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Marker
Gene Monthly Newsletter
January, 2005
Volume
5, Number 1
©
Copyright MGT, Inc., 2005. 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.
Smallpox Virus to be Labeled
with GFP.
The World Health Organization's
(WHO) External Advisory
Committee on smallpox has recommended that the two research teams still
possessing the smallpox virus insert a GFP marker gene into it for use in
testing the efficacy of new anti-smallpox therapy systems. Researchers in the United States (CDC, Atlanta, GA) and Russia (Koltsovo VECTOR Research Center) will be allowed to genetically engineer variola virus,
the main cause of smallpox, in order to accelerate drug development. There is some concern about the stability of
the new recombinant virus and the small risk of increasing virulence. But extrapolation from the expression of GFP
in other orthopox viruses [such as vaccinia] where the virus has
remained stable and, importantly, virulence hasn't
increased, should allow the research to move forward. The risk of bioterrorism has been influential in allowing this new
research. For more information about GFP
expression in viruses, the smallpox virus and GFP analysis systems, please see
the references below.
·
Ward BM (2004) “Pox,
dyes, and videotape: making movies of GFP-labeled vaccinia virus.” Methods Mol.
Biol. 269: 205-18.
·
Gouskos T., Wightman F.,
Lewin S.R., Torresi J., (2004) “Highly reproducible transient
transfections for the study of hepatitis B virus replication based on an
internal GFP reporter system.” J. Virol. Methods 121(1): 65-72.
·
Painter G.R., Hostetler
K.Y., (2004) “Design and development of oral drugs for the prophylaxis and
treatment of smallpox infection.” Trends Biotechnol. 22(8): 423-427.
·
Cosma A., Bühler S.,
Nagaraj R., Staib C., Hammarin A.L., Wahren B., Goebel F.D., Erfle V., Sutter
G., “Neutralization assay using a modified vaccinia virus Ankara vector
expressing the green fluorescent protein is a high-throughput method to monitor
the humoral immune response against vaccinia virus.” Clin. Diagn. Lab
Immunol. (2004) 11(2): 406-410.
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Specifically
detecting tyrosine or serine/threonine protein phosphatases and screening for
potential modulators of these enzymes, requires the use of a sensitive
phosphatase substrate as well as a battery of inhibitors that can
differentiate the various phosphatase activities often present in crude
cellular preparations. A cocktail of
inhibitors can be used to ensure selectivity for either tyrosine or
serine/threonine phosphatases. Marker
Gene now provides two fluorogenic phosphatase substrates for use in these
assays, M0541: 3-Phenylumbelliferone 7-O-phosphate,
hemipyridinium salt and M1034: Fluorescein di-O-phosphate, tetra-ammonium salt. Since these substrates are nonspecific
phosphatase substrates, it is necessary to add inhibitors to develop assays
for specific phosphatases. By adding
the inhibitors cantharidin, bromotetramisole and microcystin-LR, the assay
will be restricted to detection of tyrosine phosphatases. Alternately, by adding the inhibitors
postassium bisperoxo(1,10-phenanthroline)oxovanadate and bromotetramisole a
specific assay for serine/threonine phophatases is obtained. The resulting assays are insensitive to
free phosphate and are compatible with nonionic detergents and crude
preparations. For more information
about developing phosphatase assays for your particular system, see our
website or the references below:
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D-Luciferin
reacts with ATP in the presence of Mg 2++ /O2 and the enzyme, luciferase, to
produce light. Replacement of the
hydroxyl group on luciferin with an amino group to generate 6-amino-D-luciferin
does not adversely affect its binding by luciferase and the production of
light. In addition, the amine group
creates a site for attaching amino acids or peptides to produce double
substrates whose cleavage by proteases release the light emitting
substrate. Since the luciferase assay
is extremely sensitive, this chemistry allows novel enzymatic assays to be
developed with extraordinary levels of sensitivity. Development of such platforms also enable novel in vivo,
cell culture and biochemical assays for a variety of proteases with
applications in pathogen detection, discovery of protease inhibitors, probing
cell physiology and assessing protease activity in oncogenesis at
extraordinary sensitivity. Marker
Gene now manufactures the important starting material for these systems, M0352:
6-amino-D-luciferin, and can help you develop your ultrasensitive
chemiluminescent peptidase assays.
For more information about these assays, see the references below or
visit our website. ·
Monsees T., Geiger R.,
Miska W., (1995) “A novel
bioluminogenic assay for alpha-chymotrypsin.” J Biolumin
Chemilumin. 10(4): 213-8 ·
White, Emil H.;
Woerther, Helmut; Seliger, Howard H.; McElroy, William D. (1966) “Amino
analogs of firefly luciferin and biological activity thereof.” Journal of the American Chemical
Society 88(9): 2015-18. ·
Denburg, Jeffrey; Lee,
Reiko Takasaka; McElroy, W. D. (1969)
“Substrate-binding properties
of firefly luciferase. I. Luciferin-binding site. “ Archives
of Biochemistry and Biophysics 134(2): 381-94. |
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Both the tetracycline inducible system and tetracycline repressor systems have been incorporated into a variety of mammalian, plant and insect expression vectors including retroviral vector systems, that provide elegant genetic tools for controlled gene expression in eukaryotic cells. The inducibility of this system is based on regulatory elements of the Tn10 encoded tetracycline resistance operon of Escherichia coli. For generation of an inducible tet-responsive transactivator (tTA) the tet repressor of Tn10 of E.coli is fused to the transactivating domain of virion protein 16 (VP16) of herpes simplex virus. This fusion protein binds to the tetracycline-responsive operator (tet operator) region inserted as a heptamer separated by linker sequences and placed immediately upstream of a minimal CMV promoter. Mediated by the VP16 domain, the binding of tTA to the tet operators results in activation of the promoter. In the presence of tetracycline, tTA does not bind to the tet operator and therefore the promoter is inactive, whereas withdrawal of tet allows its activation. Thus, the promoter activity can be reversibly switched on and off, which allows controlled expression of an individual gene in vitro and in vivo. The tetracycline inducible system (Tet-On) is based on a reverse tetracycline-controlled transactivator, rtTA. Like tTA, rtTA is a fusion protein comprised of the TetR repressor and the VP16 transactivation domain; however, a four amino acid change in the tetR DNA binding moiety alters rtTA's binding characteristics such that it can only recognize the tetO sequences in the TRE of the target transgene in the presence of the tetracycline effector. Thus, in the Tet-On system, transcription of the TRE-regulated target gene is stimulated by rtTA only in the presence of tetracycline. Marker Gene has developed a linkage compound for use with cells containing the tet-inducer with cells that are expressing lacZ b-galactosidase activity. Upon administration of Tetracycline 10-O-b-D-Galactoside (M0176) at concentrations in the micromolar range. The LacZ positive cells release tetracycline, which in turn activates tetracycline induction or the tetracycline repression of a separate gene construct (in the same cell line or different cell line). For more information about these exciting new methods of controlling gene expression in vivo and in vitro, see our web site or the references below. ·
Gossen,M. and
Bujard,H. (1992) Tight Control of Gene Expression in Mammalian Cells by
Tetracycline-Responsive Promoters Proc. Natl Acad. Sci. USA, 89,
5547–5551. ·
Naleway, J.J.,
Howard-Till, R.A., "Compositions and Methods for Targeted Enzymatic
Release of Cell Regulatory Compounds" US Patent # 6,656,917 (2003). ·
Naleway, J.J.,
Howard-Till, R.A., Guzikowski, A.P., Schutte, R.C., Braden, M.R., Fox, B.A.,
Bashey, G., Pinhiero, N., Mehrotra, R. "Marker Gene Directed Drug-Delivery
to Recombinant Tumor Cells", FASEB J., 14(8) (2000a) A1410. Note: These products are for research use
only. A license is required for
commercial or other application.
Please contact us for more information. |
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Brassinosteroids (BS) are plant hormones of
ubiquitous distribution in the plant kingdom. These steroidal
lactones promote plant growth when applied to plants in concentrations lower
than 10-4 mg/ml. Among steroidal natural products,
castasterone and brassinolide show the highest activity in the regulation of
plant growth and development, and their biosynthesis has been a major subject
of research for many years. But
assays to measure these important phytohormones have been limited by their extremely
low natural abundance. Recent work
from the laboratory of Ales Svatos at the Max-Plank Institute of Chemical
Ecology in Germany utilized an LC-MS technique with labeling using m-Dansylaminophenylboronic acid (DAPB, M0329) which reacts with cis-vicinal diols (hydroxyl
groups on adjacent carbon atoms) to form cyclic complexes that have an
environment dependent fluorescence intensity and peak emission. DAPB also binds reversibly to cell-wall
carbohydrates and glycoproteins, as well as to glycosylated proteins and
glycolipids. Moreover, it specifically inhibits certain serine hydrolases
that have adjacent histidine residues, including subtilisin, lipoprotein
lipase, human milk lipase and the ·
Svatos A., Antonchick A., Schneider B., (2004)
“Determination of brassinosteroids in the sub-femtomolar range using
dansyl-3-aminophenylboronate derivatization and electrospray mass
spectrometry.” Rapid Commun. Mass Spectrom. 18: 816–821.
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The
2005 edition of the Marker Gene catalog is now available. Visit our website or use the link www.markergene.com/catalog2005-2006.pdf
for an electronic version. Many new
products and kits, additional literature references, data and protocols have
been included, as well as new information about our old products. If you haven’t already received yours, be
sure to add your name to our mailing list.
Visit our Customer
Information Form, or e-mail us at techservice@markergene.com
and we will have a copy sent out to you post-haste. |
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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:
Contact us by telephone at
(888) 218-4062; (541) 342-3760; (541) 912-5320 or FAX us at (541) 342-1960 or
you can write to us at 1850 Millrace Drive, Eugene, Oregon 97403-1992 or
contact us by e-mail at: techservice@markergene.com
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-lactamase
from Enterobacter cloacae P99.
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