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Marker Gene Monthly Newsletter

November, 2004

Volume 4, Number 11

© Copyright MGT, Inc., 2004. 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.

Cell Marking with EosF.

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 new fluorescent proteins from a variety of aquatic sources. In collaboration with the group of Prof. Uli Nienhaus at the Department of Biophysics, University of Ulm, Germany, these proteins have been characterized, evaluated and optimized for use as fluorescent markers. Their latest work has provided a gene encoding a fluorescent protein from the stony coral Lobophyllia hemprichii. This new protein, named EosFP, emits strong green fluorescence (516 nm) that changes to red (581 nm) upon near-UV irradiation at approximately 390 nm. This photo-induced modification is permanent because it involves a break in the peptide backbone next to the chromophore. The use of EosFP enables localized marking of individual proteins within live cells or tissues. Variants with reduced oligomerization tendency have been produced by protein engineering. Marker Gene is in discussions to provide this new protein for research use. For more information about these new fluorescent proteins, please visit our web site or see the references below.

• Wiedenmann, J., Ivanchenko, S., Oswald, F., and Nienhaus G. U. (2004) “Identification of GFP-like proteins in non-bioluminescent, azooxanthellate Anthozoa opens new perspectives for bioprospecting.” Marine Biotechnology 6: 270-277.

• Wiedenmann, J., Schenk, A., Röcker, C., Spindler, K.-D., and Nienhaus, G. U. (2002) “A far-red fluorescent protein with fast maturation and reduced oligomerization tendency from Entacmaea quadricolor (Cnidaria, Anthozoa, Actinaria).” Proc. Natl. Acad. Sci. U.S.A. 99: 11646-11651.

GUS Activity Measurements in Seeds and Seed Tissues.

Fluorescent and chromogenic analysis of GUS activity in plant leaves and seeds can be performed by a number of techniques, including the leaf-disk assay, spray application of substrates or even whole plant assays. Analysis of GUS activity in whole seed typically requires the use of an extraction buffer system, or physical manipulation of the seed tissues (lateral dissection) in order to obtain penetration of the substrates and record either fluorescence (4-MU) or photograph indigo (blue dye) chromogenic stains. Our b-Glucuronidase (GUS) Reporter Gene Activity Detection Kit (M0877) contains all of the reagents and buffers necessary to analyze seed and seed tissues using the fluorogenic substrate (4-MUG, M0240). An automated “crush and stain” method has been developed that uses a protocol equivalent to that described in our kit M0877. Please see the references below or visit our website for more information about analysis of this important marker gene in plant seed tissues.

• Y. J. Zhu, R. Agbayani and : H. Moore (2003) "Green fluorescent protein as a visual selection marker for papaya (Carica papaya L.) transformation"  Plant Cell Reports 22(9): 660 - 667.
• Stangeland, B., Mandal, A., Olsen, O.A., Salehian, Z., Aalen, R., (2003) “Isolation of GUS marker lines for genes expressed in Arabidopsis endosperm, embryo and maternal tissues.” Journal of experimental botany 54(381): 279-290.
• Tsomlexoglou E., Daulagala :W., Gooday G.W., Glover L.A., Seddon B., Allan E.J. (2003) "Molecular detection and beta-glucuronidase expression of GUS-marked Bacillus subtilis L-form bacteria in developing Chinese cabbage seedlings."  J. Appl. Microbiol. 95(2): 218-24.
• Kusnadi A.R., Evangelista R.L., Hood E.E., Howard J.A., Nikolov Z.L., (1998) "Processing of transgenic corn seed and its effect on the recovery of recombinant beta-glucuronidase." Biotechnol. Bioeng. 60(1): 44-52.
• Wang H., Cutler A.J., (1995) "Promoters from kin1 and cor6.6, two Arabidopsis thaliana low-temperature- and ABA-inducible genes, direct strong beta-glucuronidase expression in guard cells, pollen and young developing seeds."   Plant Mol. Biol. 28(4): 619-34.

lacZ Nuclear Localization Vectors.

Dr. Donald Anson and coworkers at the Department of Chemical Pathology, Women's and Children's Hospital in North Adelaide, Australia have developed several new lacZ expression vectors with high expression efficiency in mammalian cells. The coding sequence for the E. coli beta-galactosidase gene has been codon-optimised and when expressed in mammalian cells results in the expression of beta-galactosidase at levels 15-fold higher than those resulting from an analogous construct containing the native E. coli gene sequence. Two of the new vectors, pCMVblacZNLS12co (Product M1017); and pSV40blacZNLS12co (Product M1018) also contain a nuclear localization sequence that targets the expressed genes (lacZ) into the nucleus of mammalian cells. Nuclear localization signal (NLS)–containing proteins are typically transported by importins across the nuclear membrane. These new vectors contain a 12 amino acid sequence from the SV40 T antigen nuclear localization signal that is positioned directly after the methionine initiation signal. For more information on these new vector systems, see the references below or visit the Marker Gene website.

• Anson, D.S., Limberis, M., 2004. “An improved β-galactosidase reporter gene.” J. Biotechnol. 108:17-30.
• Hall, C.V. et al., 1983. “Expression and Regulation of Escherichia coli lacZ Gene Fusions in Mammalian Cells.” J. Mol. Appl. Gen. 2:101.
• Nolan G.P., Fiering S., Nicolas J.F., Herzenberg L.A., “Fluorescence-Activated Cell Analysis and Sorting of Viable Mammalian Cells Based on ß -D-galactosidase Activity after Transduction of Escherichia coli lacZ” (1988) Proc. Natl. Acad. Sci. USA 85: 2603-2607.
• Kalderon,D., Roberts,B.L., Richardson,W.D.,Smith, A.E.(1984)“A short amino acid sequence able to specify nuclear localization” Cell 39: 499-509.

• Lan Xu & Joan Massagué (2004) “Nucleocytoplasmic Shuttling of Signal Transducers” Nature Reviews Mol. Cell Biol. 5: 209 –219.

A “Read-Write” Fluorescent Protein – DRONPA.

FRAP (fluorescence recovery after photobleaching) has long been used to track protein movements inside living cells and tissues. But such systems are slow, and their ability to be reused repeatedly is limited. Recently Dr. Atsushi Miyawaki and colleagues at the Laboratory for Cell Function and Dynamics, RIKEN Brain Science Institute in Japan have isolated a new fluorescent protein from the coral Pectiniidae that has the ability to be repeatedly highlighted and erased by activation at 405 nm and 488 nm respectively, without appreciable photobleaching. These properties allow such GFP proteins to be used for intracellular protein tracing repeatedly or for protein movements within individual cells or tissues. A monomeric version of the protein with both rational and random mutations was cloned into E. coli and also mammalian cells (HeLa cells) by cloning into a pcDNA3 vector. The system has been used to monitor nuclear importation, efflux, and movement of a protein kinase (ERK) using a fusion protein approach. The protein also has potential as an information storage medium with the ability to record, erase, or reread information nondestructively. For more information about these exciting new techniques, please see the references below or visit our website.

• R. Ando, H. Mizuno, A. Miyawaki (2004) “Regulated Fast Nucleocytoplasmic Shuttling Observed by Reversible Protein Highlighting” Science, 306: 1370-1373.
• Irie M., Fukaminato T., Sasaki T., Tamai N., Kawai T., (2002) “Organic chemistry: A digital fluorescent molecular photoswitch” NATURE 420(6917): 759-760.
• Chudakov D.M., Belousov VV., Zaraisky A.G., Novoselov V.V., Staroverov D.B., Zorov D.B., Lukyanov S., Lukyanov K.A., (2003) “Kindling fluorescent proteins for precise in vivo photolabeling.” Nat. Biotechnol. 21(2):191-4.
• R. Ando, H. Hama, M. Yamamoto-Hino, H. Mizuno, and A. Miyawaki (2002) “An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein” Proc. Natl. Acad. Sci. USA 99(20): 12651-12656.

Genetic Analysis using RNAi.

RNA interference (RNAi) is a potent method of manipulating gene expression that uses only a few double-stranded RNA (dsRNA) molecules per cell to silence the expression of a target gene. Previous gene “knock-outs” using antisense DNA, dominant negative or other techniques were ineffective, but the discovery of RNAi has enabled researchers to knock out a gene in any organism efficiently. RNA silencing was first discovered in transgenic plants, where it was termed co-suppression or post transcriptional gene silencing (PTGS). The evidence for RNA silencing then emerged from experimental observation on Caenorhabditis elegans. This new approach for achieving efficient, targeted gene silencing is a quick and easy way to determine the function of a gene in a whole cell or animal system and can be up to 1,000-fold more effective than antisense techniques. RNA silencing is a sequence specific RNA degradation process that is triggered by the formation of double stranded RNA. Complementary sequences of single stranded RNA are introduced by viral methods or as transgenes. Duplexes of 21-bp nucleotide RNAs with symmetric 2-nucleotide 3’overhangs mediate the degradation of mRNA, and block expression. The method takes advantage of a natural defense mechanism in cells to prevent viral infection that triggers a cellular process for degradation. Vectors containing a variety of marker genes (lacZ, luc, amp, etc.) have been used in constructs for expression and testing of the RNAi systems. For more information about these exciting new techniques for therapeutic and research applications, see the references below or visit our website.

· Wadhwa R., Kaul S.C., Miyagishi M., Taira K., (2004) “Vectors for RNA interference.” Curr. Opin. Mol. Ther. 6(4): 367-72.

· Brown, D., Jarvis, R., Pallotta, V., Byrom, M., Ford, L. 2002 “RNA interference in mammalian cell culture: Design, execution and analysis of the siRNA effect.” Technology Notes, 9(1): 3-5.

· Brummelkamp, T.R., Bernards, R. and Agami, R. 2002 “A system for stable expression of short interfering RNAs in mammalian cells.” Science, 296(5567): 550-553.

· Caplen, N.J., Parrish, S., Imani, F., Fire, A., Morgan, R.A. (2001) “Specific inhibition of gene expression by small double stranded RNAs in invertebrate and vertebrate systems.” PNAS 98(17): 742-9747.

· Grishok, A., Tabar, H. and Mello, C.C. (2000) “Genetic requirements for inheritance of RNAi in C. elegans.” Science 287(5462): 2494-2497.

· Grosshans, H. and Slack, F.J. 2002 “Micro RNAs: small is plentiful.” J. Cell Biology 156(1): 17-21.

· Guru, T. (2000) “A silence that speaks volumes.” Nature 404(6780): 804-808.

· Hutvagner, G. and Zamore, D., (2002) “RNAi: nature abhors a double-strand.” Current Opinion in Genetics and Development. 12(2): 225-232.

· Jarvis, R.A. and Ford, L. (2001) “The siRNA target site is an important parameter for inducing RNAi in human cells.” TechNotes, 8(5): 3-5.

· Lau, N.C., Lim, L., Weinstein, E.G. and Barteli, D. (2001) “An abundant class of tiny RNAs with probable regulatory roles in C. elegans.” Science 294(5543): 858-862.

· Lee, N.S., Dohjima, T., Bauer, G., Li, H., LIi M.J., Ehasani, A., Salvaterra, Rossi, J., (2002) “Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells.” Nature Biotech. 20(5): 500-505.

· Lee, R.C. and Ambrose, V. (2001) “An extensive class of small RNAs in C. elegans.” Science 294(5545): 862-864.

· Paddison, J., Claudy, A.A., Bernstein, E., Hannon, G.J., Conklin, D.S. “Short hairpin RNAs (shRNAs) induce sequence specific silencing in mammalian cells.” Genes Dev. (2002) 16(8): 948-958.

· Roberts, J., “High thoroughput gene knockout.” The Scientist, (2002) 16(9): 35-36.

· Silhavy, D., Molnar, A., Lucioloi, A., Szittya, G., Hornyik, C., Tavazza, M., Burgyan, J. (2002) “A viral protein suppresses RNA silencing and binds silencing generated, 21 to 25 nucleotide double stranded RNAs.” EMBO Journal 21(12): 3070-3080.

· Sui, G., Sohoo, C., Affar, E.B., Gav, F., Shi, Y., Forrester, W.C., Shi, Y. (2002) “A DNA vector based RNAi technology to suppress gene expression in mammalian cells.” PNAS 99(6): 5515-5520.

· Xu, Z., Friess, H., Solioz, M., Aebi, S., Korc, M., Kleeff, J. Buchler, M.W. (2001) “BCL-X_L antisense oligonucleotides induce apoptosis and increase sensitivity of pancreatic cancer cells to gemcitabine.” Int. J. Cancer 94(2): 268-274.

· Yu, J.Y., Deruiter, S.L., Turner, D.L. (2002) “RNA interference by expression of short interfering RNAs and hairpin RNAs in mammalian cells.” PNAS 99(9): 6047-6052.

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.

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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 UO 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.
• Enzyme substrate synthesis / coupled assay design.
• Effector, Agonist, Antagonist, Inhibitor design, synthesis, and in vitro assays.
• Chromogenic, Chemiluminescent, and Fluorescence Based Technologies.
• New selection, effector and reporter uses for marker genes in Research, Cell Culture, Diagnostics and Therapeutics.
• 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; (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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