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(American Journal of Pathology. 2001;159:1239-1245.)
© 2001 American Society for Investigative Pathology

Technical Advances

Development of a Yeast Stop Codon Assay Readily and Generally Applicable to Human Genes

Akihiko Kataoka*{ddagger}, Mitsuhiro Tada{ddagger}, Masahiro Yano{ddagger}, Keiji Furuuchi{ddagger}, Santoso Cornain§, Jun-ichi Hamada{ddagger}, Gaku Suzuki{dagger}{ddagger}, Hidehisa Yamada{ddagger}, Satoru Todo* and Tetsuya Moriuchi{ddagger}

From the First Department of Surgery*
and the Third Department of Internal Medicine,{dagger}
Hokkaido University School of Medicine, Sapporo; and the Divisions of Cancer-Related Genes{ddagger}
and Gene Therapy Development,§
Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan

We established a yeast-based method to screen chain-terminating mutations that is readily applicable to any gene of interest. Based on the finding that 18- to 24-base-long homologous sequences are sufficient for gap repair in vivo in yeast, we used a strategy to amplify a test-gene fragment with addition of 24-bp sequences homologous to both cut-ends of a yeast expression vector, pMT18. After co-transformation with the amplified fragment and the linearized pMT18, each yeast (Saccharomyces cerevisiae) cell automatically forms a single-copy circular plasmid (because of CEN/ARS), which expresses a test-gene::ADE2 chimera protein. When the reading frame of the test-gene contains a nonsense or frameshift mutation, truncation of the chimera protein results in lack of ADE2 activity, leading to formation of a red colony. By using a nested polymerase chain reaction using proofreading Pfu polymerase to ensure specificity of the product, the assay achieved a low background (false positivity). We applied the assay to BRCA1, APC, hMSH6, and E-cadherin genes, and successfully detected mutations in mRNA and genomic DNA. Because this method—universal stop codon assay—requires only 4 to 5 days to screen a number of samples for any target gene, it may serve as a high-throughput screening system of general utility for chain-terminating mutations that are most prevalent in human genetic diseases.




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