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Marker Gene Monthly Newsletter

October, 2004

Volume 4, Number 10

© 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.

Monitoring Transgene Silencing.

Industrial and research applications of transgenic plants call for consistent high-level transgene expression. But independent transgenic lines generated with the same construct can often vary by more than 100-fold with respect to transgene expression levels, and gene silencing is frequently observed. Progress has been made in elucidating the molecular mechanisms of such gene silencing, which is typically caused by epigenetic effects (de novo DNA methylation, chromatin condensation, etc.) rather than any sequence alterations in the transferred gene. It is believed that the silencing may be a genome surveillance system that eliminates RNA corresponding to excessively transcribed genes, as a defense against viral infection. The genes for b–glucuronidase (GUS), streptomycin phosphotransferase (SPT), and green fluorescent protein (GFP) have all been used for analysis of such gene expression events. Compounds such as the histone deacetylase inhibitor trichostatin A, the DNA methyltransferase inhibitor 5-azacytidine (5-AzaC) or matrix-attachment regions (MARs) have been used to inhibit these effects. Marker Gene provides several products for quick estimation of marker gene levels including 4-Methylumbelliferyl-b-D-Glucuronide (MUG, M0240), the b-Glucuronidase (GUS) Reporter Gene Activity Detection Kit (M0877) and Fluorescein di-b-D-Glucuronide, di-methyl ester (M0969) for in vivo applications. For more information about these systems, see our web site or the references below.

D. Schubert, B. Lechtenberg, A. Forsbach, M. Gils, S. Bahadur, R. Schmidt (2004) ”Silencing in Arabidopsis T-DNA Transformants: The Predominant Role of a Gene-Specific RNA Sensing Mechanism versus Position Effects” The Plant Cell 16: 2561–2572.

Lorence A., Verpoorte R., (2004) “Gene transfer and expression in plants.” Methods Mol. Biol. 267: 329-50.

Holtorf S., Apel K., Bohlmann H., (1995) “Comparison of different constitutive and inducible promoters for the overexpression of transgenes in Arabidopsis thaliana.” Plant Mol. Biol. 29(4): 637-46.

Bacterial Viability Stains.

Several dyes have been used to discriminate bacterial viability and also quantitate live vs. dead bacterial cells in culture and by using flow cytometry. The ability of a flow cytometer to rapidly assess microbial viability has been investigated using several vital stains including rhodamine 123 (Rh123) (M0542), fluorescein diacetate (FDA) (M0060) and propidium iodide (M0793) for staining dead cells. Rh123 has been found to clearly differentiate viable from non-viable bacteria. Rh123 has been shown to stain and discriminate several different species of viable bacteria although this was not universal. Viable cells of Bacillus subtilis were found to stain better with FDA than with Rh123. The use of these stains allow rapid flow cytometric detection and estimation of the viability of bacterial populations. Although BacLight^TMis a popular fluorescence-based two-component stain for determining bacterial cell viability, confusing results can occur if the relative intensities of the stains or the concentration of PI relative to nucleic acid are not properly accounted for. For more information about these stains see our web site or the references below.

Diaper JP, Tither K, Edwards C., (1992) “Rapid assessment of bacterial viability by flow cytometry.” Appl Microbiol Biotechnol. 38(2): 268-72.

Stocks S.M., (2004) “Mechanism and use of the commercially available viability stain, BacLight.” Cytometry 61A(2): 189-95.

López-Amorós R., Castel S., Comas-Riu J., Vives-Rego J., (1997) “Assessment of E. coli and Salmonella viability and starvation by confocal laser microscopy and flow cytometry using rhodamine 123, DiBAC4(3), propidium iodide, and CTC.” Cytometry 29(4): 298-305.

A New Convertible Green-Red Fluorescent Protein

Research from the laboratory of Dr. Jörg Wiedenmann and colleagues at the Department of General Zoology and Endocrinology, University of Ulm, Germany have identified a number of of new fluorescent proteins from an assortment of aquatic sources. Their latest work have provided a gene encoding a fluorescent protein from the stony coral Lobophyllia hemprichii. It has been cloned into E. coli and characterized by biochemical and biophysical methods. The protein, named EosFP, emits strong green fluorescence (516 nm) that changes to red (581 nm) upon near-UV irradiation at approximately 390 nm. Because this photo-induced modification involves a break in the peptide backbone next to the chromophore, the color change is permanent, and has the potential to allow “marking” of individual proteins within living cells, individual cells or tissues. For more information about these new fluorescent proteins, please visit our web site or see the references below.

Wiedenmann J, Ivanchenko S, Oswald F, Schmitt F, Rocker C, Salih A, Spindler KD, Nienhaus GU. “EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion.” Proc Natl Acad Sci U S A. 2004 Oct 25 [pre-print].

http://www.uni-ulm.de/biologie1/Wiedenmann/

Jörg Wiedenmann, Carsten Elke, Klaus-Dieter Spindler, and Werner Funke. (2000) “Cracks in the -can: Fluorescent proteins from Anemonia sulcata (Anthozoa, Actinaria).” Proc. Natl. Acad. Sci. 97: 14091-14096.

Fluorescent GUS Detection with DMFDG.
Marker Gene, in collaboration with researchers at The University of Oregon and the USDA-ARS-Northern Crop Science Laboratory are developing a new tissue assay system for analysis of the GUS reporter gene in plant leaf disk samples. Application of the substrate to leaf tissue samples, whole plant specimens or cells in culture (protoplasts) produces a bright green fluorescence in the presence of the GUS reporter gene which codes for b-glucuronidase. The assay is quick and sensitive, and provides the ability to directly analyze leaf disk samples soaked in a perfusion buffer for GUS activity. An enhanced-permeability substrate DMFDG (M0969) is used, which improves plant cell penetration. The assay system is based upon a microtiterplate format, and can be automated for routine analyses. Please see our web site below for information on this new assay system or for information about our other GUS marker gene assay products (M0240, M0877).

Kain, S.R., Ganguly, S., Current Protocols in Molecular Biology. (1996) p. 9.6.1.

Jefferson R.A., Burgess S.M., Hirsh D., (1986) “beta-Glucuronidase from Escherichia coli as a gene-fusion marker.” Proc. Natl. Acad. Sci. USA. 83(22): 8447-51.

Jefferson R.A., Kavanagh T.A., Bevan M.W., (1987) “GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.” EMBO J. 6: 3901-3907.

Kosugi S., Suzuka I., Ohashi Y., Murakami T., Arai Y., (1991) “Upstream sequences of rice proliferating cell nuclear antigen (PCNA) gene mediate expression of PCNA-GUS chimeric gene in meristems of transgenic tobacco plants.” Nucleic Acids Res 19(7): 1571-6.

Gallagher, S.R.,ed. (1992) “GUS Protocols: Using the GUS Gene as a Reporter of Gene Expression”. Academic Press, New York, NY.

NBD-Hydrazide for Fluorescent Carbohydrate Analysis.

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. A method for the prechromatographic fluorescence derivatization of carbonyl compounds, including reducing carbohydrates, with 7-hydrazino-4-nitro-2,1,3-benzoxadiazole is under development. The reaction products with carbohydrates or aldehydes and ketones fluoresce at wavelengths from 548 to 580 nm with excitation from 450 to 470 nm. The separation and quantitation of the resultant hydrazones can be carried out by TLC and HPLC on silica gel and reversed phase (C-8 or C-18) materials. Detection limits obtained for benzaldehyde by TLC with fluorodensitometric evaluation is near 5 ng/spot and by HPLC and with fluorescence detection near 200 pg. These new techniques will complement our existing Carbohydrate Analysis Detection Kit (M0272) employing reductive amidation with 1,5-EDANS (M0273). If you would like to help us with beta-testing of these new techniques, please contact our technical assistance staff at techservice@markergene.com or visit our website for more information about these new analysis techniques.

H. Koizumi and Y. Suzuki, (1988) “High-performance liquid chromatography of aliphatic aldehydes by means of post-column extraction with fluorometric detection” Journal of Chromatography A 457: 299-307.

Guebitz, G.; Wintersteiger, R.; Frei, R. W., (1984) “Fluorogenic labeling of carbonyl compounds with 7-hydrazino-4-nitro-2,1,3-benzoxadiazole (NBD-H).” Journal of Liquid Chromatography 7(4): 839-54.

New 2005 Catalog Will Be Available Soon.

The 2005 edition of the Marker Gene catalog is in production. Many new products and kits, additional literature references, data and protocols will be included, as well as new information about our old products. 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.

Sign up now!

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.

To add your name to our WebNewsletter mailing list, please use the following link: subscribe@markergene.com.

To unsubscribe from our mailing list, please use the following link: unsubscribe@markergene.com. Your e-mail address will be deleted in 2-3 business days.


	If you are having trouble viewing this email, click on this link. To order any of our products, please go to our ORDER FORM or visit one of our distributors:
	Marker Gene Monthly Newsletter October, 2004 Volume 4, Number 10 © 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.
	Monitoring Transgene Silencing. Industrial and research applications of transgenic plants call for consistent high-level transgene expression. But independent transgenic lines generated with the same construct can often vary by more than 100-fold with respect to transgene expression levels, and gene silencing is frequently observed. Progress has been made in elucidating the molecular mechanisms of such gene silencing, which is typically caused by epigenetic effects (de novo DNA methylation, chromatin condensation, etc.) rather than any sequence alterations in the transferred gene. It is believed that the silencing may be a genome surveillance system that eliminates RNA corresponding to excessively transcribed genes, as a defense against viral infection. The genes for b–glucuronidase (GUS), streptomycin phosphotransferase (SPT), and green fluorescent protein (GFP) have all been used for analysis of such gene expression events. Compounds such as the histone deacetylase inhibitor trichostatin A, the DNA methyltransferase inhibitor 5-azacytidine (5-AzaC) or matrix-attachment regions (MARs) have been used to inhibit these effects. Marker Gene provides several products for quick estimation of marker gene levels including 4-Methylumbelliferyl-b-D-Glucuronide (MUG, M0240), the b-Glucuronidase (GUS) Reporter Gene Activity Detection Kit (M0877) and Fluorescein di-b-D-Glucuronide, di-methyl ester (M0969) for in vivo applications. For more information about these systems, see our web site or the references below. D. Schubert, B. Lechtenberg, A. Forsbach, M. Gils, S. Bahadur, R. Schmidt (2004) ”Silencing in Arabidopsis T-DNA Transformants: The Predominant Role of a Gene-Specific RNA Sensing Mechanism versus Position Effects” The Plant Cell 16: 2561–2572. Lorence A., Verpoorte R., (2004) “Gene transfer and expression in plants.” Methods Mol. Biol. 267: 329-50. Holtorf S., Apel K., Bohlmann H., (1995) “Comparison of different constitutive and inducible promoters for the overexpression of transgenes in Arabidopsis thaliana.” Plant Mol. Biol. 29(4): 637-46.
	Bacterial Viability Stains. Several dyes have been used to discriminate bacterial viability and also quantitate live vs. dead bacterial cells in culture and by using flow cytometry. The ability of a flow cytometer to rapidly assess microbial viability has been investigated using several vital stains including rhodamine 123 (Rh123) (M0542), fluorescein diacetate (FDA) (M0060) and propidium iodide (M0793) for staining dead cells. Rh123 has been found to clearly differentiate viable from non-viable bacteria. Rh123 has been shown to stain and discriminate several different species of viable bacteria although this was not universal. Viable cells of Bacillus subtilis were found to stain better with FDA than with Rh123. The use of these stains allow rapid flow cytometric detection and estimation of the viability of bacterial populations. Although BacLight^TMis a popular fluorescence-based two-component stain for determining bacterial cell viability, confusing results can occur if the relative intensities of the stains or the concentration of PI relative to nucleic acid are not properly accounted for. For more information about these stains see our web site or the references below. Diaper JP, Tither K, Edwards C., (1992) “Rapid assessment of bacterial viability by flow cytometry.” Appl Microbiol Biotechnol. 38(2): 268-72. Stocks S.M., (2004) “Mechanism and use of the commercially available viability stain, BacLight.” Cytometry 61A(2): 189-95. López-Amorós R., Castel S., Comas-Riu J., Vives-Rego J., (1997) “Assessment of E. coli and Salmonella viability and starvation by confocal laser microscopy and flow cytometry using rhodamine 123, DiBAC4(3), propidium iodide, and CTC.” Cytometry 29(4): 298-305.
	A New Convertible Green-Red Fluorescent Protein Research from the laboratory of Dr. Jörg Wiedenmann and colleagues at the Department of General Zoology and Endocrinology, University of Ulm, Germany have identified a number of of new fluorescent proteins from an assortment of aquatic sources. Their latest work have provided a gene encoding a fluorescent protein from the stony coral Lobophyllia hemprichii. It has been cloned into E. coli and characterized by biochemical and biophysical methods. The protein, named EosFP, emits strong green fluorescence (516 nm) that changes to red (581 nm) upon near-UV irradiation at approximately 390 nm. Because this photo-induced modification involves a break in the peptide backbone next to the chromophore, the color change is permanent, and has the potential to allow “marking” of individual proteins within living cells, individual cells or tissues. For more information about these new fluorescent proteins, please visit our web site or see the references below. Wiedenmann J, Ivanchenko S, Oswald F, Schmitt F, Rocker C, Salih A, Spindler KD, Nienhaus GU. “EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion.” Proc Natl Acad Sci U S A. 2004 Oct 25 [pre-print]. http://www.uni-ulm.de/biologie1/Wiedenmann/ Jörg Wiedenmann, Carsten Elke, Klaus-Dieter Spindler, and Werner Funke. (2000) “Cracks in the -can: Fluorescent proteins from Anemonia sulcata (Anthozoa, Actinaria).” Proc. Natl. Acad. Sci. 97: 14091-14096.
	Fluorescent GUS Detection with DMFDG. Marker Gene, in collaboration with researchers at The University of Oregon and the USDA-ARS-Northern Crop Science Laboratory are developing a new tissue assay system for analysis of the GUS reporter gene in plant leaf disk samples. Application of the substrate to leaf tissue samples, whole plant specimens or cells in culture (protoplasts) produces a bright green fluorescence in the presence of the GUS reporter gene which codes for b-glucuronidase. The assay is quick and sensitive, and provides the ability to directly analyze leaf disk samples soaked in a perfusion buffer for GUS activity. An enhanced-permeability substrate DMFDG (M0969) is used, which improves plant cell penetration. The assay system is based upon a microtiterplate format, and can be automated for routine analyses. Please see our web site below for information on this new assay system or for information about our other GUS marker gene assay products (M0240, M0877). Kain, S.R., Ganguly, S., Current Protocols in Molecular Biology. (1996) p. 9.6.1. Jefferson R.A., Burgess S.M., Hirsh D., (1986) “beta-Glucuronidase from Escherichia coli as a gene-fusion marker.” Proc. Natl. Acad. Sci. USA. 83(22): 8447-51. Jefferson R.A., Kavanagh T.A., Bevan M.W., (1987) “GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.” EMBO J. 6: 3901-3907. Kosugi S., Suzuka I., Ohashi Y., Murakami T., Arai Y., (1991) “Upstream sequences of rice proliferating cell nuclear antigen (PCNA) gene mediate expression of PCNA-GUS chimeric gene in meristems of transgenic tobacco plants.” Nucleic Acids Res 19(7): 1571-6. Gallagher, S.R.,ed. (1992) “GUS Protocols: Using the GUS Gene as a Reporter of Gene Expression”. Academic Press, New York, NY.
	NBD-Hydrazide for Fluorescent Carbohydrate Analysis. 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. A method for the prechromatographic fluorescence derivatization of carbonyl compounds, including reducing carbohydrates, with 7-hydrazino-4-nitro-2,1,3-benzoxadiazole is under development. The reaction products with carbohydrates or aldehydes and ketones fluoresce at wavelengths from 548 to 580 nm with excitation from 450 to 470 nm. The separation and quantitation of the resultant hydrazones can be carried out by TLC and HPLC on silica gel and reversed phase (C-8 or C-18) materials. Detection limits obtained for benzaldehyde by TLC with fluorodensitometric evaluation is near 5 ng/spot and by HPLC and with fluorescence detection near 200 pg. These new techniques will complement our existing Carbohydrate Analysis Detection Kit (M0272) employing reductive amidation with 1,5-EDANS (M0273). If you would like to help us with beta-testing of these new techniques, please contact our technical assistance staff at techservice@markergene.com or visit our website for more information about these new analysis techniques. H. Koizumi and Y. Suzuki, (1988) “High-performance liquid chromatography of aliphatic aldehydes by means of post-column extraction with fluorometric detection” Journal of Chromatography A 457: 299-307. Guebitz, G.; Wintersteiger, R.; Frei, R. W., (1984) “Fluorogenic labeling of carbonyl compounds with 7-hydrazino-4-nitro-2,1,3-benzoxadiazole (NBD-H).” Journal of Liquid Chromatography 7(4): 839-54.
	New 2005 Catalog Will Be Available Soon. The 2005 edition of the Marker Gene catalog is in production. Many new products and kits, additional literature references, data and protocols will be included, as well as new information about our old products. 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. Sign up now!
	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.
	To add your name to our WebNewsletter mailing list, please use the following link: subscribe@markergene.com. To unsubscribe from our mailing list, please use the following link: unsubscribe@markergene.com. Your e-mail address will be deleted in 2-3 business days.