Direct selection for mutators in Escherichia coli.
We have constructed strains that allow a direct selection for mutators of Escherichia coli on a single plate medium. The plate selection is based on using two different markers whose reversion is enhanced by a given mutator. Plates containing limiting amounts of each respective nutrient allow the growth of ghost colonies or microcolonies that give rise to full-size colonies only if a reversion event occurs. Because two successive mutational events are required, mutator cells are favored to generate full-size colonies. Reversion of a third marker allows direct visualization of the mutator phenotype by the large number of blue papillae in the full-size colonies. We also describe plate selections involving three successive nutrient markers followed by a fourth papillation step. Different frameshift or base substitution mutations are used to select for mismatch-repair-defective strains (mutHLS and uvrD). We can detect and monitor mutator cells arising spontaneously, at frequencies lower than 10(-5) in the population. Also, we can measure a mutator cascade, in which one type of mutator (mutT) generates a second mutator (mutHLS) that then allows stepwise frameshift mutations. We discuss the relevance of mutators arising on a single medium as a result of cells overcoming successive growth barriers to the development and progression of cancerous tumors, some of which are mutator cell lines.
Department of Microbiology and Molecular Genetics and The Molecular Biology Institute, University of California, Los Angeles, California 90095, USA. jhmiller@mbi.ucla.edu
J H
JH
Miller
A
A
Suthar
J
J
Tai
A
A
Yeung
C
C
Truong
J L
JL
Stewart
D015075:2-Aminopurine / Q000494:pharmacology; D001431:Bacteriological Techniques; D003470:Culture Media; D004260:DNA Repair; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification; D005821:Genetic Techniques; D016296:Mutagenesis; D009153:Mutagens / Q000494:pharmacology; D009876:Operon
D003470:Culture Media; D009153:Mutagens; D015075:2-Aminopurine