Total chemical synthesis and expression in Escherichia coli of a maize glutathione-transferase (GST) gene.
We have constructed a totally synthetic gene encoding a maize glutathione S-transferase (GST I). This gene, composed of 1320 nucleotides (nt) (660 bp), was assembled from only 16 synthetic oligodeoxynucleotides (average length 83 nt), using an efficient one-step annealing/ligation protocol. Sequencing was performed to verify the authenticity of the final assembled gene. Significantly, not a single mutation was found in either of the two constructs sequenced, indicating a remarkably low mutation frequency. The synthetic gene was introduced into Escherichia coli where it was successfully expressed. The biological activity of the GST I enzyme produced in E. coli was monitored by assaying bacterial extracts for the ability to conjugate [14C]atrazine in the presence of glutathione. This biologically active synthetic GST1 gene can now be introduced into plants to assess its ability to confer tolerance to the triazine class of herbicides.
Wosnick
M A
MA
Allelix Incorporated, Mississauga, Ontario, Canada.
Barnett
R W
RW
Carlson
J E
JE
D000595:Amino Acid Sequence; D001280:Atrazine / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005813:Genes, Synthetic; D005982:Glutathione Transferase / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D003313:Zea mays / Q000201:enzymology / Q000235:genetics*
D005982:Glutathione Transferase; D001280:Atrazine