The occurrence of spontaneous mutations can lead to cancer especially if a cell undergoes a certain amount of genetic changes, which differs from type to type of cancers. However, mutation rates determined in proliferating cells are not sufficient to explain the observed frequencies of multiple mutations. Consequently, it is assumed that a nameable fraction of mutations also arises from non-proliferating cells.
Adaptive mutations in the model organism Saccharomyces cerevisiae are particularly suitable to study spontaneous mutagenesis in quiescent cells. Common systems use amino acid starvation to provoke cell cycle arrest. In particular the lys2[Delta]Bgl system is well-studied, where yeast cells are starved for the essential amino acid lysine. However, some disadvantages arise from this type of starvation, with regard to stringency of cell cycle arrest, mimicking of natural circumstances and presence of glucose in the experimental setup. Therefore, in this study those limits were antagonized by replacing amino acid starvation with starvation for carbohydrates. Two target genes, FBP1 encoding the enzyme fructose-1,6-bisphosphatase and PCK1 encoding phosphoenolpyruvate carboxykinase, involved in gluconeogenesis were impaired to block the pathway. The study showed that a block in gluconeogenesis leads to a strict cell cycle arrest without residual growth from the beginning of selection on non-sugar carbon sources. Three alternative nutrients (lactate, pyruvate and glycerol) were tested in carbohydrate starvation, revealing lactate to be the most suitable one. Furthermore, a frameshift strain with a revertible mutation in the FBP1 gene was constructed to perform adaptive mutation assays. Revertant colonies could be detected with the new fbp1FS-128 system over a time course of 22 days without artifacts. The system was established successfully and was also used to study rad27[Delta] repair gene mutant, which was not feasible with previously used lysine starvation. Moreover, a fusion of the tumor suppressor gene TP53 and FBP1 was constructed to increase mutation frequency.