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Marker Gene Monthly Newsletter

July, 2007

Volume 7, 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.

Heat-Shock Promoter Elements.

Over two decades ago, puffing patterns were discovered in the chromosomes of Drosophila after they were heated to high temperatures. These puffs were found to be at positions where there was active synthesis of mRNA, and the proteins that were being produced were termed heat shock proteins. Since then it has been shown that a variety of agents, including heavy metals or anaerobic conditions, can also induce responses similar to those induced by heat, suggesting that a more appropriate name for these genes could be "stress genes". Analogous heat shock proteins have since been found in many other species including chick embryonic fibroblasts, Chinese hamster ovary cells, E. coli , yeast and plants. Heat shock proteins can provide some protection for the cell against environmental stresses by a phenomenon known as "acquired thermotolerance". Cells exposed to a single heat shock, or another stress, are protected against the effects of a second, otherwise lethal heat or oxidative shock. The exact mechanism of this protection is not fully understood, but may involve their activity as chaperone proteins, binding tightly to hydrophobic residues of partially-synthesized peptide sequences on the ribosome.

Much of the early work on heat shock proteins (hsp70, hsp90 and hsp40) has utilized marker genes for elucidation of their activities. A hybrid hsp70-lacZ gene was found to be under normal heat shock regulation when integrated into the Drosophila germ line. Deletion analysis was used to identify the Drosophila hsp70 heat shock promoter. The sequence was found to be upstream from the TATA box and have homology to the analagous sequences found in other species. The consensus sequence CT-x-GAA-xx-TTC-x-AG was used to construct synthetic oligonucleotides, based on various consensus sequences. When these were placed upstream of the TATA box of the herpes virus thymidine kinase gene (tk) (in place of the normal upstream promoter element), the resultant recombinant tk gene was heat-inducible both in monkey COS cells and in Xenopus oocytes. These methods offer the opportunity to use heat shock promoters for upregulation of cloned genes in a variety of species. For more information about heat shock proteins and their promoters, please see the references below, or visit our website.

· Steller H, Pirrotta V, (1986) “P transposons controlled by the heat shock promoter.” Mol Cell Biol. 6(5): 1640–1649.

· Pelham HR, Bienz M, (1982) “A synthetic heat-shock promoter element confers heat-inducibility on the herpes simplex virus thymidine kinase gene.” EMBO J. 1(11): 1473–1477.

· Ritossa, F. (1962) “A new puffing pattern induced by heat shock and DNP in Drosophila.” Experientia 18:571-573.

· Kelly P, Schlesinger MJ, (1978) “The effect of amino acid. analogs and heat shock on gene expression in chicken. embryo fibroblasts.” Cell 15:1277-1286.

· Bouche G, Amalric F, Caizergues-Ferrer M, Zalta JP, (1979) “Effects of heat shock on gene expression and subcellular protein distribution in Chinese hamster ovary cells “Nucleic Acids Research 7:1739-1747.

· Lemeaux, P. G. Herendeen SL, Bloch PL, Neidhardt FC. (1978) “Transient rates of synthesis of individual polypeptides in E. coli following temperature shifts.“ Cell 13:427-434.

· Miller, M. J. Xuong NH, Geiduschek EP. (1979) “A response of protein synthesis to temperature shift in the yeast Saccharomyces cerevisiae “Proc. Nat. Acad. Sci. USA 76:5222-5225.

· Barnett, T. Altschuler M., McDaniel CN, Mascarenhas JP, (1980) “Heat shock induced proteins in plant cells.” Dev. Genet. 1:331-340.

· Lis JT, Simon JA, Sutton CA, (1983) “New heat shock puffs and beta-galactosidase activity resulting from transformation of Drosophila with an hsp70-lacZ hybrid gene.” Cell 35:403-410.

Selective PMCA Inhibitor of Calcium Clearance.

5(6)-Carboxyeosin (CE, M1300) is a brominated analog of carboxyfluorescein with a number of interesting applications in cell biology. Halogenated derivatives of fluorescein dyes are known to be good photosensitizers and singlet oxygen generators. For example, carboxyeosin has a singlet oxygen yield that is approximately 19 times greater than that of fluorescein. Using this property, CE has been found to be an excellent dye for photoconverting DAB as a method for improving resolution for immunolocalization and in situ hybridization techniques in light and electron microscopy. Upon excitation, carboxyeosin labeled probes can be used to generate singlet oxygen, which in turn will oxidize diaminobenzidine (DAB) into an opaque precipitate within cells. The resulting DAB reaction provides uniform, nondiffusible staining properties for monitoring hybridization or antibody localization in cells and tissues.

Carboxyeosin has also been found to be a sensitive, non-covalently bound fluorescent probe for monitoring conformational changes in detergent-solubilized Na,K-ATPase as well as plasma membrane Ca(+2) ATPase activity. Finally, carboxyeosin is also a well-known, specific PMCA inhibitor. The plasma membrane calcium pump (PMCA) is one of the essential mechanisms to control calcium efflux across the plasma membrane, thereby keeping and restoring a low cytosolic calcium concentration. For more information about this new probe for use in these assays, please see the references below, or visit our website.

· Gatto C, Milanick MA, (1993) “Inhibition of the red blood cell calcium pump by eosin and other fluorescein analogues” Am. J. Physiol. 264: C1577–C1586.

· Fierro L, DiPolo R, Llano I, (1998) “Intracellular calcium clearance in Purkinje cell somata from rat cerebellar slices.” J. Physiol. 510: 499–512.

· Sedova M, Blatter LA (1999) “Dynamic regulation of [Ca2+] by plasma membrane Ca(2+)-ATPase and Na+/Ca2+ exchange during capacitative Ca2+ entry in bovine vascular endothelial cells.” Cell Calcium 25: 333–343.

· Deerinck TJ, Martone ME, Lev-Ram V, Green DP, Tsien RY, Spector DL, Huang S, Ellisman MH. (1994) "Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy." J. Cell Biol. 126: 901-910.

· Esmann M, (1991) “Conformational transitions of detergent-solubilized sodium-potassium-ATPase are conveniently monitored by the fluorescent probe 6-carboxy-eosin.” Biochemical and Biophysical Research Communications 174(1): 63-9.

· Shmigol A, Eisner DA, Wray S, (1998) “Carboxyeosin decreases the rate of decay of the [Ca2+]i transient in uterine smooth muscle cells isolated from pregnant rats.” Pflugers Arch 437: 158-60.

· Neckers DC, Valdes-Aguilera OM. "Photochemistry of the Xanthene Dyes." (1993) Adv. Photochem 18: 315.

· Gandin E, Lion Y, Van de Vorst A. (1983) "Quantum Yield of Singlet Oxygen Production by Xanthene Derivatives." Photochem. Photobiol. 37: 271

· Kurnellas MP, Nicot A, Shull GE, Elkabes S, (2005) “Plasma membrane calcium ATPase deficiency causes neuronal pathology in the spinal cord: a potential mechanism for neurodegeneration in multiple sclerosis and spinal cord injury” FASEB J. 19:298-300.

A Novel, Water Soluble Lipase Substrate.

The highly polar, highly water soluble lipase substrate octanoyl pyrenetrisulfonic acid, tri-sodium salt (M1296) is a derivative of HPTS (pyranine, M1236) that forms tight micelles in aqueous solutions. Alkyl esters of HPTS (sometimes called Cascade Blue) are well known for their substrate ability to measure esterase activity. Longer chain derivatives function as lipase substrates. Lipase activity can be measured by monitoring the sensitive fluorescence kinetics following the increase in emission of product HPTS (hydroxypyrene trisulfonic acid) as a function of time. The activity of a number of lipases have been measured using this substrate including wheat germ lipase, candida cylindracea lipase as well as carboxylic ester hydrolase and acylase 1. Because of its relatively low pKa value (7.3) under assay conditions, it is partially dissociated to form an anion that shows strong absorption maximizing at 460 nm and fluoresces with very high quantum yield and maximum intensity at 520 nm. Unfortunately, when excited between 440 and 480 nm, the fluorescence intensity is dependent on the pH of the solution, which requires careful pH control. However, when excited at 415 nm (the isosbestic point), no pH dependence of fluorescence intensity at 520 nm is noted. For more information about this new lipase substrate, please see the references below or visit our website.

· Koller, E, Wolfbeis, OS, (1988) “Preparation of pyrenesulfonic acid derivatives for photometric determination of enzyme activity.” Austrian Patent AT 385755.

· Baumeister B, Sakai N, Matile, S, (2001) “p-Octiphenyl b -barrels with ion channel and esterase activity.” Organic Letters 3(26): 4229-4232.

· Wolfbeis OS, Koller E, (1983) “Fluorimetric assay of hydrolases at longwave excitation and emission wavelengths with new substrates possessing unique water solubility.” Anal Biochem. 129(2):365–370.

Long-Wavelength Esterase Activity Assays.

In addition to its use as a common long-wavelength esterase substrate, in cell viability assays, or for high throughput screening of esterase activity in a live cell format, resorufin acetate (M0806) has also found utility for the analysis of several other important enzymes. It has been found to be a good substrate for sheep liver cytosolic aldehyde dehydrogenase (CAD), both from the point of view of practical spectrophotometry and in terms of information provided about the nature of the catalysis shown by this enzyme.

It has also been shown to be an attractive substrate for use in assay of chymotrypsin activity, since the absorbance of the product is several times more intense than that formed by the widely used p-nitrophenyl acetate. Furthermore, under the right conditions, resorufin acetate allows convenient observation of the burst reaction for this enzyme by conventional spectrophotometry. For more information about these assays and resorufin substrates, please see the references below, or visit our website.

· Kitson, TM, Kitson KE, (1997) “Studies of the esterase activity of cytosolic aldehyde dehydrogenase with resorufin acetate as substrate Biochem. J. 322: 701-708.

· Kazlauskas, RJ (2006) “Quantitative Assay of Hydrolases for Activity and Selectivity Using Color Changes” in Enzyme Assays, Jean-Louis Reymond, ed., (2006) Wiley-VCH Verlag GmbH & Co. KGaA

· Kitson T.M. (1996) “Comparison of Resorufin Acetate and p -Nitrophenyl Acetate as Substrates for Chymotrypsin” Bioorganic Chemistry 24(4): 331-339.

· Böttcher D, Bornscheuer T, (2006) “High-throughput screening of activity and enantioselectivity of esterases.” Nature Protocols 1: 2340 – 2343.

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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

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