sequence results. the arabidopsis information resource the national center for genome resources...

Sequence Results

The Arabidopsis Information ResourceThe National Center for Genome Resources (NCGR), Santa Fe, New MexicoThe Arabidopsis Biological Resource Center (ABRC), Ohio State UniversityCarnegie 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 AwardsThe 2010 Project:

To determine the function of all genes in Arabidopsis thaliana by the year 2010

Principal Investigator Institution TitleTotal

Award*

Total Duration (years)

Last, Robert0519740

Michigan State University Arabidopsis 2010: Understanding Chloroplast Function$3,999,985 4

Vierstra, Richard0519970

University of Wisconsin-Madison Functional Analysis of the Ubiquitin-Protein Ligase (E3) Families in Arabidopsis

$4,061,983 4

Dong, Xinnian0519898

Duke University Expression Profiling of Plant Disease Resistance Pathways$3,554,359 4

Fiscal Year 2001 AwardsThe 2010 Project:

To determine the function of 25,000 genes in Arabidopsis thaliana by the year 2010

Principal Investigator Institution TitleTotal

Award*

Total Duration (years)

Ecker, Joseph0115103

The Salk Institute for Biological Studies Arabidopsis 2010: A Sequence-Indexed Library of Insertion Mutations in the Arabidopsis Genome

$3,000,000 2

Schuler, Mary0115068

University of Illinois Urbana-Champaign Arabidopsis 2010: Functional Genomics of Arabidopsis P450s

$3,461,517 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.

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 chromosome)

What does the number At1g11680 mean?

Publications describing your gene.

Obtaining a knockout of geneAt1g11680

SIGnAL Salk Institute Genomic Analysis Laboratory

Frequently asked questions answers a lot of questions!!!

Localizing expression of geneAt1g11680

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

20µl

Qiagen miniprep DNA, approx. 200 ng/µl

1µl1µl2µl

15µl1µl

pUNI pHOST 10x BufferWaterCre

20 min37°C

Transform in DH5α.Select on Amp.

Univector Plasmid-Fusion System

β-GlucuronidaseGreen Fluorescent Protein

Luciferase

β-Glucuronidase

General localization.

X-Gluc + β-Glucuronidase =Blue

General localization andsubcellular localization.

Green Fluorescent Protein

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 enzymesLiu, 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 recombinationGregory D. Van DuyneAnnual Review of Biophysics and Biomolecular Structure Vol. 30: 87-104 (2001)