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Sequence Results The Arabidopsis Information Resource The National Center for Genome Resources (NCGR), Santa Fe, New Mexico The Arabidopsis Biological Resource Center (ABRC), Ohio State University Carnegie Institution, Department of Plant Biology, Stanford University Mission Summary: Provide research resources for the Arabidopsis research community NSF 2010 Project To determine the function of all genes in Arabidopsis thaliana by the year 2010 Fiscal Year 2005 Awards The 2010 Project: To determine the function of all genes in Arabidopsis thaliana by the year 2010 Principal Investigator Institution Title Last, Robert 0519740 Michigan State University Arabidopsis 2010: Understanding Chloroplast Function Vierstra, Richard 0519970 University of Wisconsin-Madison Functional Analysis of the Ubiquitin-Protein Ligase (E3) Families in Arabidopsis Dong, Xinnian 0519898 Duke University Expression Profiling of Plant Disease Resistance Pathways Total Award* Total Duration (years) $3,999,985 4 $4,061,983 4 $3,554,359 4 Total Award* Total Duration (years) Fiscal Year 2001 Awards The 2010 Project: To determine the function of 25,000 genes in Arabidopsis thaliana by the year 2010 Principal Investigator Ecker, Joseph 0115103 Schuler, Mary 0115068 Institution The Salk Institute for Biological Studies University of Illinois Urbana-Champaign Title Arabidopsis 2010: A Sequence-Indexed Library of Insertion Mutations in the Arabidopsis Genome Arabidopsis 2010: Functional Genomics of Arabidopsis P450s $3,000,000 $3,461,517 2 4 How To Determine Gene Function? • Gene Overview • Knockouts • Localization Getting an overview of your gene. www.arabidopsis.org P450s are membrane-associated proteins, either in the inner membrane of mitochondria or in the endoplasmic reticulum of cells where they metabolize thousands of endogenous and exogenous compounds. What does the number At1g11680 mean? At (Arabidopsis thaliana) 1,2,3,4,5 (chromosome number) or M for mitochondrial or C for chloroplast. g (gene), other letters possible for repeats etc.) 12300 (five-digit code, numbered from top/north to bottom/south of chromosom Publications describing your gene. Obtaining a knockout of gene At1g11680 SIGnAL Salk Institute Genomic Analysis Laboratory Frequently asked questions answers a lot of questions!!! Localizing expression of gene At1g11680 Cre-loxP Site-Specific Recombination (cyclization recombination) Cre-LoxP for Promoter GUS fusion Cre recombinase (Cre): A type I topoisomerase from P1 bacteriophage that catalyzes site-specific recombination of DNA between loxP sites The loxP recognition element: Recommended reaction for Cre-LoxP pUNI pHOST 10x Buffer Water Cre 1µl 1µl 2µl 15µl 1µl 37°C 20 min Transform in DH5α. Select on Amp. 20µl Qiagen miniprep DNA, approx. 200 ng/µl Univector Plasmid-Fusion System Green Fluorescent Protein β-Glucuronidase Luciferase β-Glucuronidase General localization. X-Gluc + β-Glucuronidase = Blue Green Fluorescent Protein General localization and subcellular localization. Luciferase Evaluation of gene expression/induction. In Summary: The Arabidopsis Information Resource • The primary goal of TAIR is to assist the Arabidopsis community discover gene function. • Extensive gene knockout and localization tools (T-DNA lines, full-length cDNAs, and pUNI cDNAs) serve as the core elements of what TAIR offers. • The TAIR website, its databases, and resources continue to expand, so if you are unable to find a T-DNA insertion today…try tomorrow! Syngenta Inc has now donated about 9,000 more lines to ABRC and sequences to this site at Mar. 31 2006 Key References Cre-loxP recombination vectors for the expression of Riken Arabidopsis full-length cDNAs in plants Toshiro Shigaki, Mamata Kole, John M. Ward, Heven Sze, and Kendal D. Hirschi BioTechniques Vol. 39, No. 3: pp 301-303 (Sep 2005) The Cre-loxP recombination-based reporter system for plant transcriptional expression studies Shigaki, T., R. Ravindranadha, A.B. Sivitz, J.M. Ward, H. Sze, and K.D. Hirschi Plant Mol. Biol. 58:65-73 (2005) The univector plasmid-fusion system, a method for rapid construction of recombinant DNA without restriction enzymes Liu, Q., M.Z. Li, D. Leibham, D. Cortez, and S.J. Elledge Curr. Biol. 8:1300-1309 (1998) A structural view of Cre-loxP site-specific recombination Gregory D. Van Duyne Annual Review of Biophysics and Biomolecular Structure Vol. 30: 87-104 (2001)