Marker Gene Monthly Newsletter   

July, 2004

Volume 4, Number 7

© 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.

New DAAO Positive Selection Marker.

The ability to separate and purify transgenic cells has often involved the use of negative selection systems like amp (detoxifies the antibiotic ampicillin), neo (detoxifies G-418), tet (which detoxifies tetracyclin) or various herbicide resistance genes.   But these negative selection schemes often cause widespread toxicity and affect the growth and viability of all cells in culture.  A much preferable technique involves promoting growth of the transgenic cells over non-transgenic ones.  Several new systems have been developed to promote such positive selection in cell culture and even in vivo.  Recently Dr. Torgny Näsholm and his group at the Umea Plant Science Center, Umea, Sweden have developed a novel marker system that is based on the metabolism of D-amino acids (mirror images of L-amino acids).  Their system allows both positive and negative selection, depending on the amino acids used.  They generated transgenic plants expressing D-amino acid oxidase (DAAO) from the DAO1 gene of the yeast Rhodotorula gracilis. This enzyme catalyzes deamination of several D-amino acids. 

Nontransgenic A. thaliana seedlings were arrested soon after germination in the presence of D-serine or D-alanine, whereas plants expressing DAO1 detoxify these amino acids and grow without developmental abnormalities. In addition, D-valine and D-isoleucine are nontoxic to wild-type plants but kill plantlets with the extra transgenic DAO1 gene.  Thus, the same transgene product can be used to allow both positive and negative selection, depending on the selective agent applied.  For more information about these new techniques and other selection systems, please see our website or the references below:

NOTE: The DAAO Selection Marker Technology (SELDA Technology) is available for license from BASF Plant Sciences GmbH. Please contact Dr. Andreas Ranz, BPS – Li 444, D-67117 Linburgerhof, Germany (e-mail: andreas.renz@basf-ag-de) for more information.

• “A conditional marker gene allowing both positive and negative selection in plants.” Erikson, O., Hertzberg, M. & Näsholm, T. Nat. Biotechnol. 22: 455–458 (2004).
• “Selectable marker genes in transgenic plants: applications, alternatives and biosafety.”  (2004) Miki, B., McHugh, S., J. Biotechnol. 107: 193–232.
• “Active site plasticity in D-amino acid oxidase: a crystallographic analysis.” (1997) Todone F ; Vanoni MA ; Mozzarelli A ; Bolognesi M ; Coda A ; Curti B ; Mattevi A.,  Biochemistry  36(19): 5853-60.
•  “D-amino acid oxidase: new findings.” (2000) Pilone, M.S., Cell. Mol. Life. Sci. 57: 1732–1747.
• “Chromatographic determination of L- and D-amino acids in plants.” (2003) Bruckner, H., Westhauser, T., Amino acids 24: 43–55.
  

Agrobacterium tumefaciens for Plant Transformations.

For several years now the plant parasite Agrobacterium tumefaciens (a bacterium related to the nitrogen-fixing bacteria Rhizobium found on plant roots) has been used to genetically engineer plants. Most agrobacterium have plasmids that they can inject into plant cells at wound sites. This plasmid DNA is designed to integrate into the chromosomes of the infected plant’s cellular DNA. The inserted DNA directs the plant cell to: 1) divide without growth control ("tume"-faciens" or  “tumor promoting”) and 2) make nitrogen-containing compounds such as nopaline and other things that the bacteria can feed upon. In this way the bacterium induces the plant to make it a home for it and also to synthesize its food.  But plant biologists  have taken advantage of this natural genetic engineering property of A. tumefaciens plasmids by 1) removing the tumor-inducing qualities, 2) removing the nopaline synthetase and other "bacterial" genes but they have 3) retained their ability to insert their message into chromosomal DNA.  Many new plasmids have been developed to insert genes of interest into plants.  Because most plant cells are pluripotent (i.e. they can be induced with appropriate phytohormones to regenerate into whole plants), one cell infected with a recombinant Agrobacterium plasmid can be propagated into a functioning recombinant plant that can pass on the acquired gene(s) to its progeny.  For more information about plant transformations using Agrobacterium tumefaciens please see our web site or the references below:

• Binns, A.N. and M.F. Thomashow. (1988) “Cell biology of Agrobacterium infection and transformation of plants”. Ann. Rev. Microbiol. 42: 575-606.
• Lindsey, K. & Gallois, P. “Transformation of sugar beet (Beta vulgaris) by Agrobacterium tumefaciens.” (1990) J. Exp. Bot. 41: 529–536.
• H. Ezura, K. -I. Yuhashi, T. Yasuta, K. Minamisawa (2000) “Effect of ethylene on Agrobacterium tumefaciens-mediated gene transfer to melon.” Plant Breeding 119(1): 75-79.
• Veena, Hongmei Jiang, R.W. Doerge, Stanton B. Gelvin (2003) “Transfer of T-DNA and Vir proteins to plant cells by Agrobacterium tumefaciens induces expression of host genes involved in mediating transformation and suppresses host defense gene expression”  The Plant Journal 35(2): 219-236.

FACS Blue lacZ b-Galactosidase Detection Kit.

One of the most common reporter genes used in molecular biology is the E.Coli lacZ gene that codes for an active subunit of ß-galactosidase in vivo. Since this enzyme is generally absent in normal mammalian, yeast, some bacterial and even plant cells, it can be detected at very low levels, and since the enzyme has a wide substrate specificity, monitoring lacZ expression (and therefore co-expressed genes or promoter efficiency) has become routine to the point of detection of as few as 5 copies of ß-galactosidase per cell by FACS analysis.  Although chromogenic assays of ß-galactosidase activity (i.e. X-Gal) are widely used, application of the fluorogenic substrate 3-carboxyumbelliferyl ß-D-galactopyranoside (CUG) combined with Fluorescence Activated Cell Sorting (FACS) analysis has been shown to be several orders of magnitude more sensitive.  In addition, because of its high water solubility and detection limits, the CUG substrate has found extensive use in automated ELISA type assay systems.  The FACS Blue lacZ b-Galactosidase Detection Kit (M0255) is especially useful when co-staining with another colored dye like a fluorescein-type cell label (FITC-antibody, fluorescein based substrate, etc.) for dual wavelength detection. Many new instruments now have mutli-color FACS detection capabilities (see the BD LSRII system for example) that can utilize this new kit.  Please consult your instrument manufacturer brochure for more information.  The FACS Blue lacZ ß-galactosidase detection kit provides all the reagents and a detailed protocol to perform up to 500 automated (10 x 96 well microtiterplate) assays.  See also information contained in our Products M0257 and M0250 pages and in the references below:

• “Fluorescence-Activated Cell Analysis and Sorting of Viable Mammalian Cells Based on ß -D-galactosidase Activity after Transduction of Escherichia coli lacZ”  (1988) Nolan G.P., Fiering S., Nicolas J.F., Herzenberg L.A.,  Proc. Natl. Acad. Sci. USA 85: 2603-2607.
• “Improved FACS-Gal: flow cytometric analysis and sorting of viable eukaryotic cells expressing reporter gene constructs.” (1991) Fiering SN ; Roederer M ; Nolan GP ; Micklem DR ; Parks DR ; Herzenberg LA Cytometry 12(4): 291-301.
• “FACS-Gal: Flow-Cytometric Analysis and Sorting of Cells Expressing Reporter Gene Constructs.” M. Roederer, S. Fiering, L.A. Herzenberg, Methods: Companion to Meth. Enzymol. 2 (1991) 248.
• “The Homogeneous Substrate-Labeled Fluorescent Immunoassay” Burd, J. F., Methods in Enzymology. 74: (1981) 79-86.

A New M. tuberculosis Marker

The main component of the cell walls of mycobacteria is lipoarabinomannan (LAM), a lipopolysaccharide containing mainly arabinose and mannose.   LAM plays an important role in infection by Mycobacterium tuberculosis, the bacterium that causes tuberculosis, because it helps it to invade macrophages, reduce the immune response, and protect it from oxidation.  Recently researchers at the School of Biochemistry and Molecular Biology at the University of Leeds discovered that some of the mannose end groups on the outside of the molecule carry another type of sugar, a 5-methylthiopentofuranose.   This new sugar has a five carbon structure (xylopentose) with one of its usual five hydroxyls replaced by a sulfur (thio) atom to which is also attached a methyl group. This discovery is interesting because this is the first time that a methylthiosugar has been identified as a component of a polysaccharide.  This thioether may be responsible for the protection from oxidation provided by LAM.  In addition, the xylo- configuration is unusual, since sugars with a xylo-configuration are usually found only in plants, not in bacteria.  The presence of this new sugar in LAM may help in new detection and diagnosis methods for the bacteria and may also be a good point of attack for new tuberculosis therapies that would target this new sugar’s biosynthesis.  For more information about this new detection system, visit our website or see the references below.

• “5-Methylthiopentose: a new substituent on lipoarabinomannan in Mycobacterium tuberculosis.” (2002) Treumann A., Xidong F., McDonnell L., Derrick P.J., Ashcroft A.E., Chatterjee D., Homans S.W.,  J. Mol. Biol. 316(1): 89-100.
• “Lipomannan and Lipoarabinomannan from a Clinical Isolate of Mycobacterium kansasii” (2003) Y. Guérardel, E. Maes, V. Briken, F. Chirat, Y. Leroy, C. Locht, G. Strecker, L. Kremer J. Biol. Chem. 278(38): 36637-36651.
  
  

Lysophosphatidic Acid (LPA) as an early diagnostic tool for ovarian cancer.

Ovarian cancer is usually diagnosed when the cancer has already progressed to Stage 3 or 4. Recently, Rebecca Sutphen, MD, of the H. Lee Moffitt Cancer Center and Research Institute, Tampa, Fla., in conjunction with Yan Xu, PhD, Department of Cancer Biology, Lerner Research Institute, Cleveland, Ohio, and the University of South Florida Health Sciences Center, have identified a potential ovarian cancer biomarker that may help with early detection. Their studies evaluated the level of Lysophosphatidic Acid (LPA) in ovarian cancer patients versus the level of CA-125, a common biomarker for ovarian cancer.  Almost all healthy women were found to have low levels of lysophospholipids, but most ovarian cancer patients were found to have high levels of these substances.  In 93% of the cases, the increased lysophospholipid levels were an accurate predictor of ovarian cancer and the "false positive" rate was lower than 4%.  The LPA levels were studied using chromatography/mass spectroscopy.  Lysophosphatidic acid is produced from phosphatidyl choline by the action of two phospholipase enzymes.  For more information about these new early detection markers, see our website or the references below:

• Sutphen R., Xu Y., Wilbanks G.D., Fiorica J., Grendys E.C. Jr, LaPolla J.P., Arango H., Hoffman M.S., Martino M., Wakeley K., Griffin D., Blanco R.W., Cantor A.B., Xiao Y.J., Krischer J.P., "Lysophospholipids Are Potential Biomarkers of Ovarian Cancer." (2004) Cancer Epidemiol Biomarkers Prev. 13(7): 1185-91.
• Xu Y, Shen Z, Wiper DW, Wu M, Morton RE, Elson P, Kennedy AW, Belinson J, Markman M, Casey G. (1998) “Lysophosphatidic acid as a potential biomarker for ovarian and other gynecologic cancers.” JAMA. 280(8): 719-23.
• Shen Z., Wu M., Elson P., Kennedy A.W., Belinson J., Casey G., Xu Y., (2001) “Fatty acid composition of lysophosphatidic acid and lysophosphatidylinositol in plasma from patients with ovarian cancer and other gynecological diseases.”  Gynecol Oncol. 83(1): 25-30.
  
  

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.

Sign up now!

 

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. 

To add your name to our WebNewsletter mailing list, please use the following link: subscribe@markergene.com.

To unsubscribe from our mailing list, please use the following link: unsubscribe@markergene.com. Your e-mail address will be deleted in 2-3 business days.