Marker Gene Monthly Newsletter   

May, 2003

Volume 3, Number 5

© 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.

Apoptosis vs. Necrosis by FACS Analysis.

Distinction of the apoptosis and necrosis death pathways, especially by fluorescence-activated cell sorting (FACS) analysis is an ongoing field of investigation.   Apoptosis, also referred to as “programmed cell death,” is accompanied by cell dehydration leading to a change of cell shape and size: i.e. cells become elongated and generally are smaller, accompanied by characteristic “blebbing” of the cell membranes. Chromatin condensation, beginning at the nuclear periphery, is accompanied by loss of the nuclear envelope and is followed by nuclear fragmentation.  Nuclear fragments, together with the constituents of the cytoplasm (including intact organelles), are packaged into “apoptotic bodies,” which are shed from the dying cell.  When apoptosis occurs in vivo apoptotic bodies are phagocytized by neighboring cells, typically without triggering an inflammatory response.  Light scattering analysis by FACS has been used as a measure of cell morphology changes in apoptosis.

Necrosis frequently represents a cell’s response to gross injury and can be induced by application of cytotoxic agents. The early event of necrosis is mitochondrial swelling followed by rupture of the plasma membrane and release of the cytoplasmic constituents, which include proteolytic enzymes.  This process does induce an inflammatory reaction in the tissue and often results in scar formation. DNA degradation is not as extensive during necrosis as in the case of apoptosis, and the products of degradation are heterogenous in size, not forming characteristic discrete bands on electrophoretic DNA gels.  Marker Gene sells several reagents that can be used to help differentiate apoptotic vs. necrotic cells by FACS analysis, including Dihydrorhodamine 123 (DHR123, M0545), which measures intracellular production of reactive oxygen species (ROS), Fluorescein di-acetate and carboxyfluorescein di-acetate (M0060 and M0011) which can be used to measure membrane integrity, as well as a variety of new Caspase enzyme assay kits (M0815-M0838).  Please see our Web site and the references below for more information about these techniques.

• Kalai, M., Loo, G.V., Vanden Berghe, T., Meeus, A., Burm, W., Saelens, X., Vandenabeele, P., (2002) “Tipping the balance between necrosis and apoptosis in human and murine cells treated with interferon and dsRNA.” Cell Death and Diff. 9: 981-994.
• Simon, H.U., Haj-Yehia, A., Levi-Schaffer, F., (2000) “Role of reactive oxygen species (ROS) in apoptosis induction.” Apoptosis 5: 415-418.
• Compton M.M., (1992) “A biochemical hallmark of apoptosis: Internucleosomal degradation of the genome.” Cancer Metast. Rev .11:105-119.
• Wyllie AH: (1992) “Apoptosis and the regulation of cell numbers in normal and neoplastic tissues: An overview.” Cancer Metast. Rev.11: 95-103.
• Oltvai Z.N., Korsmeyer S.J., (1994) “Checkpoints of dueling dimers foil death wishes.” Cell 79:189-192.
  

Promoter Analysis Using Marker Genes.

Gene expression and regulation are mediated by DNA sequences, in most instances, directly upstream of a gene coding sequence, which recruit transcription factors, regulators, and an RNA polymerase in a spatially defined fashion. Few nucleotides within a promoter actually make contact with the bound proteins. The minimal set of nucleotides that can recruit a protein factor is called a cis-acting element.  Methods to identify the promoter region and its functional elements include the use of multiple mutations to modify this region, or by insertion of fragments of the promoter region into a vector containing a marker gene.  The marker gene allows for easy analysis of gene expression, since its expression can be easily monitored by chromogenic or fluorogenic assays.  Among the marker genes used in this context, are bop (purple/orange screen) (Baliga and DasSarma, 1999, 2000; dhfr (trimethoprim resistance) (Danner and Soppa, 1996); cat (chloramphenicol resistance) (Colgan and Manley, 1995);  bgaH b-Galactosidase) (Patenge et al., 2000) GUS (Tucker, et al, 2002) and lacZ (Watanabe, et al., 1998). For more information about these techniques, see the references below:

• Baliga NS, DasSarma S. (1999) “Saturation mutagenesis of the TATA box and upstream activator sequence in the haloarchaeal bop gene promoter.” J Bacteriol. 181(8):2513-2518.
• Baliga NS, Dassarma S. (2000) “Saturation mutagenesis of the haloarchaeal bop gene promoter: identification of DNA supercoiling sensitivity sites and absence of TFB recognition element and UAS enhancer activity.” Mol Microbiol ;36(5):1175-1183.
• Danner S, Soppa J. Characterization of the distal promoter element of halobacteria in vivo using saturation mutagenesis and selection. (1996) Mol. Microbiol. 19(6):1265-1276.
• Colgan J, Manley JL. (1995) “Cooperation between core promoter elements influences transcriptional activity in vivo.” Proc Natl Acad Sci U.S.A. 92(6):1955-1959.
• Patenge N, Haase A, Bolhuis H, Oesterhelt D. (2000) “The gene for a halophilic beta-galactosidase (bgaH)ofHaloferax alicantei as a reporter gene for promoter analyses in Halobacterium salinarum.” Mol Microbiol. 36(1):105-113.
• Tucker, M.L., Whitelaw, C.A., Lyssenko, N.N., Nath, P., (2002) “Functional Analysis of Regulatory Elements in the Gene Promoter for an Abscission-Specific Cellulase from Bean and Isolation, Expression and Binding Affinity of Three TGA-Type Basic Leucine Zipper Transcription Factors.”  Plant Physiol. 130: 1487-1496.
• Watanabe, H., Zoli, M., Changeux, J.P. (1998) “Promoter analysis of the neuronal nicotinic acetylcholine recepter a4 gene: methylation and expression of the transgene.” Eur. J. Neurosci. 10: 2244-2253.

Cathepsin Protease Detection Kits

Cathepsins are ubiquitous lysosomal proteases that are classified according to their active site.  Cathepsins play an important role in the turnover of intracellular proteins and extracellular proteins via endocytosis. Extracellularly they have been implicated in tumor invasion and metastasis and, recently, as a positive mediator of apoptosis induced by gamma-interferon, Fas/APO-1, and TNF-alpha. Cathepsins secreted by invading tumor cells can degrade collagen and elastin, thereby destroying the basal laminar region.  In normal cells, following their synthesis, cathepsins are transported into the lysosomal compartment.  However, in tumor cells, they are secreted into the surrounding medium. The presence of cathepsins in the extracellular matrix may be employed as a method of determining the tumor invasiveness and the clinical outcome of cancer chemotherapy.  Marker Gene now carries a variety of Cathepsin detection kits for Cathepsins B, K and L (M0839, M0841, M0843).  Please see our Web site or the references below for more information.

• Friedrich B.,  Jung K., Lein M., Türk I., Rudolph B., Hampel G., Schnorr D., Loening S.A., (1999) “Cathepsins B, H, L and cysteine protease inhibitors in malignant prostate cell lines, primary cultured prostatic cells and prostatic tissue.” Eur. J. Cancer 35(1): 138-44.
•   Westley B.R., May F.E., (1999) “Prognostic value of cathepsin D in breast cancer.” Br. J. Cancer 79(2): 189-90.
• Krepela E., Procházka J., Kárová B.,  (1999) “Regulation of cathepsin B activity by cysteine and related thiols.” Biol. Chem. 380(5): 541-51.
• Kos J., Nielsen H.J., Krasovec M., Christensen I.J., Cimerman N.,  Stephens R.W., Brünner N., (1998) “Prognostic values of cathepsin B and carcinoembryonic antigen in sera of patients with colorectal cancer.”  Clin. Cancer Res. 4(6): 1511-6.
• Duffy M.J., (1996) “Proteases as prognostic markers in cancer.” Clin. Cancer Res. 2(4): 613-8.
• Deiss L.P., Galinka H., Berissi H., Cohen O., Kimchi A., (1996) “Cathepsin D protease mediates programmed cell death induced by interferon-gamma, Fas/APO-1 and TNF-alpha.” EMBO J. 15(15): 3861-70.
• Cavallo-Medved D., Sloane B.F., (2003) “Cell-surface cathepsin B: understanding its functional significance.” Curr. Top. Dev. Biol. 54:313-41.

Carbohydrate Analysis/Detection Kit

Most native proteins contain post-transcriptional glycosylation patterns whose structures are dependent both on species and cell type. The characterization of the complex oligosaccharides obtained from these glycoproteins has proven a difficult and time-consuming endeavor. Our Carbohydrate Analysis/Detection Kit (M0272) is capable of quickly estimating and/or comparing the composition of the carbohydrates in such samples. The Kit provides reagents and protocols for analyzing these carbohydrates through covalent labeling with a fluorescent reagent (1,5-EDANS).  The principle involves enzymatic removal of the oligosaccharides from a native protein (or mixture of reducing sugars), reductive amination of the reducing sugars and analysis of the resultant glycanamines using two-dimensional PAGE analysis or by other well-established techniques.  The advantages of using the 1,5 EDANS fluorophore include its low detection limit, water solubility, pH fluorescence invariance, stability, distinctive fluorescence from protein chromophores, and ability to be detected using normal phase chromatography techniques.  For more information about carbohydrate analysis, see the references below:

• Jackson, P., (1996) “The analysis of fluorophore-labeled carbohydrates by polyacrylamide gel electrophoresis.” Mol Biotechnol. 5(2): 101-23
• Morimoto K., Maeda N., Abdel-Alim A.A., Toyoshima S., Hayakawa T., (1999) “Structural characterization of recombinant human erythropoietins by fluorophore-assisted carbohydrate electrophoresis.” Biol. Pharm. Bull. 22(1): 5-10.
• Cottaz S., Brasme B., Driguez H., (2000) “A fluorescence-quenched chitopentaose for the study of endo-chitinases and chitobiosidases.” Eur. J. Biochem. 267(17): 5593-600.
  

NPTII Assays in Plants

The neomycin phosphotransferase gene (from E. coli) (NPTII) is one of the most widely used selectable markers for plant transformation.  It is also used in gene expression and regulation studies in different organisms in part because N-terminal fusions can be constructed that retain enzymatic activity. In animal cells, G418 and neomycin are used as selectable agents.  NPTII protein activity can be detected by enzymatic assay. The enzyme NPTII inactivates by phosphorylation a number of aminoglycoside antibiotics such as kanamycin, neomycin, geneticin (or G418) and paromomycin.  In detection methods, the modified substrates (the phosphorylated antibiotics) can be detected by thin-layer chromatography.  Dot-blot analysis or polyacrylamide gel electrophoresis of the enzyme or RNA can also be used for detection. Plants such as maize, cotton, tobacco, Arabidopsis, flax, soybean and many others have been successfully transformed with the NPTII gene. In plants, kanamycin is the most commonly used selective agent, normally in concentrations ranging from 50 to 500 mg/l, effective in inhibiting the growth of untransformed cells. In rice, however, kanamycin seems to interfere with the regeneration of the transformed cells into green plants and paromomycin is often used for selecting NPTII-transformed rice cells. Therefore, the choice of the selective agent is important and based on the plant species to be transformed.  For more information about these assays and techniques, see the references below:

• Henderson, L., Rao, A.G., Howard, J. (1991) “An Immunoaffinity Immobilized Enzyme Assay for Neomycin Phosphotransferase II in Crude Cell Extracts.” Analytical Biochemistry. 194: 64 – 68.
• Y.-J. Eu, M.-H. Lee, H.-S. Chang, T. H. Rhew, H. Y. Lee, C.-H. Lee, (1998) “Chlorophyll fluorescence assay for kanamycin resistance screening in transgenic plants” Plant Cell Reports 17(3): 189-194.
• Jaiwal P. K., Kumari Ragini, Ignacimuthu S., Potrykus I., Sautter C., “Agrobacterium tumefaciens-mediated genetic transformation of mungbean (Vigna radiata L. Wilczek): A recalcitrant grain legume.” (2001) Plant Science 161(2): 239-247.
  
  

Over 100 Products and Kits in our New Catalog!

The 2003-2004 edition of the Marker Gene catalog is now available.  Many new products and kits, additional literature references, data and protocols have been included, as well as new information about our old products.  Don’t miss out on this resource for Marker Gene detection.  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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