Mutant profiles of selectable genetic elements.
A method is presented that allows simultaneous analysis of the effects of all possible point mutations within a specific mutation window of at least 50 base pairs on a DNA fragment that codes for a selectable function. It relies on the detection of mismatched base pairs with hydroxylamine and osmium tetroxide. A mutant plasmid library of randomly distributed point mutations within the lacZ' gene of Escherichia coli was selected for functional alpha-complementation by growth on lactose. The DNA fragments of the selected and unselected library were each heat denatured and again renatured, thereby generating a randomly distributed set of all possible mismatches within the mutagenesis window. Cytidine-containing mismatches were then detected with hydroxylamine, and thymidine-containing mismatches were detected with osmium tetroxide. When this procedure was performed for both DNA strands, all mismatches could be detected. A comparison of the results of the unselected and selected library leads to an estimation of the effects of each detectable mutation on alpha-complementation in vivo. This method, called "mutant profiling," should be applicable to all selectable genetic elements.
Wurst
H
H
Fakultät für Biologie, Universität Konstanz, Federal Republic of Germany.
Pohl
F M
FM
D000595:Amino Acid Sequence; D001482:Base Composition; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D015723:Gene Library; D005816:Genetic Complementation Test; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics / Q000302:isolation & purification; D009838:Oligodeoxyribonucleotides; D009993:Osmium Tetroxide / Q000494:pharmacology; D010957:Plasmids; D015183:Restriction Mapping; D014169:Transformation, Bacterial; D001616:beta-Galactosidase / Q000235:genetics
D004269:DNA, Bacterial; D006898:Hydroxylamines; D009692:Nucleic Acid Heteroduplexes; D009838:Oligodeoxyribonucleotides; D019811:Hydroxylamine; D001616:beta-Galactosidase; D009993:Osmium Tetroxide