DETECTION OF APC MUTATIONS BY A YEAST-BASED PROTEIN TRUNCATION TEST (YPTT)

APC gene mutations play a role in the initiation step of colorectal ca rcinogenesis in both familiar adenomatous polyposis (FAP) and non-FAP patients. almost all of the APC mutations are nonsense or frameshift m utations, which truncate the APC protein and al-e thought to inactivat e normal APC function. We show a novel method for detecting nonsense a nd frameshift APC gene mutations by using Saccharomyces cerevisiae. Po lymerase chain reaction (PCR)-amplified APC fragments are cloned direc tly into yeast expression vectors in vivo, and the yeast expresses a h emagglutinin epitope (HA)-tagged APC peptide;When an APC fragment cont ains a nonsense or frameshift mutation, HA-tagged truncating APC pepti de can be detected by Western blotting using an anti-HA antibody, We i dentified both germ-line and somatic APC mutations in patients with FA P and non-FAP colorectal tumors, respectively, This method, called the yeast-based protein truncation test (YPTT)lis simple and fairly cheap , and it can be applied to any genes that are inactivated by protein t runcating mutations. (C) 1998 Wiley-Liss, Inc.