Apoptotic protease-activating factor 1 (Apaf-1) as a liable gene for spontaneous mutations in vitro

The apoptotic protease-activating factor 1 (Apaf-1) receives the death signal in the intrinsic or mitochondrial pathway of apoptosis. Upon the releasing of cytochrome c from the intermembrane space of mitochondria and binding to Apaf-1 molecules, a heptameric apoptosome complex is formed and triggers the downstream cascade of caspases. Here, for the first time we present spontaneous mutations and recombinations of the Apaf-1 gene and its neighbouring sequences. We sequence 48 colonies containing pcDNA3.1 vector with Nluc/Apaf1 and Cluc/Apaf1 obtained through the quick-change site-directed mutagenesis method, transforming to DH5-α and XL10-Gold at two temperatures, 18 and 37ºC. In 21 of these cases, we found 38 different mutations. Our data suggest that there is a direct relationship between bacterial incubation temperatures and the number of unwanted spontaneous mutations. During our experiment we found that the Apaf-1 gene is much less susceptible to spontaneous mutations when it is transformed into XL10-Gold at 18 ºC. In contrast, a large number of spontaneous mutations were found when the gene of interest transformed into DH5α at 37ºC.