Abstract
The Arabidopsis AHL gene encodes a 3′(2′),5′-bisphosphate nucleotidase (BPNTase) involved in the reductive sulfate activation pathway. A bacterial expression vector containing AHL cDNA was randomly mutagenized with hydroxylamine and transformed into the E. coli cysteine auxotrophic mutant cysQ. Bacterial colonies that did not show evidence of complementation, i.e. those that exhibited slower growth on cysteine-free medium, were selected for further study. Sequencing of the AHL cDNA in one such clone revealed the conversion of cytosine 635 (C635) to thymine, resulting in an Alanine (A212) to Valine substitution. This microbial complementation procedure is useful in BPNTase structure-activity studies for biotechnological applications.
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Acknowledgments
We thank Dr. Howard Goodman (Harvard Medical School, retired), Dr. Doil Choi (KRIBB) and Ms. Suna Sohn for grateful assistances in initiating this work. This work was supported by a grant (CG2112) from the Crop Functional Genomics Center funded by the Korea Ministry of Science and Technology, the grant KRF-2004-005-F00013 from the Korea Research Foundation, and in part by a grant from the BioGreen21 program of the Rural Development Administration. Fellowship support from the Ministry of Education through the Brain Korea 21 Project is also acknowledged.
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Cheong, JJ., Hwang, I., Rhee, S. et al. Complementation of an E. coli cysteine auxotrophic mutant for the structural modification study of 3′(2′),5′-bisphosphate nucleotidase. Biotechnol Lett 29, 913–918 (2007). https://doi.org/10.1007/s10529-007-9324-7
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DOI: https://doi.org/10.1007/s10529-007-9324-7