Marker Gene Monthly Newsletter   

September, 2005

Volume 5, Number 9

© 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.

Live Plant GUS Assay Systems.

The most useful marker genes code for an enzyme that is not found in the organism being studied.  The E. coli ß-glucuronidase gene (GUS) represents one of the most popular such genes, and it has been used to measure gene expression levels in bacteria, animals, plants and, more recently, in yeasts and filamentous fungi. This protein has a molecular weight of 68,200 and appears to function as a tetramer.  It is very stable, and will tolerate many detergents, widely varying ionic conditions, and general abuse, but when isolated is most active in the presence of thiol reducing agents such as ß mercaptoethanol or DTT.  It can be assayed at any physiological pH, with an optimum between 5.2 and 8.0. The GUS gene is usually incorporated into a gene fusion vector in combination with either a promoter or gene of interest.  

Sensitive measurement of GUS activity in live tissues is possible using the fluorescent substrate 4-methylumbelliferyl b-D-glucuronide (MUG) (M0240).   Non-destructive GUS assay protocols using MUG have been described for staining cultured plant tissues and calli as well as seedlings grown in culture.  Basically these involve spraying or staining in media containing 0.5-2mM MUG, incubating 1-6 hours at 30-37^oC, transferring to a fresh medium (no MUG) adding 0.3 M Na₂CO₃ to the tissues or plants (developer), and evaluating the fluorescent staining patterns after about 20 min., either using a UV light source or fluorescence microscopy.  The latter developer treatment is not necessary when spraying plants with a substrate solution.  These protocols are incorporated into our b-Glucuronidase (GUS) Reporter Gene Activity Detection Kit (M0877).   Although the kit is primarily designed for staining tissue homogenates or lysed samples, addition of Triton X-100 (0.5%) (a non-ionic detergent), polyvinylpolypyrolidone with b-mercaptoethanol or metabisulphite or vacuum treament of live tissues while bathing in or spraying with a substrate solution, has been known to improve permeation of live plant tissues.  For more information about non-destructive fluorescent GUS assay systems, see the references below or visit our website.

• Martin, T., Wohner, R.V., Hummel, S., Willmitzer, L., Fromer, W.B., "The GUS Reporter System as a Tool to Study Plant Gene Expression" in "Gallagher, S.R., ed., "GUS Protocols: Using the GUS Gene as a Reporter of Gene Expression." (1992) Academic Press, San Diego, CA pp. 23-43.
• Gould, J.H., Smith, R.H., (1989) A Non-destructive assay for GUS in the media of Plant Tissue Cultures" Plant Mol. Biol. Rep. 7: 209-216. 
• Lorincz M, Roederer M, Diwu Z, Herzenberg L.A., Nolan G.P., "Enzyme-generated intracellular fluorescence for single-cell reporter gene analysis utilizing Escherichia coli beta-glucuronidase." Cytometry 24, 321-329 (1996).
• Côté C.,  Rutledge, R. G. (2003) "An improved MUG fluorescent assay for the determination of GUS activity within transgenic tissue of woody plants" Plant Cell Rep 21: 619 .624.
• Li, W., Yourman, L. F., Leong, S. A., Spear, R. N., Andrews J. H. (2003) “Assay of ß-glucuronidase activity in intact transformed Aureobasidium pullulans spores” Fungal Genetics Newsletter 44: 29-32.
• http://www.umanitoba.ca/afs/plant_science/COURSES/39-768/l04/l04.2.html
  

Ultrasensitive Homogeneous Immunoassays using D-Luciferin-6-O-Galactopyanoside (Luc-Gal).

b-Galactosidase is widely used as a marker gene for measuring gene expression levels, as well as for protein-protein interaction studies based on the yeast two-hybrid enzyme complementation assays.  In the latter assays, the bacterial enzyme b-galactosidase is genetically engineered into two inactive fragments; a donor and an acceptor. When mixed together, the two fragments combine to form active enzyme, in a process termed “complementation”.  Sensitive measurement of protein interactions can be made by attaching two different proteins to these two fragments, usually as fusion proteins with the donor (ED) and acceptor (EA) fragments produced through cloning into appropriate vector systems.  Recent work from the laboratory of Dr. Joe Andrade and co-workers at the University of Utah, Department of Bioengineering and Dr. Rueyming Loor at Microgenics Corp. (Freemont, CA) have further refined these assays for high-throughput chemiluminescent immunoassay.  By using an analyte specific antibody, and binding an analyte to the ED fragment, measurement of analyte concentration in a sample can be made in a competition assay for binding to a limited number of antibody sites.  It is important that such CEDIA^® (cloned enzyme donor immunoassay) analyses have the ED conjugated to the analyte without affecting the ability of ED to bind to EA and of the functional enzyme.  When an antibody is used, that is directed against the analyte, it prevents the ED-analyte conjugate from interacting with the EA to form a functional enzyme. But any analyte present in the sample competes with the ED-analyte conjugate for antibody binding sites.  Thus, the β-galactosidase enzyme activity is directly proportional to the amount of analyte in the sample tested.  Dr. Andrade found that the most sensitive detection substrate is the new chemiluminescent compound, D-Luciferin-6-O-b-D-Galactopyanoside (Luc-Gal) (also called Beta-Glo^®).  Using a coupled enzyme reaction, where added firefly luciferase is used to develop the light signal, picomolar concentrations of analyte can be measured, without washing steps, in a microplate assay format.  Marker Gene will now be providing this high-purity, ultrasensitive substrate and will be available to help with development of your assay methods.  For more information about these assays and the new Luc-Gal substrate, please see the references below, visit our website, or contact us for more information.

• Henderson, D.R. Friedman, S.B. Harris, J.D. Manning, W.B. Zoccoli, M.A., (1986)”CEDIA, a new homogeneous immunoassay system” Clin.Chem.32: 1637 –1641.
• Yang,Y., Janatova, J., Andrade, J.D., (2005) “Homogeneous enzyme immunoassay modified for application to luminescence-based biosensors” Anal. Biochem. 33: 102 –107.
• Khanna, P.L. Dworschack R.T., Manning W.B., Harris J.D.,  (1989) “A new homogeneous enzyme immunoassay using recombinant enzyme fragments.” Clin. Chim. Acta. 15: 231–9.
• Geiger R.,  Schneider E.,  Wallenfels K.,  Miska W., (1992)  “A new ultrasensitive bioluminogenic enzyme substrate for beta-galactosidase”     Biol. Chem. Hoppe-Seyler  373(12): 1187-91.
• Ugarova, N. N.; Voznyi, Ya. V.; Kutuzova, G. D.; Dement'eva, E. I.   (1991) “Bioluminescent assay of b-galactosidase using D-luciferin-o-b-galactoside.” Biolumin. Chemilumin. Proc. Int. Symp., 6th  (1991),  Meeting Date 1990,   pp. 511-14.  Publisher: Wiley Intersceince, Chichester, UK  Editor(s): Stanley, Philip E.; Kricka, Larry J.  
• Beta-Glo^® is a registered trademark of Promega, Corporation (Madison, WI). 

TMA-DPH, a Fluorescent Probe of Membrane Interiors.

1,6-Diphenyl-1,3,5-hexatriene (DPH) is a common fluorescent reagent used to monitor cell membrane interiors.  Time-resolved fluorescence anisotropy and polarized fluorescence measurements of oriented samples have shown DPH to be mainly oriented parallel to the lipid bilayer axis, but it can also reside in the center of the lipid bilayer parallel to the surface.  In order to improve the localization of DPH in the membrane, the polar derivative TMA-DPH (M1091) and DMA-DPH (M1088) have been developed that contains a cationic trimethylammonium substituent which provides a membrane surface anchor. 

DPH and its derivatives, TMA-DPH (N,N,N-Trimethyl-4-(6-phenyl-1,3,5-hexatrien-1-yl)phenylammonium p-toluenesulfonate, M1091) and DMA-DPH (1-[4-(Dimethylamino)phenyl]-6-phenylhexatriene, M1088)  are cylinder-shaped and have fluorescence emission transition dipoles that are basically aligned parallel to their long molecular axis.  As such, they are very sensitive to reorientation resulting from interactions with surrounding lipids.  For this reason, they are used in fluorescence polarization studies of rotational motion and for membrane fluidity measurements. Interestingly, DPH and it’s derivatives are mainly sensitive to only the angular reorientation of lipid acyl chains which is a motion that does not necessarily correlate with other dynamic processes such as lateral diffusion in membranes.  They have also found interesting uses in measurement of rotational motion in polymers and nanoparticles.  For more information about these exciting new membrane probes, please see our website or the references below. 

• Kaiser R.D., London E., (1998) "Location of diphenylhexatriene (DPH) and its derivatives within membranes: comparison of different fluorescence quenching analyses of membrane depth." Biochemistry 37: 8180-8190
• Prendergast, F.G., Haugland R.P., Callahan P.J., (1981)"1-[4-(Trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene: synthesis, fluorescence properties, and use as a fluorescence probe of lipid bilayers." Biochemistry 20: 7333-7338.
• "Multidrug resistance in Staphylococcus aureus due to overexpression of a novel multidrug and toxin extrusion(MATE) transport protein." Kaatz, G.W., McAleese F., Seo, S.M., Antimicrobial Agents and Chemotherapy 49(5): 1857-1864 (2005).
• "Incorporation of fluorescent probes into PAMAM dendrimers." Domanski D.M., Klajnert B., Bryszewska M., Bioelectrochemistry 63(1-2): 193-197 (2004).
• "Lipid phase separation correlates with activation in platelets during chilling." Tsvetkova N.M., Mol Membr Biol 17: 209-18 (2000).
• "Reversal of multidrug resistance phenotype by surfactants: relationship to membrane lipid fluidity." Dudeja P.K., Anderson K.M., Harris J.S., Buckingham L., Coon J.S., Arch Biochem Biophys 319: 309-15 (1995).
• "Internalization of the lipophilic fluorescent probe trimethylamino-diphenylhexatriene follows the endocytosis and recycling of the plasma membrane in cells." Biochim Biophys Acta 1030: 73-81 (1990).

Marker Gene Adds Helix Research Fluorescent Reagents.

Marker Gene now offer products from Helix Research Laboratories.  Helix Research currently produces over 200 High-Quality Research Products that include a wide base of fluorescent reagents and reactive dyes, membrane labeling reagents, aminodextrans and dextran conjugates and a variety of cell biology reagents.  Marker Gene will be adding these reagents and will also incorporate them into a variety of new kits and allied detection systems for easy use.  Check our website for updates and more information.

Plant “Florigen” factor identified.

In a follow-up to a previous NewsLetter article on Plant Flowering Mechanisms (Vol. 5(8), 2005), researchers at the Umea Plant Science Centre in Sweden and the Universite Joseph Fourier in France, believe they have identified the elusive “florigen” signaling compound that sends the signals from the leaves to the shoot apex to induce flowering in plants.  The messenger RNA for the flowering locus T (FT) gene appears to be this trigger for initiation of flowering.  The group constructed transgenic plants expressing the FT gene and the marker gene GUS under control of a heat-shock inducible promoter (Hsp) from soybean as well as the FT promoter (pFT).  When microdissected plant tissues, whole plants or individual leaves were subjected to heating, gene expression levels could be monitored throughout the plants.  In this way they were able to follow expression levels of FT in various tissues and confirm movement of the FT-induced signal from leaf to apex tissues.  For more information about these elegant techniques for monitoring gene expression in vivo please see our web site or the references below.

• Huang, T., Bohlenius, H., Eriksson, S., Parcy, F., Nilsson, O., (2005) “The mRNA of the Arabidopsis Gene FT Moves front Leaf to Shoot Apex and Induces Flowering” Science 309(5741):

• Abe, M. Kobayashi, Y., Yamamoto, S., Daimon, Y., Yamaguchi, A.,  Ikeda, Y., Ichinoki, H., Notaguchi, M., Goto, K., Araki^, T.., (2005) ^“FD, a bZIP Protein Mediating Signals from the Floral Pathway Integrator FT at the Shoot Apex” Science, 309(5737): 1052-1056.
• Wigge, P.A., Kim, M.C., Jaeger, K.E., Busch, W., Markus Schmid, M., Lohmann, J.U., Weigel, D.,  (2005) “Integration of Spatial and Temporal Information During Floral Induction in Arabidopsis” Science 309(5737): 1056-1059.
• Hoffmann-Benning, S., Zeevaart, J.A.D. “Searching for Florigen” Plant Physiology Online 24:2 (2003) http://www.plantphys.net/article.php?ch=e&id=288
• Ayre, B.G., “Florigen and a Genetic Approach to Long-Distance Signaling Through the Phloem” Plant Physiology Online 24:3 (2005) http://www.plantphys.net/article.php?ch=e&id=291
• Miguel A. Blázquez (2005) “The Right Time and Place for Making Flowers” Science 309 (5737): 1024-1025.
  
  

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

Marker Gene Accepts Major Credit Cards.

Place your orders now, using Master Card or Visa and save time and money!  Our Customer Assistance Staff can now accept either Master Card or Visa Credit Card orders, securely by telephone (toll-free) at 1-888-218-4062 (Domestic orders only).   We will continue to accept Institutional Purchase Orders for our products, online or by FAX at 1-541-342-1960.  International customers should contact us by e-mail, post or telephone for more information about International Distributors and ordering.  For information on pricing for individual products, or for a quote on bulk quantities of our products or kits, please contact our technical assistance staff at techservice@markergene.com.   We will be happy to assist you. 

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