Results from high-throughput DNA cloning of Arabidopsis thaliana target genes using site-specific recombination
Abstract
The Center for Eukaryotic Structural Genomics (CESG) was founded as a collaborative effort to develop technologies for the rapid and economic determination of protein three-dimensional structures. The initial focus was on the genome of the model plant Arabidopsis thaliana. Protocols for high-throughput cloning of Arabidopsisopen reading frames into Escherichia coli expression vectors are presented along with an analysis of results from ~2000 cloning experiments. Open reading frames were chosen on the likelihood that they would represent important unknown regions of protein conformation and fold space or that they would elucidate novel fold–function relationships. The chosen open reading frames were amplified from a cDNA pool created by reverse transcription of RNA isolated from an Arabidopsis callus culture. A novel GatewayTM protocol was developed to insert the amplified open reading frames into an entry vector for storage and sequence determination. Sequence verified entry clones were then used to create expression vectors again via the GatewayTM system.
Keywords
eukaryotes Gateway high-throughput cloning structural genomicsPreview
Unable to display preview. Download preview PDF.
References
- 1.Aceti, D.J., Blommel, P.G., Endo, Y., Fox, B.G., Frederick, R.O., Hegeman, A.D., Jeon, W.B., Kimball, T.L., Lee, J.M., Newman, C.S., Peterson, F.C., Sawasaki, T., Seder, K.D., Sussman, M.R., Ulrich, E.L., Wrobel, R.L., Thao, S., Vinarov, D.A., Volkman, B.F. and Zhao, Q. (2002) In Biopolymers (Steinbüchel, A., ed.). Wiley-VCH, Weinheim, pp. 469–496. Google Scholar
- 2.Zolnai, Z., Lee, P.T., Li, J., Chapman, M.R., Newman, C.S., Phillips, G.N., Rayment, I., Ulrich, E.L., Volkman, B.F., Markley, J.L. 2003J. Struct. Funct. Genom.41123Google Scholar
- 3.Sambrook, J.Fritsch, E.F.Maniatis, T. eds. 2001Molecular Cloning, A Laboratory Manual3Cold Spring Harbor Laboratory PressCold Spring Harbor, NYGoogle Scholar
- 4.Strausberg, R.L., Feingold, E.A., Klausner, R.D., Collins, F.S. 1999Science286455457Google Scholar
- 5.Wootton, J.C., Federhen, S. 1996Methods Enzymol266554571Google Scholar
- 6.Dunker, A.K., Brown, C.J., Obradovic, Z. 2002Adv. Protein Chem.622549Google Scholar
- 7.Axelos, M., Curie, C., Mazzolini, L., Bardet, C., Lescure, B. 1992Plant Physiol. Biochem.30123128Google Scholar
- 8.Kang, B., Busse, J.S., Dickey, C., Rancour, D.M., Bednarek, S.Y. 2001Plant Physiol.1264756Google Scholar
- 9.Murashige, T., Skoog, F. 1962Plant Physiol.15473497Google Scholar
- 10.Doyle, J.J., Doyle, J.L. 1987Phytochem. Bull.191115Google Scholar
- 11.Keleman, B.R., Klink, T.A., Behlke, M.A., Eubanks, S.R., Leland, P.A., Raines, R.T. 1999Nucleic Acids Res.2736963701Google Scholar
- 12.Kapust, R.B., Waugh, D.S. 1999Protein Sci.816681674Google Scholar
- 13.Landy, A. 1989Annu. Rev. Biochem.58913949Google Scholar
- 14.Hartley, J.L., Temple, G.F., Brasch, M.A. 2000Genome Res.1017881795Google Scholar
- 15.Ohara, O., Temple, G. 2001Nucleic Acids Res29E22CrossRefGoogle Scholar
- 16.Stevens, R.C. 2001Nat. Rev. Gen.2801809Google Scholar
- 17.The Arabidopsis Genome Initiative (2000) Nature 408, 796–815.Google Scholar