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 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 .