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.
Miller
J H
JH
Department of Microbiology and Molecular Genetics and The Molecular Biology Institute, University of California, Los Angeles, California 90095, USA. jhmiller@mbi.ucla.edu
Suthar
A
A
Tai
J
J
Yeung
A
A
Truong
C
C
Stewart
J L
JL
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