title: Complementation of an E. coli cysteine auxotrophic mutant for the structural modification study of 3'(2'),5'-bisphosphate nucleotidase.
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.
authors: School of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University, Seoul, Korea. cheongjj@snu.ac.kr
authors: Jong-Joo
authors: JJ
authors: Cheong
authors: Ingyu
authors: I
authors: Hwang
authors: Sangkee
authors: S
authors: Rhee
authors: Tae Wha
authors: TW
authors: Moon
authors: Yang Do
authors: YD
authors: Choi
authors: Hawk-Bin
authors: HB
authors: Kwon
mesh_terms: D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D003596:Cytosine / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D009708:Nucleotidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013941:Thymine / Q000737:chemistry / Q000378:metabolism; D014633:Valine / Q000235:genetics
chemical_list: D029681:Arabidopsis Proteins; D003596:Cytosine; C510507:CysQ protein, E coli; D009708:Nucleotidases; D010744:Phosphoric Monoester Hydrolases; C053354:bisphosphoadenylate 3'-nucleotidase; D014633:Valine; D003545:Cysteine; D000409:Alanine; D013941:Thymine