Detection of APC mutations by a yeast-based protein truncation test (YPTT)

✍ Scribed by Takao Suzuki; Chikashi Ishioka; Satoshi Kato; Yasushi Mitachi; Hideki Shimodaira; Masato Sakayori; Akira Shimada; Mitsuo Asamura; Ryunosuke Kanamaru

• Book ID: 101261730
• Publisher: John Wiley and Sons
• Year: 1998
• Tongue: English
• Weight: 154 KB
• Volume: 21
• Category: Article
• ISSN: 1045-2257
• DOI: 10.1002/(sici)1098-2264(199804)21:4<290::aid-gcc2>3.0.co;2-u

✦ Synopsis

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.