Marker Gene Monthly Newsletter   

June, 2004

Volume 4, Number 6

© 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.

Monitoring mRNA in Living Cells.

Recent developments in imaging analysis have made it possible to monitor the movements of individual mRNA molecules in the cytoplasm of living cells. These techniques take advantage of several reporter genes (GFP’s and lacZ) tagged to RNA binding proteins (e.g. MS2) as well as several fluorescent probes (CY3, CY5, RPF (red fluorescent protein), YFP (yellow fluorescent protein) or CFP (cyan fluorescent protein) bound to complementary proteins (e.g. the lac-repressor). The system can elegantly monitor movement of the mRNA-protein complexes (mRNP’s) from transcriptional induction, nuclear transport and movement along microtubules or in the cytoplasm. FRAP (fluorescence recovery after photobleaching) was used for time-lapse monitoring of mRNP movements inside the cell. Movies of mRNA trafficking are available on the web (see references below). Circulation of mRNP’s present in the vicinity of the transcription site can also be monitored by use of a photoactivatable form of green fluorescent protein (GFP) fused to the MS2 protein in a "visible pulse-chase" experiment. A short pulse of activating light (405 nm) confined only to the transcription site fluorescently labels RNP’s being actively transcribed or just released. The fluorescent mRNAs, upon release from the transcriptional machinery, were found to diffuse away from the transcription site in all directions. For more information about these exciting new techniques, see the references below:

• “Dynamics of Single mRNPs in Nuclei of Living Cells” (2004) Y. Shav-Tal, X. Darzacq, S. M. Shenoy, D. Fusco, S. M. Janicki, D. L. Spector, R. H. Singer, Science 18: 1797-1800.
• Single mRNA molecules demonstrate probabilistic movement in living mammalian cells.” (2003) Fusco D, N. Accornero, B, Lavoie, S.M. Shenoy, J.M. Blanchard, R.H. Singer, E. Bertrand, Curr. Biol. 13(2):161-7.
• Hirschberg, K., Miller, C.M, Presley, J.F., Ellenberg, J., Zaal, K., Cole, N.B., Siggia, E., Phair, and Lippincott-Schwartz, J. (1998) Kinetic and morphological analysis of secretory protein traffic in living cells. J. Cell Biol. 143: 1485-1503.
• Patterson, G.H., Lippincott-Schwartz, J., (2002) A Photoactivatable GFP for Selective Photolabeling of Proteins and Cells. Science 297(5588): 1873-1876.
• http://www.current-biology.com/cgi/content/full/13/2/161/DC1/
  

Mammalian Two-Hybrid Systems.

Two-hybrid systems are extremely powerful methods for detecting protein:protein interactions in vivo. The most common two-hybrid systems utilize the GAL4 binding domain.  Transcription factors, such as GAL4, consist of two discrete modular domains: a DNA- binding domain (BD) and the activation domain (AD). In GAL4 Two-Hybrid Systems, one gene is expressed as a fusion to BD, while another gene (or cDNA library) is expressed as a fusion to AD. When the fusion proteins interact, the BD and AD are brought into close proximity, thus reconstituting GAL4 and activating transcription of a reporter gene (typically lacZ b-galactosidase or luciferase). Function of these marker genes can be easily detected using FDG (Product M0250) or luciferin (Product M0237). An even simpler assay uses intracistronic complementation of two subunits of lacZ forming a functional b-galactosidase enzyme. Restoration of LacZ activity can occur only when the two fragments of the enzyme are expressed as chimeras with the interacting proteins, as the lacZ fragments alone have too low an affinity for each other to form a stable complex otherwise. For more information about these exciting new assays and their use in screening of protein libraries, please see our website or the references below:

• Antiviral drug discovery strategy using combinatorial libraries of structurally constrained peptides.” (2004) Real E ; Rain J.C., Battaglia V., Jallet C., Perrin P., Tordo N., Chrisment P., D'Alayer J., Legrain P., Jacob Y., J. Virol.  78(14): 7410-7.
• "A novel genetic system to detect protein-protein interactions." (1989) S. Fields, O.-K. Song, Nature, 340: 254-6.
• "Genome-wide protein interaction maps using two-hybrid systems." (2000) P. Legrain, L. Selig, FEBS Letters, 480: 32-6.

Transfection Efficiency using neo.

Many vector constructs contain the neo gene from Tn5 that encodes an aminoglycoside 3’-phosphotransferase, 3’ APH II.  This gene confers resistance to the antibiotic G418, an aminoglycoside used as a selective agent of transfected mammalian, yeast, plant and bacteria cells.  G418 is related to Gentamycin, Kanamycin and Neomycin. The neo gene can also be used as another method of measuring transfection efficiency. Several PCR and other assays are available for measurement of neo levels in cells, providing an alternate method of measuring transfection efficiency. NPTII protein activity can also be detected by enzymatic assay as the modified substrates -the phosphorylated antibiotics- are detected by thin-layer chromatography, dot-blot analysis or polyacrylamide gel electrophoresis. Although these assays may be time-consuming, data can be correlated with the data for use in monitoring complementation assays.  For more information about these assays, see our web site or the references below:

• Platt SG, Yang NS., "Dot assay for neomycin phosphotransferase activity in crude cell extracts."  Anal Biochem. (1987) 162(2):529-35.
• “New gentamicin-resistance and lacZ promoter-probe cassettes suitable for insertion mutagenesis and generation of transcriptional fusions.” (1995) Becker A., Schmidt M., Jager W., Puhler A. Gene. 162(1): 37-9.
• “An Immunoaffinity Immobilized Enzyme Assay for Neomycin Phosphotransferase II in Crude Cell Extracts” (1991) Henderson, L., Rao, A.G., Howard, J., Analytical Biochemistry. 194: 64 – 68.
• Sambrook, J., Fritsh, E. F. and Maniatis, T., Molecular Cloning, Cold Spring Harbour Laboratory Press, New York, 1989, 2nd ed.

New Plant Live:Dead Assays.

Marker Gene is working with scientists at several biotechnology companies to develop a simple, inexpensive assay system for monitoring plant cell viability by fluorescent staining.  The new kit will be similar to our existing Live:Dead Assay Kit  (Product M0795) which contains the esterase substrate 5(6)-carboxyfluorescein diacetate (Product M0011) and the membrane impermeant DNA stain, propidium iodide (Product M0793).   Both of these dyes can be excited at the same wavelength (485 nm) for easy measurement or microscopic analysis (propidium iodide EX 535nm/EM 617 nm e=~60K (when bound to dsDNA).  The ability to extend these assays for use in FACS analysis, high-throughput assay systems and cytotoxicity/viability assays is under investigation.   Please visit our web site or see the references below for more information about this assay system.

• “Preparation of single cells from aggregated Taxus suspension cultures for population analysis.” (2004) Naill M.C.,  Roberts S.C.,  Biotechnol. Bioeng.  86(7): 817-26.
• “Two-dimensional fluorescence spectroscopy - a new tool for the determination of plant cell viability.” Vankova, R., Gaudinova, A., Vanek, T., Sussenbekova, H.,  Kuncova, G., Opatrna, J., Plant Cell Reports 20(1): 41-47.
• “Aluminum-induced cell death in root-tip cells of barley.”, Pan, J.W., (2001) Environmental and experimental botany, 46 (1): 71-79.
• “Brief exposure to low-pH stress causes irreversible damage to the growing root in Arabidopsis thaliana: pectin-Ca interaction may play an important role in proton rhizotoxicity.”, (2001) Koyama, H., Journal of Experimental Botany, 52 (355): 361-368.
  
  

Cell Counts with CFDA-NHS.

CFDA-SE (Carboxyfluorescein Diacetate Succinimidyl ester) (Product M0013) is a non-toxic, lipophilic dye that irreversibly labels cell membranes. When labeled cells are allowed to grow, each daughter cell will contain half as much dye as the parent and will be half as bright. After each cell division the level of fluorescence will halve. Measurement of the green fluorescence can therefore be used to monitor the number of cell divisions. Data analysis of up to 6 generations of cells, using flow cytometry, have been accomplished.  A computer integration program can be used to determine how many cells belong to each generation.  Similar cell assays can be performed using our new biotin labeling reagents, like biotin-X, succinimidyl ester (M0783) when combined with a fluorescently labeled avidin or streptavidin secondary detection molecule.  For more information about these techniques, see our web site or the references below:

• "Comparison of proliferation and rapid cytokine induction assays for flow cytometric T-cell epitope mapping." (2003) Tesfa L., Volk H.D., Kern F. Cytometry 52A: 36-45.
• “Imatinib mesylate (STI571) inhibits growth of primitive malignant progenitors in chronic myelogenous leukemia through reversal of abnormally increased proliferation” (2002) M. S. Holtz, M.L. Slovak, F. Zhang, C. L. Sawyers, S. J. Forman, and R.I. Bhatia,  Blood  99(10): 3792-3800.
• "Blockage of voltage-gated K+ channels inhibits adhesion and proliferation of hepatocarcinoma cells." (2003) Zhou Q., Kwan H.Y., Chan H.C., Jiang J.L., Tam S.C., Yao X., Int. J. Mol. Med. 11: 261-6.
  
  

2004-2005 Catalog Will Be Available Soon.

The 2004-2005 edition of the Marker Gene catalog is in production.  Many new products and kits, additional literature references, data and protocols will be included, as well as new information about our old products.  Be sure to add your name to our mailing list.  Please visit our Web site and fill out our Customer Information Form, or e-mail us at techservice@markergene.com and we will have a copy sent out to you.
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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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