Marker Gene Monthly Newsletter   

August, 2003

Volume 3, Number 8

© 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.

Intron splicing of GUS used to track Agrobacterium plant transformation.

Agrobacterium tumefaciens is a commonly used vehicle for transforming dicot plants. The underlying mechanisms of transformation however are still not very well understood.  Researchers at the Institute for Gene Biology in Berlin recently used a GUS gene containing a portable intron (IV2 from the ST-LS1 gene) that could only be removed by eukaryotic splicing apparatus, not present in the agrobacterium (a prokaryote).  Upon transformation, the levels of GUS activity were monitored and indicated splicing from gene transfer and utilization throughout the whole cotyledon within 36 hours.  These data indicated efficient gene transfer and splicing in Arabidopsis with a GUS containing vector system.   pIG121 or pIG221 or pCAMBIA 1201, 1301, 2301 series vectors can be used to obtain the intron GUS.  It has also been reported that introns can enhance of gene expression. (see our WebNewsletter Jan. 2003). Methods used to detect GUS activity in plants and plant extracts include 4-Methylumbelliferyl-glucuronide (M0240), Carboxyumbelliferyl-glucuronide (M0256), and our new b-Glucuronidase (GUS) Reporter Gene Activity Detection Kit (M0877) and Chemiluminescent GUS b-Glucuronidase Detection Kit (M0856).  See the references below for more information.

• Vancanneyt, G., Schmidt, R., O'Connor-Sanchez, A., Willmitzer, L., Rocha-Sosa, M., “Construction of an intron-containing marker gene: splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation.”  Mol. Gen. Genet. (1990) 220(2):245-50.
  

LacZ Two-Hybrid System for Tracking Protein Interactions Inside Living Cells.

Two hybrid systems provide a simple and sensitive means of detecting the interaction between two proteins or peptides inside living cells. Their use has resulted in the isolation of many new proteins, and has facilitated the identification of important targets for pharmaceutical intervention of diseases and aided in the development of new drugs.  A recent extrapolation of this technique utilizes two lacZ ß-Gal mutants (Da and Dw) which are complementation fragments that, when interacting, provide a functional ß-galactosidase enzyme that can easily be analyzed within living cells using standard fluorescence techniques, i.e. FDG (fluorescein di-galactoside, M0250) or Res-Gal (Resorufin-Galactoside, M0203) staining and FACS analysis.  The method involves fusing the reading frames for two proteins to these sections of lacZ ß-galactosidase that, upon complementation (binding) provide a functional enzyme that can turn over the fluorogenic substrates.  Random cDNA sections can be used for coding one of the protein sequences, providing a genomics approach to protein identification.  For more information on these techniques, see our Web site or the references below:

• D. Thormeyer O. Ammerpohl O. Larsson Y. Xu A. Asinger C. Wahlestedt Z. Liang “Characterization of lacZ ComplementationDeletions Using Membrane Receptor Dimerization”, BioTechniques (2003) 34:346-355
• Rossi, F., C.A. Charlton, and H.M. Blau., “Monitoring protein-protein interactions in intact eukaryotic cells by β -galactosidaseComplementation”, (1997) Proc. Natl. Acad. Sci. USA 94: 8405-8410.
• Mohler, W.A. and H.M. Blau. “Gene expression and cell fusion analyzed by lacZ complementation in mammalian cells. (1996) Proc.Natl. Acad. Sci. USA 93:12423-12427.

Neutrophil Function and Chronic granulomatous disease Assays.

Neutrophils make up the first line of defense of the human body against infections. Neutrophils engulf invading bacteria in a process called "phagocytosis." The primary means by which they destroy the bacteria is the production of hydrogen peroxide and toxic oxygen radicals; this production is called "oxidative burst." Acquired defects in phagocytosis or oxidative burst can allow localized or generalized infections to develop. Such defects can be caused by toxins, drugs (including adrenocorticosteroids), and/or radiation.  Chronic granulomatous disease (CGD) is an inherited disorder with both X- linked and autosomal recessive forms. Clinical manifestations usually appear in very early childhood, but may not present themselves until later in life, especially with the autosomal recessive form. Patients with this disorder are susceptible to infections with organisms that may be non-pathogenic in the normal host. Early diagnosis of this disorder together with aggressive therapy, including the use of interferon gamma prophylaxis, has improved the prognosis of these patients. The X-linked carrier of CGD has been described and the female carriers usually remain clinically asymptomatic, although a proportion of their cells do not undergo the respiratory burst that can be detected in the neutrophil respiratory burst assay.

Dihydrorhodamine 123 (DHR) (M0545) is a dye that oxidizes to form the brightly fluorescent compound rhodamine 123. Neutrophils can oxidize DHR following stimulation by a phorbol ester subsequent to the metabolic burst generated through the hexose monophosphate (HMP) shunt. The precise mechanism of DHR oxidation is not clearly defined, but the phenomenon is closely allied to a series of metabolic events in the respiratory burst following phagocytosis which include increased HMP activity, increased oxygen consumption, and increased superoxide, hydroxyl radical, and hydrogen peroxide formation.

The measure of DHR oxidation is a useful assay in the diagnosis of CGD, and is also a useful means to determine overall metabolic integrity of phagocytising neutrophils. The assay is performed as a flow cytometric method using lysed whole blood, DHR dye, catalase, and the stimulant phorbol 12-myristate 13-acetate (PMA). Granulocytes are isolated based on flow cytometric parameters. Forward light scatter and side-angle light scatter denote relative size and granularity, respectively. If the granulocytes are functionally intact, the DHR will be oxidized to rhodamine 123 and the red (585 nm) fluorescence can be measured by flow cytometry. If the patient has X-linked CGD, the cells are unable to oxidize the DHR dye, and no fluorescence is measured relative to the negative control. Unique profiles of DHR fluorescence also characterize autosomal recessive CGD and the X-linked carrier status.

Validation studies comparing the neutrophil oxidative burst assay and the nitroblue tetrazolium (NBT) dye reduction assay showed 100% specificity and 100% sensitivity (n=46). Additionally, flow cytometry allows greater accuracy when defining the X-linked CGD carrier status, which is complicated by the presence of both normal and abnormal cells. It is also capable of detecting the characteristic pattern associated with autosomal recessive CGD. Furthermore, flow cytometry allows a larger number of cells (10,000) to be counted and eliminates the need for subjective differentiation of cell type in the microscopic NBT assay.

A substantial increase in dihydrorhodamine fluorescence, relative to baseline, is associated with normal NADPH oxidase complex function and is an essential tool in the diagnosis of chronic granulomatous disease (CGD).

Using flow cytometric fluorescence histograms of patient and control samples, stimulation indices are determined by the ratio of the mean fluorescence intensity of the PMA stimulated cells to the mean fluorescence intensity of the HBSS stimulated cells (negative control for sample). The stimulation indices of the patient sample and client submitted control sample are reported with an accompanying interpretation: normal granulocyte dihydrorhodamine fluorescence, abnormal granulocyte dihydrorhodamine fluorescence compatible with X-linked CGD, abnormal granulocyte dihydrorhodamine fluorescence compatible with the carrier state of X-linked CGD, and abnormal granulocyte dihydrorhodamine fluorescence compatible with autosomal recessive CGD.

Test results alone are not diagnostic. Results should be used in conjunction with other clinical findings to make a diagnosis of CGD. A client control drawn from a normal individual near the time of patient drawing must be included with the patient sample.  For more information about these assay and techniques, see our Web site or the references below:

• Vowells,S.J.,Fleisher,T.A., Sekhsaria,S.,Alling,D.W., Maquire,T.E.,and Mallech, H.L.(1996) “Genotype-dependent variability in flow cytometry evaluation of reduced nicotinamide adenine dinucleotide phosphate oxidase function on patients with chronic granulomatous disease.” Journal of Pediatrics. January 1996.
• Vowells,S.J.,Sekhsaria,S., Malech,H.L.,Shalit,M.,and Fleisher,T.A.(1995) “Flow cytometric analysis of the granulocyte respiratory burst:a comparison study of fluorescent probes. J of Immunological Methods. 179: 89.
• Prince,H.E.and Lape-Nixon,M. (1995) “Influence of specimen age and anticoagulant on flow cytometric evaluation of granulocyte oxidative burst generation.” J of Immunological Methods. 188: 129.
• Gorman M.R.G.and Corrochano,V.(1995) “Rapid whole-blood flow cytometry assay for diagnosis of chronic granulomatous disease.” Clin. Diagn.Lab.Immunol. 2: 227.

New GUS Kit for recombinant Plant Analysis.

The ß-glucuronidase (GUS) gene isolated from E. coli (EC 3.2.1.31) has been well documented to provide desirable characteristics as a marker gene in transformed plants.  Our new ß-Glucuronidase (GUS) Reporter Gene Activity Detection Kit (M0877) provides all the reagents, buffers, and a detailed protocol for easy quantitative measure of GUS enzyme activity in transformed plants or plant cells, through use of the fluorogenic substrate 4-methylumbelliferyl b-D-glucuronic acid (M0240).   Plants or other cell types are extracted with GUS extraction buffer containing phosphate-EDTA, pH 7.0 and detergents. The extracted b-glucuronidase hydrolyzes the 4-MUG to the fluorescent compound 4-MU (pKa 8.2) and glucuronic acid. The reaction is stopped with sodium carbonate buffer because 4-MU exhibits maximal fluorescence at pH values above its pKa. 4-MU can be excited at 365nm with emission maximum at 455nm.  Please see our Web site or the references below for more information:

• Jefferson, R.A., et al., Proc. Natl. Acad. Sci. USA. 86 (1986) 8447-8451.
• Jefferson, R.A., et al., EMBO J. 6 (1987) 3901-3907.
• Kosugi, S., et al., Plant Sci. 70 (1990) 130-140.
• Naleway, J.J. Chap. 4. in “GUS Protocols: Using the GUS Gene as a Reporter of Gene Expression”. Gallagher, S.R., ed. Acad/ Press, NY, (1992).
  
  

Quenched Peptidase Substrates for Many Applications.

Substrates that become fluorescent upon cleavage of a specific peptide sequence separating a fluorescent donor and a nonfluorescent quencher were first developed in 1972.  The quest for HIV-1 protease inhibitors led researchers at Abbott Laboratories to develop fluorogenic HIV and renin protease substrates labeled with the fluorophore EDANS (M0273) and the nonfluorescent acceptor DABCYL.  This fluorophore/quencher pair is extremely well suited for such assays because of the large spectral overlap of DABCYL absorption and EDANS fluorescence (λ_max 490 nm).   EDANS/DABCYL-labeled substrates have since been developed for numerous other proteases, including human cytomegalovirus protease, cathepsin D. carboxypeptidase A, Kex2, and IL-1 bCE.  Our staff at Marker Gene can help you develop your specific protease assay system.  Contact us for more information at techservice@markergene.com or by calling us at 1-888-218-4062.  See also the references below for more information.

•  “Fluorescende determination of carboxypeptidase A activity based on electronic energy transfer.”  Latt S.A., Auld D.S., Vallee B.L., Anal. Biochem. (1972) 50(1): 56-62.
•   “Novel fluorogenic substrates for assaying retroviral proteases by resonance energy transfer.” Matayoshi E.D., Wang G.T., Krafft G.A., Erickson J.,  Science (1990) 247(4945): 954-8.
• "A continuous fluorescence assay of renin activity." Wang GT, Chung CC, Holzman TF, Krafft GA. Anal Biochem 210, 351-359 (1993)
• "Design of sensitive fluorogenic substrates for human cathepsin D." Gulnik S.V., Suvorov L.I., Majer P., Collins J., Kane B.P., Johnson D.G., Erickson J.W., FEBS Lett., (1997) 413: 379-384.
• "Convenient fluorometric assay for matrix metalloproteinase activity and its application in biological media." Beekman B, Drijfhout JW, Bloemhoff W, Ronday HK, Tak PP, te Koppele JM. FEBS Lett (1996) 390: 221-225.
• "Site-directed double fluorescent tagging of human renin and collagenase (MMP-1) substrate peptides using the periodate oxidation of N-terminal serine. An apparently general strategy for provision of energy-transfer substrates for proteases.” Geoghegan, Michael J. Emery, William H. Martin, Alexander S. McColl, and Gaston O. Daumy, 4(6): 537-544.
• "Internally consistent libraries of fluorogenic substrates demonstrate that Kex2 protease specificity is generated by multiple mechanisms." Rockwell NC, Wang GT, Krafft GA, Fuller RS. Biochemistry 36, 1912-1917 (1997).
• "Synthesis of a fluorogenic interleukin-1 beta converting enzyme substrate based on resonance energy transfer." Pennington MW, Thornberry NA. Pept Res 7, 72-76 (1994)
• "Design and Synthesis of New Fluorogenic HIV Protease Substrates Based on Resonance Energy Transfer." Wang G.T., et al., Tetrahedron Lett. 31: 6493 (1990).
• "A general method for the preparation of internally quenched fluorogenic protease substrates using solid-phase peptide synthesis." Maggiora L.L., Smith C.W., Zhang Z.Y.. J. Med. Chem. 35: 3727-3730 (1992).
  

Over 200 Products and Kits in our New Catalog!

The 2003-2004 edition of the Marker Gene catalog will be mailed in September to over 3000 laboratories and research institutions.  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 the chance to get your copy of this resource for Marker Gene detection and use.  Be sure to add your name to our mailing list.  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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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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