Marker Gene Monthly Newsletter   

May, 2006

Volume 6, 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.

Tyrosinase as a Marker Gene in Fungal and Mammalian Cell Lines.

Many marker gene systems have been developed to allow investigators to visually identify transformed cells and to follow the transcriptional activity of promoter regions preceding the cloned gene constructs.  Marker genes typically encode protein products that can be easily assayed and detected by either histochemical, chemiluminescent or fluorescence techniques in the transformed cell.  Among the common marker genes in use, the E. coli beta-galactosidase and E. coli beta-glucuronidase genes have been extensively used for analysis of transformed cells or tissues in mammalian or plant cells. The presence of these proteins can be determined by staining with X-Gal or X-GlcU, respectively, and the enzyme levels can be easily quantified by independent enzymatic assay.  Although widely used, these two systems have some drawbacks for the study of gene expression in eukaryotic systems. Since the X-Gal and X-GlcU substrates are toxic, assays performed on live cells often result in cell death or disruption. Also, these reporters are derived from E. coli sources, where the codon usage is quite different from that found within the eukaryotic cell. Recently, the use of a Neurospora tyrosinase gene as a reporter has been introduced as an alternative marker gene that can be monitored chromogenically (visually) and can overcome the toxicity difficulties for analysis in fungal cell types.

Researchers at the Department of Biological Sciences, SUNY/Buffalo in Buffalo, NY have developed a vector which allows for transfer of the Neurospora crassa tyrosinase gene as a reporter system for transformation experiments. The tyrosinase gene encodes a 75Kd protyrosinase peptide that is subsequently proteolytically cleaved to give active tyrosinase.  Tyrosinase catalyses the formation of melanin (an insoluble black pigment) from tyrosine or DOPA. The cloned Neurospora tyrosinase gene has the appropriate codon usage, translation start site, intron splice sites, and polyadenylation/termination site for expression in fungi.  Since tyrosine can be easily translocated into cells, there is no need to lyse the cells in order to stain for tyrosinase activity. Furthermore, the levels of tyrosinase activity can be easily assayed visually or spectrophotometrically. Under most growth conditions the endogenous tyrosinase gene is repressed and the level of background tyrosinase activity from the endogenous gene is very low.  This is in sharp contrast to wild-type strains, where this enzyme is only expressed in situations of starvation or sexual differentiation. 

Additionally, since tyrosinase activity (melanin production) is highly elevated in certain melanoma tumors, the composite synthetic promoters derived from human or murine tyrosinase can be used to drive a strong and selective expression of a second reporter (e.g. lacZ) or therapeutic gene specifically in melanoma cells, providing a new powerful tool for potential gene therapy of melanomas.  For more information about these assays and gene expression systems, please see the references below or visit our website. 

• Kupper U. , Linden M., Cao K.Z ., Lerch K., (1990)  “Expression of tyrosinase in vegetative cultures of Neurospora crassa transformed with a metallothionein promoter/protyrosinase fusion gene” Current genetics 18(4): 331-5.
• Kothe, G., Deak, M., Free. S.J., (1993) “Use of the Neurospora tyrosinase gene as a reporter gene in transformation experiments.” Fungal Genet. Newsl. 40:43-45.
•   Vile R.G., Hart I.R., (1994) “Targeting of cytokine gene expression to malignant melanoma cells using tissue specific promoter sequences.” Annals of Oncology 5(4): 59-65.
• Niedermann D.M., Schilling B.C., Lerch K.,  (1990) “ATP-induced protyrosinase synthesis and carboxyl-terminal processing in Neurospora crassa. “ Pigment Cell Research 3(4): 207-213.
• Vile R.G., Hart I.R., (1993) “In vitro and in vivo targeting of gene expression to melanoma cells.” Cancer Res. 53(5): 962-967. Schedl A., Montoliu, L., Kelsey G., Schütz G., (1993) “A yeast artificial chromosome covering the tyrosinase gene confers copy number-dependent expression in transgenic mice.” Nature 362: 258 – 261.
  

Biotin Labeling for Analysis or Purification.

Biotin (vitamin H) is a very versatile reagent that can be used to label individual molecules for detection within highly complex biological samples.  The binding of avidin or streptavidin to biotin is one of the strongest known non-covalent biological interactions with dissociation constants (K_d) of approximately 10^-15 M between the protein and biotin. The high affinity and specificity of the avidin-biotin interaction has been utilized for enriching biotin-containing proteins from mixtures, for detection of DNA hybridization in DNA or RNA microarrays, for monitoring antibody labeling and for use in protein detection in gels and intracellularly.  Reactive N-hydroxysuccinimide (NHS) derivatives of biotin are compatible with most biological systems, providing the opportunity to label proteins, lipids, carbohydrates or DNA/RNA from biological samples with biotin in purification or detection protocols.  Marker Gene provides a number of biotinylation reagents including Biocytin Hydrazide (M0128) for labeling carbohydrates and glycoproteins, Biotin Succinimidyl Ester (Biotin SE) (M0785), and 6-((biotinoyl)amino)hexanoic acid, succinimidyl ester (Biotin-X, SE) (M0783) for labeling peptides, proteins or DNA, and the new MarkerGene^TM Biotin-X Antibody/ Protein Labeling Kit (M1138), which includes a gel filtration spin-column to purify the biotinylated protein from excess biotin, reagents for quantitating the degree of biotinylation and a control.  We also provide an Avidin Sulforhodamine 101 (Texas Red^TM) conjugate (M1124) and a Biotin-Dextran (70,000 MW) conjugate (M0788) for use in coupled assays. 

 

The biotinylation of proteins has also been used to isolate and identify proteins expressed in cells or at the cell surface.  After labeling, immobilized tetrameric avidin is used to capture the biotinylated proteins.  The proteins are then eluted using 8 M guanidine HCl at pH 1.5.  Recently, immobilized monomeric avidin with a lower binding affinity for biotin (K_d approximately 10^-8 M) has become commercially available.  This reagent permits recovery of biotinylated proteins and peptides using milder elution conditions, thus preserving native protein conformation and enzyme activity.  In experiments to biotinylate proteins using biotin conjugates with different spacer arm lengths, differential protein recovery has been observed when immobilized polymeric or monomeric avidins were used.  In addition, cells labeled with mixtures of the short chain and long-chain biotinylation reagents have yielded significantly more isolated proteins than detected when either reagent was used independently.  These results illustrate the importance of selecting the optimal combination of biotinylation and avidin reagents for the recovery of the proteins of interest in proteomics studies. For more information about these techniques, please see the references below, or visit our website. 

 

• Green, N.M. (1963) Avidin.3. “The nature of biotin-binding site.” Biochem. J. 89: 585.
• Sabarth, N., Lamer, S., Zimny-Arndt, U., Jungblut, P.R., Meyer, T.F., Bumann D. (2002) “Identification of surface proteins of Helicobacterpylori by selective biotinylation, affinity purification, and two-dimensional gel electrophoresis.” J. Biol. Chem. 277: 27896-27902.
• Henrikson, K.P.,. Allen, S.H.G., Maloy W.L., (1979) “An avidin monomer affinity column for the purification of biotin-containing enzymes.” Anal. Biochem. 94: 366-370.
• Khare T., Giometti C.S., (2006) “Differential recovery of biotinylated microbial proteins using monomeric or polymeric avidin”  BioTechniques  40(5): 584-588.
• Hofmann K., Titus G., Montibeller J.A., Finn F.M. (1982) "Avidin binding of carboxyl-substituted biotin and analogues." Biochemistry 21: 978-984.
• Antikainen N.M. , Smiley R.D., Benkovic S.J., Hammes G.G. (2005)"Conformation coupled enzyme catalysis: single-molecule and transient kinetics investigation of dihydrofolate reductase." Biochemistry 44: 16835-43

Cytochrome P450 Chemiluminescent Assays.

Cytochrome P450 enzymes (P450s) catalyze the oxidative metabolism of a vast number of foreign hydrophobic chemicals including many therapeutic drugs, inside target cells. The inhibition of cytochrome P450s by drugs is an important consideration in drug discovery.  It can alter drug disposition and cause adverse drug-drug interactions.  For example, if a first drug inhibits the metabolism of a second co-administered drug, the second drug may accumulate to a toxic level. There are currently over 50 cytochrome P450 genes that have been identified in humans, but enzymes encoded by only five of them, CYP1A2, 2C9, 2C19, 2D6 and 3A4, account for nearly 90% of cytochrome P450-dependent drug metabolism. 

   Recently several new assays have been developed for analysis of cytochrome P450 enzymes using a chemiluminescent system based upon use of 6’-O-ether derivatives of D-luciferin combined with the enzyme firefly luciferase [EC 1.13.12.7].   Although these ether derivatives of luciferin are normally inhibitors of the luciferase enzyme, cytochrome P450s can act to remove the ether group, and activate the substrate for light emission.  These new substrates, therefore, provide a highly sensitive method of screening drugs and molecules for cP450 inhibition, in a high-throughput manner. 

   For some P450s, including CYP1A2, 2C9 and 3A4, a 6’-O-modification is sufficient.  But for CYP2C19 and 2D6, it also appears to be necessary to modify the carboxy group at the 1-position, typically as an alkyl ester.  The role of the ester group in the activity of CYP2D6 toward the 6’-O-methoxy position of D-luciferin can be explained in part by the neutralization of an acidic group in the substrate.  Natural CYP2D6 substrates are typically neutral or cationic.  Three dimensional molecular modeling studies of the CYP2D6 enzyme have identified two acidic residues (Asp 301 and Glu 216) with their negative charges near the active site.  The 1-carboxy esterified luciferin is neutral in this area, while the  nonesterified form still carries a negative charge and would be repelled from the active site of a CYP2D6 enzyme by the negatively charged residues.  Increased hydrophobicity of luciferin esters over the carboxylic forms also improve efficiency in CYP2C19 and 2D6 cP450 reactions because there is generally a positive correlation between hydrophobicity and affinity for these P450 active sites.  Marker Gene now provides several D-luciferin analogs including D-Luciferin, 6' Methyl Ether (M0236) and D-Luciferin, Ethyl Ester (M0906) that can be used in these new chemiluminescent cytochrome P450 assays.  For more information about these new assays and these new cP450 substrates, please see the references below, or visit our website. 

• Yueh M.F., Kawahara M., Raucy J.,  (2005) “High volume bioassays to assess CYP3A4-mediated drug interactions: induction and inhibition in a single cell line.” Drug Metab Dispos. 33(1): 38-48.
• Guengerich, F.P. (2001) “Common and uncommon cytochrome P450 reactions related to metabolism and chemical toxicity.“ Chem. Res. Toxicol. 14: 611–50.
• Bjornsson T.D., Callaghan J.T., Einolf H.J., Fischer V., Gan L., Grimm S., Kao J., King SP., Miwa G., Ni L., Kumar G., McLeod J., Obach R.S., Roberts S., Roe A., Shah A., Snikeris F., Sullivan J.T., Tweedie D., Vega J.M., Walsh J., Wrighton S.A  (2003) “The conduct of in vitro and in vivo drug-drug interaction studies: a Pharmaceutical Research and Manufacturers of America (PhRMA) perspective. “ Drug Metab. Dispos. 31:  815–32.
• Nelson, D.R. Cytochrome P450 Homepage: http://drnelson.utmem.edu/CytochromeP450.html
• Williams, J.A. Hyland R., Jones B.C., Smith D.A., Hurst S., Goosen T.C., Peterkin V., Koup J.R., Ball S.E  (2004) Drug Metab. Dispos. 32, 1201–8.
• Guengerich, F.P. Hanna I.H., Martin M.V., Gillam E.M.,  (2003) “Role of glutamic acid 216 in cytochrome P450 2D6 substrate binding and catalysis.” Biochemistry 42: 1245–53.
• Hanna I.H.,  Kim M.S., Guengerich, F.P. (2001) “Heterologous expression of cytochrome P450 2D6 mutants, electron transfer, and catalysis of bufuralol hydroxylation: the role of aspartate 301 in structural integrity. “Arch. Biochem. Biophys. 393: 255–61.
• de Groot, M.J. Ackland M.J., Horne V.A., Alex A.A., Jones B.C  (1999) “Novel approach to predicting P450-mediated drug metabolism: development of a combined protein and pharmacophore model for CYP2D6.” J. Med. Chem. 42, 1515–24.

Discreet PEG linkers for Biological Applications.

Some of the most problematic obstacles in drug and diagnostic development have included non-specific interactions, cell/tissue/membrane permeability, aqueous solubility, unexpected accumulation and/or toxicity, and improvements in drug-drug conjugation methodologies.  Recently, modification with polyethylene glycol (PEG) has been found quite useful in mediating most of these parameters.  But discreet PEG molecules for use in such conjugation, modification or formulation of drugs or for use in linking two or more drug molecules together, have been generally unavailable.  Recently, a wide variety of these discreet PEG molecules have become commercially accessible for use in drug development, or for other research and development applications from QuantaBioDesign, Inc. of Powell, Ohio.  These molecules can be used  to develop and produce new drug derivatives and other compounds based on modification with discreet PEG’s that have individual properties.  The design of new compounds incorporating the power and versatility of the dPEG™’s is under development.  Marker Gene is working with QuantaBioDesign, Inc., the primary manufacturer of our dPEG™’s; to provide these new linkers and modification reagents.  QuantaBioDesign and Marker Gene are  committed to developing new technologies that meet the very high product standards required by regulated environments, while at the same time diligently working towards processes that are economical.  For more information about these new reagents and applications, please see the references below or visit our website and the QuantaBioDesign’s website at (http://www.quantabiodesign.com). 

• Greenwald R.B, Choe Y.H, McGuire J., Conover C.D., (2003) “Effective drug delivery by PEGylated drug conjugates.” Adv. Drug Deliv. Rev. 55(2): 217-50.
• Li, L., Yazaki, P.J., Anderson, A.L., Crow, D., Colcher, D., Wu, A.M.,  Williams, L.E.,  Wong, J.Y.C., Raubitschek, A., Shively J.E.,  (2006) “Improved Biodistribution and Radioimmunoimaging with Poly(ethylene glycol)-DOTA-Conjugated Anti-CEA Diabody. Bioconjugate Chemistry 17(1): 68-76.
• Fujimoto, Y., Kimura, E., Murata, S., Kusumoto, S., Fukase K., (2006) “Synthesis and bioactivity of fluorescence- and biotin-labeled lipid A analogues for investigation of recognition mechanism in innate immunity.” Tetrahedron Letters47: 539-543.
• Kornilova, A.Y., Bihel, F., Das,C., Wolfe M.S.,  (2005) “The initial substrate-binding site of gamma-secretase is located on presenilin near the active site. Proc. Natl. Acad. Sci. USA, 10(9):  3230-3235.
• Maina, T.,  Nock, B.A., Zhang, H., Nikolopoulou, A., Waser, B., Reubi, J.C. Maecke, ,H.R., (2005) “Species Differences of Bombesin Analog Interactions with GPR-R Define the Choice of Animal Models in the Development of GRP-R-Targeting Drugs.” The Journal of Nuclear Medicine 46(5): 823-830.
  
  

Analysis of Lipid Rafts in Cell and Tissue Membranes.

Cell plasma and Golgi membranes have been found to contain small microdomains or “rafts” that are enriched in specific lipids, cholesterol and proteins. Due to their ability to sequester these membrane components, rafts have been shown to be involved in many key cellular functions, including signal transduction, membrane fusion, organization of the cytoskeleton, lipid sorting, protein trafficking, and localization and activity of specific membrane channels.  Rafts have also been shown to exist in lipid bilayers containing lipid compositions approximating those of plasma membranes.  In both natural and bilayer membranes, rafts have been characterized by their insolubility at low temperatures in detergents such as Triton X-100, and it has been found that detergent resistant membranes (DRMs) are enriched in specific lipids, including sphingomyelin (SM) and cholesterol.  A combination of fluorescent probes that have differential lipid phase partition behavior have been valuable in monitoring lipid rafts and lipid membranes.  DiO-C18(3) favors disordered fluid lipid phases, whereas DiI-C20:0 prefers the more spatially ordered lipid phases found in rafts.  Marker Gene now provides several of the key carbocyanine labeling reagents for monitoring these rafts, including 3,3'-dioctadecyloxacarbocyanine perchlorate ('DiO'; DiOC₁₈(3))  (M1197).  For more information about these new assays and the methods involved in staining cell systems, please see the references below or visit our website.

• Simons K.,  Ehehalt R.,  (2002) “Cholesterol, lipid rafts, and disease” J. Clin. Invest. 110:597-603.
• Vidal A., McIntosh T.J., “Transbilayer Peptide Sorting Between Raft and Non-Raft Bilayers: Comparison of Detergent Extraction and Confocal Microscopy” Biophysical Journal 89: 1102-1108 (2005).
•   Sondermann J., ( 1971) “Darstellung Oberflachenaktiver Polymethincyanin-Farbstoffe mit Langen N-Alkyl-Ketten." J. Liebigs Ann. Chem. 749: 183.
• Yguerabide J, Stryer L. (1971) "Fluorescence spectroscopy of an oriented model membrane." Proc Natl Acad Sci USA 68: 1217-1221.

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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