Date: 02 Mar 2005

Results from high-throughput DNA cloning of Arabidopsis thaliana target genes using site-specific recombination

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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.

This revised version was published online in March 2005 with corrections to the affiliations.