Development of a yeast stop codon assay readily and generally applicable to human genes.
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.
Kataoka
A
A
First Department of Surgery, Hokkaido University School of Medicine, Sapporo, Japan.
Tada
M
M
Yano
M
M
Furuuchi
K
K
Cornain
S
S
Hamada
J
J
Suzuki
G
G
Yamada
H
H
Todo
S
S
Moriuchi
T
T
D025601:Adenomatous Polyposis Coli Protein / Q000235:genetics; D001943:Breast Neoplasms / Q000235:genetics; D015820:Cadherins / Q000235:genetics; D018388:Codon, Terminator / Q000235:genetics*; D003110:Colonic Neoplasms / Q000235:genetics; D004268:DNA-Binding Proteins; D005091:Exons / Q000235:genetics; D005260:Female; D005656:Fungal Proteins / Q000235:genetics; D019398:Genes, BRCA1 / Q000235:genetics; D005821:Genetic Techniques; D006801:Humans; D015335:Molecular Probes / Q000737:chemistry; D009154:Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D013728:Terminator Regions, Genetic / Q000235:genetics*; D014407:Tumor Cells, Cultured
D025601:Adenomatous Polyposis Coli Protein; D015820:Cadherins; D018388:Codon, Terminator; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C098790:MSH6 protein, S cerevisiae; D015335:Molecular Probes; D029701:Saccharomyces cerevisiae Proteins