Article thumbnail
Location of Repository

Identification of frame-shift intermediate mutant cells

By Christoph Gasche, Christina L. Chang, Loki Natarajan, Ajay Goel, Jennifer Rhees, Dennis J. Young, Christian N. Arnold and C. Richard Boland

Abstract

Frame-shift mutations at microsatellites occur as a time-dependent function of polymerase errors followed by failure of postreplicational mismatch repair. A cell-culture system was developed that allows identification of intermediate mutant cells that carry the mutation on a single DNA strand after the initial DNA polymerase errors. A plasmid was constructed that contained 13 repeats of a poly(dC-dA)⋅poly(dG-dT) oligonucleotide immediately after the translation initiation codon of the enhanced GFP (EGFP) gene, shifting the EGFP gene out of its proper reading frame. The plasmid was introduced into human mismatch repair-deficient (HCT116, hMLH1-mutated) and mismatch repair-proficient (HCT116+chr3, hMLH1 wild type) colorectal cancer cells. After frame-shift mutations occurred that restored the EGFP reading frame, EGFP-expressing cells were detected, and two distinct fluorescent populations, M1 (dim cells) and M2 (bright cells), were identified. M1 cell numbers were stable, whereas M2 cells accumulated over time. In HCT116, single M2 cells gave rise to fluorescent colonies that carried a 2-bp deletion at the (CA)(13) microsatellite. Twenty-eight percent of single M1 cells, however, gave rise to colonies with a mixed fluorescence pattern that carried both (CA)(13) and (CA)(12) microsatellites. It is likely that M1 cells represent intermediate mutants that carry (CA)(13)⋅(GT)(12) heteroduplexes. Although the mutation rate in HCT116 cell clones (6.2 × 10(−4)) was 30 times higher than in HCT116+chr3 (1.9 × 10(−5)), the proportion of M1 cells in culture did not significantly differ between HCT116 (5.87 × 10(−3)) and HCT116+chr3 (4.13 × 10(−3)), indicating that the generation of intermediate mutants is not affected by mismatch-repair proficiency

Topics: Biological Sciences
Publisher: The National Academy of Sciences
Year: 2003
DOI identifier: 10.1073/pnas.0437965100
OAI identifier: oai:pubmedcentral.nih.gov:149933
Provided by: PubMed Central
Download PDF:
Sorry, we are unable to provide the full text but you may find it at the following location(s):
  • http://dx.doi.org/10.1073/pnas... (external link)
  • Suggested articles


    To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.