Detection of APC mutations by a yeast-based protein truncation test (YPTT).
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.
Suzuki
T
T
Department of Clinical Oncology, Tohoku University, Sendai, Japan.
Ishioka
C
C
Kato
S
S
Mitachi
Y
Y
Shimodaira
H
H
Sakayori
M
M
Shimada
A
A
Asamura
M
M
Kanamaru
R
R
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*
D025601:Adenomatous Polyposis Coli Protein; D003598:Cytoskeletal Proteins