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

Abstract

The apoptotic protease-activating factor 1 (Apaf-1) receives the death signal in the intrinsic ormitochondrial pathway of apoptosis. Upon the releasing of cytochrome c from theintermembrane space of mitochondria and binding to Apaf-1 molecules, a heptamericapoptosome complex is formed and triggers the downstream cascade of caspases. Here, for thefirst time we present spontaneous mutations and recombinations of the Apaf-1 gene and itsneighbouring sequences. We sequence 48 colonies containing pcDNA3.1 vector withNluc/Apaf1 and Cluc/Apaf1 obtained through the quick-change site-directed mutagenesismethod, transforming to DH5-α and XL10-Gold at two temperatures, 18 and 37oC. In 21 ofthese cases, we found 38 different mutations. Our data suggest that there is a direct relationshipbetween 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 spontaneousmutations when it is transformed into XL10-Gold at 18 oC . In contrast, a large number ofspontaneous mutations were found when the gene of interest transformed into DH5α at 37oC .