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Detection of ATM gene mutation in human glioma cell line M059J by a rapid frameshift/stop codon assay in yeast
Rika Tsuchida1, Takayuki Yamada, Masatoshi Takagi
1Department of Pediatrics and Developmental Biology, Postgraduate Medical School, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan.
Abstract:
A yeast-based frameshift/stop codon assay for examining ATM (ataxia telangiectasia mutated) mutations was established. Each of six fragments of a PCR-amplified coding sequence for ATM is inserted in frame by homologous recombination into a yeast URA3 fusion protein gene, and the transformants are assayed for growth in the absence of uracil. The usefulness of this assay was verified in a panel of cell lines derived from individuals with homozygous and heterozygous ATM mutations. The assay was also shown to distinguish between specimens with wild-type alleles and those with truncating mutations: a frameshift mutation or an inserted stop codon. Using this assay M059J cells, which fail to express the catalytic subunit of DNA-dependent protein kinase (PRKDC, also known as DNA-PKcs) and are hypersensitive to ionizing radiation, were found to express two different aberrant ATM transcripts: one characterized by 4776 del 133, which corresponds to the deletion of exon 33, and the other by 4909 ins 116. Subsequent analysis of the intron sequences revealed that 4909 ins 116 is comprised of a nucleotide sequence corresponding to 84013-84128 in intron 33 with a cryptic splice site. Thus the radiosensitive phenotype of M059J cells appears to be due to a defect in PRKDC and a truncating ATM mutation.
Insights
A novel yeast assay detects ataxia telangiectasia mutated (ATM) gene mutations, including frameshifts and stop codons. This tool identified two aberrant ATM transcripts in radiosensitive M059J cells, revealing a potential dual cause for their phenotype.
Area of Science:
- Genetics
- Molecular Biology
- Cancer Research
Background:
- Ataxia telangiectasia mutated (ATM) is a critical gene involved in DNA damage response.
- Mutations in ATM are linked to various cancers and genetic disorders.
- Accurate detection of ATM mutations is crucial for diagnosis and treatment.
Purpose of the Study:
- To establish a yeast-based assay for detecting frameshift and stop codon mutations in the ATM gene.
- To validate the assay's efficacy using cell lines with known ATM mutations.
- To investigate the genetic basis of radiosensitivity in M059J cells.
Main Methods:
- Development of a yeast URA3 fusion protein gene assay.
- Insertion of ATM gene fragments via homologous recombination.
- Analysis of ATM transcripts and intron sequences in M059J cells.
Main Results:
- The yeast assay successfully identified frameshift and stop codon mutations in ATM.
- Two aberrant ATM transcripts (4776 del 133 and 4909 ins 116) were detected in M059J cells.
- The 4909 ins 116 mutation involved a cryptic splice site in intron 33.
Conclusions:
- The developed yeast assay is effective for detecting ATM truncating mutations.
- M059J cells exhibit a radiosensitive phenotype due to a PRKDC defect and a truncating ATM mutation.
- The study highlights the utility of the yeast assay in cancer mutation analysis.
