Results from high-throughput DNA cloning of Arabidopsis thaliana target genes using site-specific recombination.
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 Arabidopsis open reading frames into Escherichia coli expression vectors are presented along with an analysis of results from approximately 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 Gateway 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 Gateway system.
Thao
Sandy
S
Center for Eukaryotic Structural Genomics and Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706, USA.
Zhao
Qin
Q
Kimball
Todd
T
Steffen
Eric
E
Blommel
Paul G
PG
Riters
Megan
M
Newman
Craig S
CS
Fox
Brian G
BG
Wrobel
Russell L
RL
D017360:Arabidopsis / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D018076:DNA, Complementary / Q000737:chemistry; D018744:DNA, Plant / Q000235:genetics*; D023281:Genomics; D016366:Open Reading Frames; D010957:Plasmids / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic
D017931:DNA Primers; D018076:DNA, Complementary; D018744:DNA, Plant