonaphthalene-1-Sulfonyl)-3-Aminobenzene Boronic Acid as an Active-Site-Directed Fluorescent Probe of Bile-Salt-Stimulated Human Milk Lipase." J. Mol. Catalysis 52: 317  .
• Goldberger G, Paz MA, Torrelio BM, Okamoto Y, Gallop PM. (1987) "Effect of hydroxyorganoboranes on synthesis, transport and N-linked glycosylation of plasma proteins." Biochem. Biophys. Res. Commun. 148: 493-499.
• Hayashi Y, Makino M. (1985) "Fluorometric measurement of glycosylated albumin in human serum." Clin. Chim. Acta. 149: 13-19.
•   Vainio P. (1983) "N-(5-dimethylaminonaphthalene-1-sulfonyl)-3-aminobenzene boronic acid as an active-site-directed fluorescent probe of lipoprotein lipase." Biochim. Biophys. Acta 746: 217-219.
• Gallop PM, Paz MA, Henson E. (1982) "Boradeption: a new procedure for transferring water-insoluble agents across cell membranes." Science 217: 166-169.
• Philipp M, Maripuri S. (1981) "Inhibition of subtilisin by substituted arylboronic acids." FEBS Lett. 133: 36-38.
• Burnett TJ, Peebles HC, Hageman JH. (1980) "Synthesis of a fluorescent boronic acid which reversibly binds to cell walls and a diboronic acid which agglutinates erythrocytes." Biochem. Biophys. Res. Commun. 96: 157-162.

Apoptosis Detection in Plant Cells.

In plants, apoptosis (Programmed Cell Death, PCD) is a common response to stress, injury or pathogen attack.  Many dying plant cells at a late stage undergo morphological and biochemical changes similar to those in apoptotic mammalian cells, including chromatin condensation, DNA ladder formations, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL)-positive response and the formation of apoptotic-like bodies.  But analysis of the early events of apoptosis in plants has been difficult.  Recently, the involvement of mitochondria in plant apoptosis has been demonstrated in a number of systems. A mitochondrial oxidative burst appears to be an important regulator of the plant cell death response.  Among the methods used to monitor the mitochondrial membrane function of protoplasts, the probe DiOC6(3) has been used to correlate mitochondrial activity and depolarization in conjunction with the production of NADH by FACS analysis. The ultraviolet (UV)-excited blue autofluorescence of NADH reflects the cellular NADH content, which is lowered in apoptotic cells.  In plant protoplasts, however, both NADH and NADPH may contribute to UV-excited blue autofluorescence. The timing and magnitude of changes of DiOC6(3) fluorescence during cell death are highly correlated with changes in UV-excited blue autofluorescence levels.  Additional cell co-staining with a cell viability probe, like fluorescein di-acetate (FDA, M0060) as well as monitoring changes in nuclear morphology by counterstaining the nuclei with DAPI are also suggested for confirmation of apoptosis.  For more information about these general methods and systems for monitoring apoptosis in plant cells, please visit our website or see the references below.

• Poot, M., Pierce, R.H. (1999) “Detection of changes in mitochondrial function during apoptosis by simultaneous staining with multiple fluorescent dyes and correlated multiparameter flow cytometry.” Cytometry, 35: 311–317.
• Balk, J. and Leaver, C. (2001) “The PET1-CMS mitochondrial mutation in sunflower is associated with premature programmed cell death and cytochrome c release.” Plant Cell 13: 1803–1818.
• Yao N., Eisfelder B.J., Marvin J., Greenberg, J.T.,  “The mitochondrion – an organelle commonly involved in programmed cell death in Arabidopsis thaliana.” The Plant Journal (2004) 40: 596–610.
• Yao, N., Tada, Y., Sakamoto, M., Nakayashiki, H., Park, P., Tosa, Y., Mayama, S. (2002) “Mitochondrial oxidative burst involved in apoptotic response in oats.” Plant J. 30: 567–579.
• Simeonova, E., Garstka, M., Koziol-Lipinska, J., Mostowska, A. (2004) “Monitoring the mitochondrial transmembrane potential with the JC-1 fluorochrome in programmed cell death during mesophyll leaf senescence.” Protoplasma 223: 143–153.
  

Compare Our Quality. 

Marker Gene strives to offer our customers products of the highest quality and at the best possible prices.  Our years of experience allow us to provide timely products for less cost to you.  See our latest Price Comparison Chart that compares our prices with those from several alternate sources, to see if you can save money by switching to Marker Gene (http://www.markergene.com/crossref.htm).  Or visit our website at www.markergene.com and click on the link “COMPARE”.  We think you will appreciate our efforts to keep costs low and maintain excellent quality of our products for your research.  For more information about any of our products, simply telephone us toll free at 1-888-218-4062 or contact us by e-mail at techservice@markergene.com.  We will be happy to send you more about our products and their specifications.

CONTRACT  RESEARCH@markergene.com

Marker Gene Technologies, Inc. has the expertise to perform contract research with you on your project. We have worked with many biotechnology and pharmaceutical companies on successful, proprietary and patented projects.

Contract Research and Development Capabilities in the following areas:

• Established in 1993 at the University of Oregon Riverfront Research Park.
• Screening Assay Development for HTS and uHTS
• Chemical and Cellular Assays – High-Content Screening.
• DNA/RNA (genomics) and protein (proteomics) labeling and assay development.
• Pharmaceutical Intermediates - design, synthesis, and in vitro testing in mammalian cell culture.
• Specializing in Carbohydrate, Lipid, Peptide, and Nucleic Acid Chemistries.
• Fully equipped laboratories (Biochemistry, Chemical Synthesis, Tissue Culture, Analytical).
• Confidentiality, help in patent preparation and filings.

Contact us by telephone at (888) 218-4062 or (541) 342-3760 or FAX us at (541) 342-1960 or you can write to us at  Contract Research, Marker Gene Technologies, Inc., 1850 Millrace Drive, Eugene, Oregon 97403-1992 or contact us by e-mail at: techservice@markergene.com

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