The sequence spectrum of frameshift reversions obtained with a novel adaptive mutation assay in Saccharomyces cerevisiae

Research on the mechanisms of adaptive mutagenesis in resting, i.e. non-replicating cells relies on appropriate mutation assays. Here we provide a novel procedure for the detection of frameshift-reverting mutations in yeast. Proliferation of non-reverted cells in this assay is suppressed by the lack of a fermentable carbon source. The test allele was constructed in a way that the reversions mimic microsatellite instability, a condition often found in cancer cells. We show the cell numbers during these starvation conditions and provide a DNA sequence spectrum of a representative set of revertants. The data in this article support the publication "Glucose starvation as a selective tool for the study of adaptive mutations in Saccharomyces cerevisiae" (Heidenreich and Steinboeck, 2016) [1].


How data was acquired
Cell counts by microscopy; DNA sequencing

Data format Raw, analyzed Experimental factors
Random choice of revertants from different time points (for sequencing)

Experimental features
A novel mutation assay under glucose-limited conditions was performed. Cell numbers during glucose starvation were assessed. The test allele of a representative number of revertants was sequenced.

Data source location
Vienna, Austria Data accessibility Data are presented in this article

Value of the data
We share the protocol for a new adaptive mutation assay in Saccharomyces cerevisiae that will be the new standard assay in our lab. We want to encourage others to use it as well.
As a part of quality assurance, we demonstrate the lack of proliferation during protracted starvation.
We provide the first sequence spectrum of reversions of the novel fbp1MS2 test allele to serve as a benchmark for future comparisons.

Data
DNA replication is the major source of mutations in proliferating cells. However, mutations also arise in resting cells. The mechanisms responsible for the latter are less well understood than replication-dependent mutagenesis. The most interesting subset of mutations in resting cells are such that appear to be adaptive, i.e. that provide a selective advantage to the mutants by enabling a resumption of proliferation. For the study of such adaptive mutations, a combination of a useful test allele and appropriate cell cycle-arresting but non-lethal conditions is necessary [2]. In this article, we present data on the implementation of a novel adaptive mutation assay ( Fig. 1 and Table 1) as a tool to generate adaptive revertants for further analysis. We monitored the quality of the induced cell cycle arrest (Fig. 2) and provide a sequence analysis of representative sets of revertants ( Fig. 3 and Table 2).

Components of the mutation assay
The yeast strain YFMS2 carries a custom-designed microsatellite sequence as a target for spontaneous reversions (Fig. 1). Details of the construction are presented in [1]. Translation of the resulting fbp1MS2 allele is truncated by a frameshift. Starvation for glucose (on lactate medium) drives YFMS2 cells into a cell cycle arrest that permits the selection for revertants able to resume proliferation by a mutational reversion of the fbp1MS2 frameshift. The media used (YPD and SC) have been described by Sherman [3]. SC/Lactate is SC medium with 3% DL-lactate instead of 2% glucose. The detailed experimental procedure of the new adaptive mutation assay is listed in Table 1.

Data on the strictness of starvation-induced cell cycle arrest
Any variation in cell numbers during starvation for glucose was monitored by counting the cells washed from representative parallel plates treated identical to the mutation assay. Data obtained with the assay strain YFMS2 were compared to data obtained with a FBP1 knockout strain named EHDF1 (Fig. 2). A leaky cell cycle arrest would be evident by an increase in cell numbers. Prepare about 50 SC/Lactate plates. 32 plates of similar thickness are needed (mean weight þ/ À 5%, to provide a similar amount of nutrients on each plate). Determination of cell numbers and viability: Rinse cells off the plates A6 and B6 (in parallel) with a total of 4 ml sterile water (add 2 ml sterile water, release cells with a Drigalski spatula, transfer suspension to a 15 ml tube, repeat twice with 1 ml water). Measure the volume of the collected liquid with the help of a 5 ml glass pipet. Directly determine cell density with a hemocytometer. Prepare appropriate dilutions and plate about 100 cfu´s on each of three YPD plates for survival determination. Calculate the mean of the two counts Transfer all plates to a humidified (e.g. cell culture) incubator (30°C)

DAYS 4 to 15
Count and mark newly emerging colonies (larger than 0.5 mm diameter) on all plates

DNA sequence data
Revertant colonies obtained in fbp1MS2 mutation assays performed according to Table 1 were isolated and their genomic DNA prepared. A fragment containing the FBP1 allele was amplified by PCR and after purification sequenced by a contractor (VBC genomics, Vienna, Austria). The clones have been randomly chosen among those appearing either on day 4 (i.e. replication-dependent revertants carried over from preculture), day 8 (early adaptive mutants) or days 11-13 (late adaptive revertants). The type and the location of the genetic alterations of 58 revertants is shown in Fig. 3. Table 2 lists the distribution of mutation types among the three temporal groups. With this number of clones, there is neither a significant difference in the mutational spectrum between the three temporal groups nor between the replication-dependent revertants and the total of adaptive revertants (p 40.05 with an algorithm by Adams and Skopek [4]).   3. Mutational spectrum of 58 revertants of the fbp1MS2 allele. Numbering above the sequence is in relation to the translation start (start codon is shaded), deleted stretches are indicated by (Δ), a gain of one TC repeat in the engineered TC microsatellite and one GA repeat in an innate GA repeat is indicated by an arrow ( * ). The reversion window, where potential reversions have to be located is underlined.

Table 2
Incidence of the mutation types shown in Fig. 3 grouped by their origin (day of colony appearance during mutation assay indicated).