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Marker Gene Monthly Newsletter

March, 2003

Volume 3, Number 3

© 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.

a-Galactosidase Marker Gene for Yeast.

The yeast two-hybrid screen is a genetic method of detecting protein-protein interactions in vivo. Positive clones are selected by their ability to activate the transcription of a reporter gene, which also enables them to grow on nutritionally selective media. Most two-hybrid methods use the E. coli lacZ gene as the reporter gene. Usually the colonies growing on the selection plates are assayed for the activation of the reporter gene lacZ by a filter-lift assay. Marker Gene currently produces several substrates that are useful for b-Gal detection in yeast strains (M0250, FDG; M0203, Resorufin-Gal; M0252, TFMU-Gal, M0257, CUG). Recently yeast strains producing the a-galactosidase marker gene have been developed and used to assay GAL4-based two-hybrid interactions directly on nutritional selection plates with the chromogenic substrate X-a-Gal. Look for new products from Marker Gene in this area in the near future. For more information about these techniques, see the references below.

S. Aho, A. Arffman, T.Pummi and J. Uitto, A novel reporter gene MEL1 for the yeast two-hybrid system. Anal. Biochem., 253, 270-272 (1997).

P. Chevalier, D. Roy and and L. Savoie, “X-a-gal-based medium for simultaneous enumeration of bifidobacteria and lactic acid bacteria in milk.”, J.Microbiol. Meth., 13, 75 (1991).

R. Gossrau and Z. Lojda, “Histochemical detection of a-D-galactosidase with 5-Br-4-Cl-3-indoxyl a-D-galactoside.” Acta Histochem., 85, 213 (1989).

R.S. Tubb and P.L. Liljestrom, “A colony-colour method which differentiates a-galactosidase positive strains of yeast.” J. Inst. Brew. 92, 588, (1986).

New 800 Number for Marker Gene!

Marker Gene now has a toll-free telephone number to help you to contact us. Give us a call with your questions about using new and robust marker genes, selection markers or reporter gene systems in your live cell, tissue, primary cell culture or in in vitro assays or for other information about your molecular biology or enzymology protocols. We are happy to help! Call us at:

1-888-218-4062

Of course, you can still contact us at techservice@markergene.com, by FAX at 1-541-687-7963; 1-541-342-1960: 1-541-344-1624 or by visiting our Web site at http://www.markergene.com for more information about our products, contract research or custom synthesis capabilities at MGT or for additional contact information. Let us help with your assay development today.

Toward a Marker Gene for Colorectal Tumors.

The long-standing hope of finding specific reporter genes that are upregulated in tumors has recently shown renewed promise in work published by Dr. Andrew Feinberg and his group at Johns Hopkins University School of Medicine. By examining the DNA methylation patterns of patients with genetic predisposition to colorectal cancer or who have been diagnosed with the disease, they found statistically higher levels of gene silencing in the gene IGF2 (insulin-like growth factor 2) in these patients (5X or 21X higher respectively). These assays were performed using a reverse transcription of mRNA isolated by biopsy from colonoscopy tissue samples, using a PCR format with specific primers. The levels of methylation were analyzed by a bisulfite genomic sequencing method. For more information about these techniques, please see the references below.

H. Cui, M. Cruz-Correa, F. M. Giardiello, D. F. Hutcheon, D. R. Kafonek, S. Brandenburg, Y. Wu, X. He, N. R. Powe, and A. P. Feinberg, “Loss of IGF2 Imprinting: A Potential Marker of Colorectal Cancer Risk” Science 299 (2003) 1753-55.

H. Uejima, M. P. Lee, H. Cui, and A. P. Feinberg, Nature Genet. 25, 375-376 (2000).

Feinberg, AP: “Genomic imprinting and cancer. In The Metabolic and Molecular Bases of Inherited Disease”, 8th ed. Scriver CR, Beaudet AR, Sly W, Valle D (eds), McGraw-Hill, New York, pp 525-537, 2001.

Hydrophobic Stains for 2D-SDS-PAGE.
The ability to stain proteins inside SDS-PAGE gels offers a significant advantage over chemical labeling techniques or blotting / post-electrophoresis staining and destaining methods. John Shultz and Gregg Larson (Promega Corp., Madison, WI) recently introduced a number of hydrophobic analogs of fluorescent dyes (fluorescein, Dansyl chloride, carbocyanines, etc.) that can be used for direct and quantitative labeling of proteins inside polyacrylamide gels. The stains bind to the SDS hydrophobic protein coat and exhibit low background fluorescence. For more information about these stains and techniques, please see the references below.
Larson, Gregg A.; Shultz, John W., “Applications Of The Chromaphor Protein Recovery System.” BioTechniques, 15:316-323.

Shultz, John W., Larson, Gregg A.., “Protein Staining Compositions and Methods”, US Patent 5,705,649 (1998).

Kendrick, N., “Laser scanning quantification of 2-D gel spots using ChromaPhor^TM Green Stain”, Promega Notes Magazine 37 (1992) 11.

AHAS Reporter Gene in Plants.

Acetohydroxyacid synthase (AHAS, EC 4.1.3.18) also known as acetolactate synthase, is an herbicide-resistance selection marker used in plant cell studies. It catalyzes the first step in branched-chain amino-acid (valine, leucine and isoleucine) biosynthesis, and when a modified version is cloned into plants, detoxifies the inhibition of the enzyme by sulfonylurea herbicides slufometuron methyl and chlorosuluron (marketed by Du Pont as the active ingredients in Oust and Glean, respectively) and the inidazolinone herbicides 2-(4-isopropyl-4-methyl-5-oxo-2-inidazolin-2-yl)-nicotinic acid and 2-(4-isopropyl-4-methyl-5-oxo-2-inidazolin-2-yl)-3-quinolinecarboxylic acid (marketed by American Cyanamid as the active ingredients in Arsenal and Scepter, respectively). The recombinant plants carry a mutation (changing the serine (coded by AGT) at position 621 to asparagine (AAT), which provides a relief of the inhibition by these herbicides. An assay of this AHAS reporter has been developed, involving conversion of the enzymatic product acetolactate to acetoin, followed by detection as a complex with creatine and naphthol. For more information about this selection marker gene in plants see the references below.

Singh, B.K., Stidham, M.A., Shaner, D.L., “Assay of Acetohydroxyacid Synthase” Anal. Biochem. 171 (1988) 173-179.

Hill, C.M., Pang, S.S., Duggleby, R.G., “Purification of E.Coli acetohydroxyacid synthase isoenzyme II and reconstitution of active enzyme from its individual pure subunits.” Biochem. J. 327 (1997) 891-898.

Schloss, J.V., Van Dyk, D.E., Vasta,. J.F., Kutny, R.M., “Purification and Properties of Salmonella typhimurium Acetolactate Synthase Isozyme II from E. Coli HB101/pDU9”, Biochemistry 24 (1985) 4952-4959.

Zhu T, Peterson DJ, Tagliani L, St. Clair G, Baszczynski CL, and Bowen B. 1999. “Targeted manipulation of maize genes in vivo using chimeric RNA/DNA oligonucleotides.” Proceedings of the National Academy of Science 96:8768-8773.

Zhu T, Mettenburg K, Peterson DJ, Tagliani L, and Baszczynski CL. 2000. “Engineering herbicide-resistant maize using chimeric RNA/DNA oligonucleotides.” Nature Biotechnology 18:555-558.

Over 50 New Products and Kits in the New Catalog!

The 2003-2004 edition of the Marker Gene catalog is due from the printers in a few weeks. 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 March, 2003 Volume 3, Number 3 © 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.
	a-Galactosidase Marker Gene for Yeast. The yeast two-hybrid screen is a genetic method of detecting protein-protein interactions in vivo. Positive clones are selected by their ability to activate the transcription of a reporter gene, which also enables them to grow on nutritionally selective media. Most two-hybrid methods use the E. coli lacZ gene as the reporter gene. Usually the colonies growing on the selection plates are assayed for the activation of the reporter gene lacZ by a filter-lift assay. Marker Gene currently produces several substrates that are useful for b-Gal detection in yeast strains (M0250, FDG; M0203, Resorufin-Gal; M0252, TFMU-Gal, M0257, CUG). Recently yeast strains producing the a-galactosidase marker gene have been developed and used to assay GAL4-based two-hybrid interactions directly on nutritional selection plates with the chromogenic substrate X-a-Gal. Look for new products from Marker Gene in this area in the near future. For more information about these techniques, see the references below. S. Aho, A. Arffman, T.Pummi and J. Uitto, A novel reporter gene MEL1 for the yeast two-hybrid system. Anal. Biochem., 253, 270-272 (1997). P. Chevalier, D. Roy and and L. Savoie, “X-a-gal-based medium for simultaneous enumeration of bifidobacteria and lactic acid bacteria in milk.”, J.Microbiol. Meth., 13, 75 (1991). R. Gossrau and Z. Lojda, “Histochemical detection of a-D-galactosidase with 5-Br-4-Cl-3-indoxyl a-D-galactoside.” Acta Histochem., 85, 213 (1989). R.S. Tubb and P.L. Liljestrom, “A colony-colour method which differentiates a-galactosidase positive strains of yeast.” J. Inst. Brew. 92, 588, (1986).
	New 800 Number for Marker Gene! Marker Gene now has a toll-free telephone number to help you to contact us. Give us a call with your questions about using new and robust marker genes, selection markers or reporter gene systems in your live cell, tissue, primary cell culture or in in vitro assays or for other information about your molecular biology or enzymology protocols. We are happy to help! Call us at: 1-888-218-4062 Of course, you can still contact us at techservice@markergene.com, by FAX at 1-541-687-7963; 1-541-342-1960: 1-541-344-1624 or by visiting our Web site at http://www.markergene.com for more information about our products, contract research or custom synthesis capabilities at MGT or for additional contact information. Let us help with your assay development today.
	Toward a Marker Gene for Colorectal Tumors. The long-standing hope of finding specific reporter genes that are upregulated in tumors has recently shown renewed promise in work published by Dr. Andrew Feinberg and his group at Johns Hopkins University School of Medicine. By examining the DNA methylation patterns of patients with genetic predisposition to colorectal cancer or who have been diagnosed with the disease, they found statistically higher levels of gene silencing in the gene IGF2 (insulin-like growth factor 2) in these patients (5X or 21X higher respectively). These assays were performed using a reverse transcription of mRNA isolated by biopsy from colonoscopy tissue samples, using a PCR format with specific primers. The levels of methylation were analyzed by a bisulfite genomic sequencing method. For more information about these techniques, please see the references below. H. Cui, M. Cruz-Correa, F. M. Giardiello, D. F. Hutcheon, D. R. Kafonek, S. Brandenburg, Y. Wu, X. He, N. R. Powe, and A. P. Feinberg, “*Loss of IGF2* Imprinting: A Potential Marker of Colorectal Cancer Risk” Science 299 (2003) 1753-55.** H. Uejima, M. P. Lee, H. Cui, and A. P. Feinberg, Nature Genet. 25, 375-376 (2000). Feinberg, AP: “Genomic imprinting and cancer. In The Metabolic and Molecular Bases of Inherited Disease”, 8th ed. Scriver CR, Beaudet AR, Sly W, Valle D (eds), McGraw-Hill, New York, pp 525-537, 2001.
	Hydrophobic Stains for 2D-SDS-PAGE. The ability to stain proteins inside SDS-PAGE gels offers a significant advantage over chemical labeling techniques or blotting / post-electrophoresis staining and destaining methods. John Shultz and Gregg Larson (Promega Corp., Madison, WI) recently introduced a number of hydrophobic analogs of fluorescent dyes (fluorescein, Dansyl chloride, carbocyanines, etc.) that can be used for direct and quantitative labeling of proteins inside polyacrylamide gels. The stains bind to the SDS hydrophobic protein coat and exhibit low background fluorescence. For more information about these stains and techniques, please see the references below. Larson, Gregg A.; Shultz, John W., “Applications Of The Chromaphor Protein Recovery System.” BioTechniques, 15:316-323. Shultz, John W., Larson, Gregg A.., “Protein Staining Compositions and Methods”, US Patent 5,705,649 (1998). Kendrick, N., “Laser scanning quantification of 2-D gel spots using ChromaPhor^TM Green Stain”, Promega Notes Magazine 37 (1992) 11.
	AHAS Reporter Gene in Plants. Acetohydroxyacid synthase (AHAS, EC 4.1.3.18) also known as acetolactate synthase, is an herbicide-resistance selection marker used in plant cell studies. It catalyzes the first step in branched-chain amino-acid (valine, leucine and isoleucine) biosynthesis, and when a modified version is cloned into plants, detoxifies the inhibition of the enzyme by sulfonylurea herbicides slufometuron methyl and chlorosuluron (marketed by Du Pont as the active ingredients in Oust and Glean, respectively) and the inidazolinone herbicides 2-(4-isopropyl-4-methyl-5-oxo-2-inidazolin-2-yl)-nicotinic acid and 2-(4-isopropyl-4-methyl-5-oxo-2-inidazolin-2-yl)-3-quinolinecarboxylic acid (marketed by American Cyanamid as the active ingredients in Arsenal and Scepter, respectively). The recombinant plants carry a mutation (changing the serine (coded by AGT) at position 621 to asparagine (AAT), which provides a relief of the inhibition by these herbicides. An assay of this AHAS reporter has been developed, involving conversion of the enzymatic product acetolactate to acetoin, followed by detection as a complex with creatine and naphthol. For more information about this selection marker gene in plants see the references below. Singh, B.K., Stidham, M.A., Shaner, D.L., “Assay of Acetohydroxyacid Synthase” Anal. Biochem. 171 (1988) 173-179. Hill, C.M., Pang, S.S., Duggleby, R.G., “Purification of E.Coli acetohydroxyacid synthase isoenzyme II and reconstitution of active enzyme from its individual pure subunits.” Biochem. J. 327 (1997) 891-898. Schloss, J.V., Van Dyk, D.E., Vasta,. J.F., Kutny, R.M., “Purification and Properties of Salmonella typhimurium Acetolactate Synthase Isozyme II from E. Coli HB101/pDU9”, Biochemistry 24 (1985) 4952-4959. Zhu T, Peterson DJ, Tagliani L, St. Clair G, Baszczynski CL, and Bowen B. 1999. “Targeted manipulation of maize genes in vivo using chimeric RNA/DNA oligonucleotides.” Proceedings of the National Academy of Science 96:8768-8773. Zhu T, Mettenburg K, Peterson DJ, Tagliani L, and Baszczynski CL. 2000. “Engineering herbicide-resistant maize using chimeric RNA/DNA oligonucleotides.” Nature Biotechnology 18:555-558.
	Over 50 New Products and Kits in the New Catalog! The 2003-2004 edition of the Marker Gene catalog is due from the printers in a few weeks. 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.