Rose H. Lin

University of California, Los Angeles

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Malgorzata M. Slupska

University of California, Los Angeles

Jean Lee Stewart

University of California, Los Angeles

Emily F. Mao

University of California, Los Angeles

Angela G. King

University of Plymouth

Chantal A. Lackey

University of Washington

Louise I. Lu

University of California, Los Angeles

Jeffrey H Miller

University of California, Los Angeles

Claudia Baikalov

Amgen (United States)

Pauline Funchain

Stanford University

Ju‐Huei Chiang

University of California, Los Angeles

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University of California, Los Angeles

Molecular Sciences Institute

California Institute of Technology

Hawaii Department of Health

Kapiolani Medical Center for Women and Children

University of Hawaii System

Department of Public Health and Social Services

University of Plymouth

University of Exeter

Queen Mary University of London

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The Consequences of Growth of a Mutator Strain of Escherichia coli as Measured by Loss of Function Among Multiple Gene Targets and Loss of Fitness

Genetics (2000)

Pauline Funchain Annie Yeung Jean Lee Stewart Rose H. Lin Malgorzata M. Slupska

Abstract We have examined the composition of members of mutator populations of Escherichia coli by employing an extensive set of phenotypic screens that allow us to monitor the function of >700 genes, constituting ~15% of the genome. We looked at mismatch repair deficient cells after repeated cycles of single colony isolation on rich medium to generate lineages that are forced through severe bottlenecks, and compared the results to those for wild-type strains. The mutator lineages continued to accumulate mutations rapidly with each increasing cycle of colony isolation. By the end of the 40th cycle, after ~1000 generations, most of the lineages had reduced colony size, 4% had died out, 55% had auxotrophic requirements (increasing to 80% after 60 cycles), and 70% had defects in at least one sugar or catabolic pathway. In addition, 33% had a defect in cell motility, and 26% were either temperature-sensitive or cold-sensitive lethals. On the other hand, only 3% of the wild-type lineages had detectable mutations of any type after 40 cycles. By the 60th cycle, the typical mutator cell carried 4–5 inactive genes among the 15% of the genome being monitored, indicating that the average cell carried at least 24–30 inactivated genes distributed throughout the genome. Remarkably, 30% of the lineages had lost the ability to utilize xylose as a carbon source. DNA sequencing revealed that most of the Xyl− mutants had a frameshift in a run of eight G's (GGGGGGGG) in the xylB gene, either adding or deleting one -G-. Further analysis indicated that rendering E. coli deficient in mismatch repair unmasks hypermutable sites in certain genes or intergenic regions. Growth curves and competition tests on lineages that passed through 90 cycles of single colony isolation showed that all lineages suffered reduced fitness. We discuss these results in terms of the value of mutators in cellular evolution.

10.1093/genetics/154.3.959

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Examination of the Role of DNA Polymerase Proofreading in the Mutator Effect of Miscoding tRNAs

Journal of Bacteriology (1998)

Malgorzata M. Slupska Angela G. King Louise I. Lu Rose H. Lin Emily F. Mao

We previously described Escherichia coli mutator tRNAs that insert glycine in place of aspartic acid and postulated that the elevated mutation rate results from generating a mutator polymerase. We suggested that the proofreading subunit of polymerase III, epsilon, is a likely target for the aspartic acid-to-glycine change that leads to a lowered fidelity of replication, since the altered epsilon subunits resulting from this substitution (approximately 1% of the time) are sufficient to create a mutator effect, based on several observations of mutD alleles. In the present work, we extended the study of specific mutD alleles and constructed 16 altered mutD genes by replacing each aspartic acid codon, in series, with a glycine codon in the dnaQ gene that encodes epsilon. We show that three of these genes confer a strong mutator effect. We have also looked for new mutator tRNAs and have found one: a glycine tRNA that inserts glycine at histidine codons. We then replaced each of the seven histidine codons in the mutD gene with glycine codons and found that in two cases, a strong mutator phenotype results. These findings are consistent with the epsilon subunit playing a major role in the mutator effect of misreading tRNAs.

Proofreading

10.1128/jb.180.21.5712-5717.1998

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Citations (29)