Mutations in alpha-subunit of Escherichia coli F1-ATPase obtained by hydroxylamine-mutagenesis of plasmids carrying the uncA gene.
In order to generate mutants randomly in the Escherichia coli uncA gene (encoding the alpha-subunit of F1-ATPase), plasmids carrying uncA were treated in vitro with hydroxylamine. Restriction fragments of the mutated uncA gene were then reconstructed into plasmid pDP34, which expresses all of the F1F0 structural genes, and the reconstructed mutant plasmids were expressed in a strain carrying a deletion of chromosomal uncA. Each of the mutations was characterized by DNA sequencing, growth assays, and biochemical assays of membrane preparations. Three nonsense and one frameshift mutation were identified and their properties were studied briefly. Eight new missense mutations were identified and characterization of their properties is described. These eight mutations were R139H, A177V, R210C, R303C, A306V, T343I, G351S, and P370L.
Pagan
J
J
Department of Biochemistry, University of Rochester Medical Center, New York 14642.
Senior
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AE
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000201:enzymology; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial / Q000187:drug effects*; D005838:Genotype; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000187:drug effects*; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000235:genetics*; D015183:Restriction Mapping; D013696:Temperature
D006898:Hydroxylamines; D046911:Macromolecular Substances; D019811:Hydroxylamine; D006180:Proton-Translocating ATPases