Marker Gene Monthly Newsletter   

March, 2004

Volume 4, Number 3

© 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.

Honing in on SARS Detection and Treatment.

Reports began to emerge from Guangdong Province in China in 2003 of a fatal atypical pneumonia defined as severe acute repiratory syndrome (SARS).   The genetic sequence of the coronavirus, which is now understood to be the cause of SARS, reveals it has some 30,000 base pairs and is a new type of coronavirus (SARS-CoV).  Several PCR-based assays have been developed for SARS detection, as well as viral RNA-level and IgG assays and even tomographic analysis.  Recent identification of the angiotensin converting enzyme receptor (ACE-2) as a critical binding site used by the SARS coronavirus for entry into the host cells has led to a number of new potential therapeutic and detection strategies for the SARS virus.  By utilizing the soluble carboxypeptidase enzyme as well as antibodies for ACE-2, it has been possible to block the virus attachment to the SARS S1 binding domain.   For more information about these techniques see the references below:

• W. Li, M. J. Moore, N. Vasilieva, J. Sui, S. K. Wong, M. A. Berne, M. Somasundaran, J. L. Sullivan, K. Luzuriaga, T. C. Greenough, H. Choe, M. Farzan (2003) “Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus” Nature 426: 450 – 454.
• Marra MA, Jones SJM, Astell CR et al. “The genome sequence of the SARS-associated coronavirus.” (2003) Science, 300(5624): 1399-1404.
• Wu X ., Cheng G., Di B., Yin A., He Y., Wang M., Zhou X., He L., Luo K., Du L.,  “Establishment of a fluorescent polymerase chain reaction method for the detection of the SARS-associated coronavirus and its clinical application.” (2003) Chin Med J 116(7): 988-90.
• Poon L.L., Chan K.H., Wong O.K., Cheung T.K., Ng I., Zheng B., Seto W.H., Yuen K.Y., Guan Y., Peiris J.S.,  “Detection of SARS coronavirus in patients with severe acute respiratory syndrome by conventional and real-time quantitative reverse transcription-PCR assays.” (2004) Clin Chem 50(1): 67-72.
  

The Cah / Nitrile Hydratase Selection Marker.

Several groups are developing new selection systems for recombinant plants that utilize a fungal enzyme to degrade the popular herbicide cyanamide into urea (H₂N-CN + H₂O -> H₂N-CO-NH₂), which the plants can then use as a nitrogen source.  Dr. J. Troy Weeks and his team at the Agricultural Research Service (Lincoln, Nebraska) developed this new selection marker system for use in wheat plants by utilizing the Cah (cyanamide hydratase) gene isolated from the fungus Myrothecium verrucaria.  The cah marker gene enables callus (clumps of plant tissue) grown in petri dishes to convert cyanamide into the nutrient, urea.   Dr. Guido Hartmann and collegues at the Institut fur Biochemie, Ludwig-Maximilians-Universitat, Munich first isolated the enzyme from the fungus and cloned it into E. coli.  For more information about these new marker genes, and their use as selection markers, see the references below:

• Maier-Greiner UH, Obermaier-Skrobranek BM, Estermaier LM, Kammerloher W, Freund C, Wulfing C, Burkert UI, Matern DH, Breuer M, Eulitz M, et al. (1991) “Isolation and properties of a nitrile hydratase from the soil fungus Myrothecium verrucaria that is highly specific for the fertilizer cyanamide and cloning of its gene.” Proc Natl Acad Sci U S A. 88(10): 4260-4.  
• J.T. Weeks, K.Y. Koshiyama, U. Maier-Greiner, T. Schäeffner, O.D. Anderson (2000) “Wheat Transformation Using Cyanamide as a New Selective Agent” Crop Science 40: 1749-1754.  http://crop.scijournals.org/cgi/content/abstract/40/6/1749

TAMRA labeling of DNA, Proteins and Live Cells.

Under the name TAMRA, the carboxylic acid derivative of tetramethylrhodamine (5(6)-Carboxytetramethylrhodamine, M0962) has achieved prominence because of its high extinction coefficient, stability and pH insensitivity, and is routinely used as a dye for oligonucleotide labeling, automated DNA sequencing applications, carbohydrate and protein labeling.  It has even been used for direct labeling of live cells.  Detection of TAMRA can be easily observed via direct fluorescence measurement (EX 540 nm; EM: 565 nm) of the labeled analog using classical methods or can be used with two fluorescent dyes in combination (i.e. fluorescein labeling, EX: 488nm; EM520 nm)), via fluorescence resonance energy transfer (FRET).  The fluorescence of TAMRA conjugates can also be efficiently quenched using the new QSY 7® quencher.  Marker Gene now sells this popular fluorescent labeling reagent as its highly fluorescent mixed isomer (M0962).  For more information about TAMRA and its use for conjugation to proteins, carbohydrates and oligonucleotides, see our web site or the references below.

• Carol A Casey, Cheryl R Baldwin, Jacy L Kubik, Agnes M Hindemith and Benita L McVicker (2004)  “Use of Flow Cytometric Analysis to Examine the Uptake of Apoptotic Bodies by Healthy Hepatocytes” Comparative Hepatology 3 (Suppl 1):S40.  Farinas J,
• Verkman AS. (1999) “Receptor-mediated targeting of fluorescent probes in living cells.” J. Biol Chem. 274(12): 7603-6.
• Oefner P.J., Huber C.G., Umlauft F., Berti G.N., Stimpfl E., Bonn G.K., (1994) "High-resolution liquid chromatography of fluorescent dye-labeled nucleic acids." Anal Biochem 223, 39-46
• Zhao J.Y., Diedrich P., Zhang Y., Hindsgaul O., Dovichi N.J., (1994) "Separation of aminated monosaccharides by capillary zone electrophoresis with laser-induced fluorescence detection." J Chromatogr B Biomed Appl 657, 307-313
• Ravdin P, Axelrod D "Fluorescent tetramethyl rhodamine derivatives of alpha-bungarotoxin: preparation, separation, and characterization.". Anal Biochem 80, 585-592 (1977)

Mutagenicity Testing in Live Plants with GUS.

A transgenic plant–based assay to study the genetic effects of toxins and heavy metals has been developed by Dr. Igor Kovalchuk and co-workers at the Friedrich Miescher Institue in Basel, Switzerland. Arabidopsis thaliana plants carrying a ß-glucuronidase (GUS) marker gene that contains either a point mutation or a recombination site, were used to analyze the frequency of somatic point mutations and homologous recombination in whole plants.  Previous studies have shown that these transgenic lines respond to ionizing and UV-C irradiation and to methyl methanesulfonate treatment with an increased frequency of homologous recombination or prevention of repair and increased numbers of point-mutations, respectively.   Restoration of GUS activity was used to easily measure these events.  This system presents itself as a simple and convenient alternative to other (i.e. Ames test) types of mutagenicity testing, especially in open environment studies.  Marker Gene provides several newer methods of sensitively measuring GUS activity in whole plant or plant extracts, including the ß-Glucuronidase (GUS) Reporter Gene Activity Detection Kit (Product M0877), the Chemiluminescent ß-Glucuronidase (GUS) Detection Kit (Product M0856) as well as several substrates for ß-glucuronidase activity (M0240 and M0256).  For more information about these techniques, see our web site or the references below:

•   O. Kovalchuk, V. Titov, B. Hohn, I. Kovalchuk  (2001) “A sensitive transgenic plant system to detect toxic inorganic compounds in the environment” Nature Biotechnology 19: 568 – 572.
• Kovalchuk, I., Kovalchuk, O., Hohn, B. (2000). “Genome-wide variation of the somatic mutation frequency in transgenic plants.” EMBO J. 19: 4431–4438
• Puchta, H., Swoboda, P., Hohn, B. (1995) “Induction of homologous DNA recombination in whole plants.” Plant J. 7: 203–210.
• Schaaper, R.M. & Dunn, R.L. Spontaneous mutation in the Escherichia coli lacI gene. Genetics 129: 317–326 (1991).
• Lewin, D.E. & Ames, B.N. Classifying mutagens as to their specificity in causing the six possible transitions and transversions: a simple analysis using the Salmonella mutagenicity assay. Environ. Mutagen. 8:  9–28 (1986).
• Rossman, T.G. et al. Performance of 133 compounds in the lambda prophage induction endpoint of the Microscreen assay and a comparison with S. typhimurium mutagenicity and rodent carcinogenicity assays. Mutat. Res. 260: 349–367 (1991).
• Sacco, M.G. et al. A transgenic mouse model for the detection of cellular stress induced by toxic inorganic compounds. Nat. Biotechnol. 15: 1392–1397 (1997).
  
  

Depurination and DNA Strand Breakage.

Depurination of DNA results in the loss of a purine from the DNA backbone and DNA strand breakage.  Such DNA lesions are often the result of ionizing radiation, free radicals, or alkylating reagents that destabilize the N-glycosidic bond.  Left unchecked, they have the potential to cause mutagenesis and carcinogenesis.  Depurination is also a well-known side reaction in DNA synthesis and in isolation of DNA or RNA samples from gels, when the oligonucleotide is subjected to weak acid conditions.  During synthetic  deprotection of trityl protecting groups, depurination can cause chain cleavage producing fragmentation.  Depurination is especially troublesome in oligonucleotides that are either long or A-rich.  Upon release of the base (purine), a reducing sugar (ribose) is left on the backbone.  Analysis of these abasic sites can take advantage of the aldehyde thus produced to quantitate depurination.  These include reactive biotin (ARP) or fluorescein (FARP) labeling compounds which incorporate a carboxymethylhydroxylamine function.  Marker Gene is developing new labeling reagents for depurination detection, with improved sensitivity and stablity.  For more information about these techniques, see the references below. 

• T. Suzuki, S. Ohsumi, K. Makino “Mechanistic studies on depurination and apurinic site chain breakage in oligodeoxyribonucleotides.” (1994) Nucleic Acids Res.  22 (23): 4997–5003.
• Ide H., Akamatsu K., Kimura Y., Michiue K., Makino K.,  Asaeda A.,  Takamori Y., Kubo K., (1993) “Synthesis and damage specificity of a novel probe for the detection of abasic sites in DNA.” Biochemistry 32(32): 8276-83.
• Makrigiorgos G.M., Chakrabarti S., Mahmood A. (1998) “Fluorescent labelling of abasic sites: a novel methodology to detect closely-spaced damage sites in DNA.” Int J Radiat Biol 74(1): 99-109.
  
  

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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