title: Detection of APC mutations by a yeast-based protein truncation test (YPTT).
abstract: APC gene mutations play a role in the initiation step of colorectal carcinogenesis in both familial adenomatous polyposis (FAP) and non-FAP patients. Almost all of the APC mutations are nonsense or frameshift mutations, which truncate the APC protein and are thought to inactivate normal APC function. We show a novel method for detecting nonsense and frameshift APC gene mutations by using Saccharomyces cerevisiae. Polymerase chain reaction (PCR)-amplified APC fragments are cloned directly into yeast expression vectors in vivo, and the yeast expresses a hemagglutinin epitope (HA)-tagged APC peptide. When an APC fragment contains a nonsense or frameshift mutation, HA-tagged truncating APC peptide can be detected by Western blotting using an anti-HA antibody. We identified both germ-line and somatic APC mutations in patients with FAP and non-FAP colorectal tumors, respectively. This method, called the yeast-based protein truncation test (YPTT), is simple and fairly cheap, and it can be applied to any genes that are inactivated by protein truncating mutations.
authors.affiliation: Department of Clinical Oncology, Tohoku University, Sendai, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Suzuki
authors.forename: C
authors.initials: C
authors.lastname: Ishioka
authors.forename: S
authors.initials: S
authors.lastname: Kato
authors.forename: Y
authors.initials: Y
authors.lastname: Mitachi
authors.forename: H
authors.initials: H
authors.lastname: Shimodaira
authors.forename: M
authors.initials: M
authors.lastname: Sakayori
authors.forename: A
authors.initials: A
authors.lastname: Shimada
authors.forename: M
authors.initials: M
authors.lastname: Asamura
authors.forename: R
authors.initials: R
authors.lastname: Kanamaru
mesh_terms: D025601:Adenomatous Polyposis Coli Protein; D015153:Blotting, Western; D003598:Cytoskeletal Proteins / Q000235:genetics* / Q000302:isolation & purification*; D016368:Frameshift Mutation; D017491:Genes, APC; D005820:Genetic Testing; D006801:Humans; D009154:Mutation / Q000235:genetics*; D017354:Point Mutation; D012441:Saccharomyces cerevisiae / Q000235:genetics*
chemical_list: D025601:Adenomatous Polyposis Coli Protein; D003598:Cytoskeletal Proteins