Product Focus: pTARGET™ Vector---Reverse Transcriptase---PolyATtract^® mRNA Isolation System

Creation of genome-wide protein expression libraries using random activation of gene expression.

This paper describes a technique for generating normalized, genome-wide transcript and expression libraries termed Random Activation of Gene Expression (RAGE). The method uses a series of specialized integration vectors to express protein from endogenous genes in mammalian cells. The RAGE vectors contain a promoter linked to an exon and an unpaired splice donor site. The pTARGET™ Vector^(a) (Cat.# A1410) was used as the backbone for the RAGE vectors, pRIG-1 and derivatives, and pRIG-20. When transfected, the vector can integrate into the host cell genome via nonhomologous recombination. Random integration of the vector is promoted by low-dose irradiation of cells prior to transfection. If integration of the vector occurs either within or upstream of an endogenous gene, the vector will produce a chimeric transcript in which one or more of the exons of the endogenous gene is replaced by the vector-encoded exon. To test the activation of genes using the RAGE vectors, a library of 5 x 10⁶ clones in human HT1080 cells was screened. A set of genes consisting of two transcriptionally active genes and 19 genes that are silenced in HT1080 cells was surveyed for activation. Despite undetectable levels of expression in parental HT1080 cells, all 19 of the silenced genes were activated in at least one pool of clones, and most were present in multiple pools. Since normally silent genes were activated in the RAGE library, the technique was assessed to determine its utility in detecting previously unknown genes. A 5-million clone library was produced, the library was divided into pools of approximately 25,000 clones each, and vector-tagged cDNA molecules were cloned and sequenced from these pools to produce RAGE sequence tags (RSTs). Out of an initial group of 75,003 clones sequenced at random, 19,547 unique gene clusters were identified, with 53% of those gene clusters identified as novel when compared to sequences in GenBank^®. These libraries also appear to be highly normalized, as a majority of the unique clone isolates appeared no more than twice in the screen of 75,000 clones. This suggests that most genes will be isolated at similar frequencies using the RAGE method. Besides using pTARGET™ Vector as the backbone of the RAGE vectors, this group used Promega's M-MLV Reverse Transcriptase^(b) (Cat.# M1705) in RT-PCR to confirm expression of known genes in clone pools, and the PolyATtract^® mRNA Isolation System^(b) (Cat.# Z5300) to purify mRNA from RAGE libraries for generation of RSTs.

Harrington, J.J.*, Sherf, B., Rundlett, S., Jackson, P.D., Perry, R., Cain, S., Leventhal, C., Thornton, M., Ramachandran, R., Whittington, J., Lerner, L., Costanzo, D., McElligott, K., Boozer, S., Mays, R., Smith, E., Veloso, N., Klika, A., Hess, J., Cothren, K., Lo, K., Offenbacher, J., Danzig J. and Ducar, M. (2001) Nature Biotechnol. 19, 440–445.

Athersys, Inc., 3201 Carnegie Ave., Cleveland, OH  44115 USA.

*To whom correspondence should be addressed. jharrington@athersys.com

Product Focus: Anti-ACTIVE^® MAPK Antibodies

A hairpin binding site in tobacco plasma membranes mediates activation of the pathogenesis-related gene HIN1 independent of extracellular calcium but dependent on mitogen-activated protein kinase activity.

Plant cell defensive responses to bacterial pathogens are activated by bacterial effector proteins. One group of these proteins are the hairpins. The role of this type of protein has been unclear, but hairpins have been shown to elicit disease-resistance responses such as the hypersensitive reaction (HR), accumulation of pathogenesis-related (PR) transcripts and systemic acquired resistance when infiltrated into nonhost plants. Tobacco cell response to bacterial effector proteins was studied to determine the pathways activated in disease-associated responses. An effector protein from Pseudomonas syringae pv phaseolicola (hairpin Psph) was shown to bind to the plasma membrane of tobacco cells and activate the pathogenesis related HIN1 gene. Hairpin Psph induction of HIN1 was compared to the activity of another known inducer of HIN1 expression, the Phytophthora megasperma-derived b-elicitin, b-megaspermin. b-megaspermin induces HIN1 via an increase in cytoplasmic free calcium concentrations. In contrast, hairpin Psph activation of the salicylic acid-responsive MAPK (SIPK) and HIN1 transcript accumulation was independent of extracellular calcium concentrations. Hairpin Psph-treated cells incubated with the MEK Inhibitor U0126 (Cat.# V1121) showed a decrease in HIN1 and other PR gene expression levels. The Anti-ACTIVE^® MAPK pAb (Cat.# V8031) was used to show that SIPK activity was activated rapidly and transiently by hairpin Psph stimulation of tobacco cells.

Lee, J.¹, Klessig, D.F.², and Nürnberger, T.^1* (2001) Plant Cell 13, 1079–1093.

¹Department of Stress and Developmental Biology, Leibniz Institute of Plant Biochemistry, Weinberg 3, D-06120 Halle/Saale, Germany, ²Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, NJ  08854-8020 USA.

*To whom correspondence should be addressed. tnuernbe@ipb-halle.de

Product Focus: RiboMAX™ Large Scale RNA Production System

A Drosophila IkappaB kinase complex required for relish cleavage and antibacterial immunity.

The Rel family of proteins is essential in human innate immunity and in the Drosophila immune response. In Drosophila, two pathways have been identified that lead to activation of the immune response. The antifungal response proceeds using the Toll receptor pathway, eventually resulting in the nuclear localization of the Rel proteins Dif and Dorsal. The antibacterial response involves another Drosophila Rel protein, Relish, a homolog of the precursor of NFkappaB p50 protein. This paper identifies and characterizes a Drosophila IkappaB kinase (IKK) complex that is activated by lipopolysaccharide (LPS) and that is required for the activation of antibacterial immune response genes and LPS-dependent cleavage of Relish. An IKK protein with homology to human IKK beta was isolated from a cDNA library; an interacting IKK was isolated by a yeast two-hybrid screen. These two Drosophila IKKs show similarity to human IKK beta and gamma, and they were designated DmIKKbeta and DmIKKgamma, respectively. In vitro interaction of these proteins was confirmed using in vitro-translated full-length DmIKKbeta and flag-tagged DmIKKgamma in immunoprecipitation assays. IKKs transiently transfected into Schneider cells were also shown to interact in vivo by immunoprecipitation. RNA interference (RNAi) was used to show that these DmIKKs are necessary for LPS-induced cleavage of Relish and for activation of the bacterial immune response proteins Attacin, Cecropin and Diptericin. Double-stranded RNA for RNAi was produced using the RiboMAX™ Large Scale RNA Production System^(a,b) (Cat.# P1300) from IKK coding templates flanked on each side by the T7 promoter. RNAi experiments were also used to show that neither of these IKKs have an effect on the Toll signaling pathway, as monitored by transcriptional activation of the antifungal Drosomycin.

Silverman, N.¹, Zhou, R.¹, Stoven, S.², Pandey, N.¹, Hultmark, D.¹, and Maniatis, T.^1* (2000) Genes Dev. 14, 2461–2471.

¹Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA; ²Umeå Center for Molecular Pathogenesis, Umeå University, S-901 87 Umeå, Sweden.

*To whom correspondence should be addressed. maniatis@biohp.harvard.edu

Product Focus: RiboMAX™ Large Scale RNA Production System

Vasopressin mRNA localization in nerve cells: Characterization of cis-acting elements and trans-acting factors.

The localization of mRNA species in neuronal cells is not well understood. Nerve cells can transport mRNA transcripts to locations outside the main cell body, often sorting defined transcripts into the dendrites. This may occur to facilitate local translation of proteins within the dendrites, although definitive evidence for local translation in the dendrites has not yet been seen. Sorting of the vasopressin (VP) mRNA transcript to dendrites has been shown to be dependent on a 395-nucleotide long cis-acting dendritic localization sequence. These researchers describe the purification and identification of a trans-acting protein factor that specifically binds to the dendritic localization sequence of the VP mRNA. UV crosslinking experiments defined a protein within rat brain extracts that specifically bound to the VP localization sequence. This protein was purified by affinity chromatography. Biotinylated full-length VP RNA that lacked the poly(A) tail was produced using the RiboMAX™ Large Scale RNA Production System^(a,b) (Cat.# P1300), and then captured using the Streptavidin MagneSphere^® Paramagnetic Particles^(b) (Cat.# Z5481). Heparin column fractions of cytosolic brain extracts were incubated with the biotinylated transcripts and paramagnetic particles, washed, and eluted, resulting in isolation of a specifically bound 78kDa protein. The isolated protein was microsequenced and cloned by use of degenerate PCR. Sequencing of the cDNA showed that the VP-RNA binding protein was rat Poly(A) binding protein.

Mohr, E.^1*, Prakash, N.², Vieluf, K.¹, Fuhrmann, C.¹, Buck, F.¹, and Richter, D.¹ (2001) Proc. Natl. Acad. Sci. USA 98, 7072–7079.

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