Contrasting properties of gene-specific regulatory, coding, and copy number mutations in Saccharomyces cerevisiae: frequency, effects, and dominance

Abstract

Genetic variation within and between species can be shaped by population-level processes and mutation; however, the relative impact of "survival of the fittest" and "arrival of the fittest" on phenotypic evolution remains unclear. Assessing the influence of mutation on evolution requires understanding the relative rates of different types of mutations and their genetic properties, yet little is known about the functional consequences of new mutations. Here, we examine the spectrum of mutations affecting a focal gene in Saccharomyces cerevisiae by characterizing 231 novel haploid genotypes with altered activity of a fluorescent reporter gene. 7% of these genotypes had a nonsynonymous mutation in the coding sequence for the fluorescent protein and were classified as "coding" mutants; 2% had a change in the S. cerevisiae TDH3 promoter sequence controlling expression of the fluorescent protein and were classified as "cis-regulatory" mutants; 10% contained two copies of the reporter gene and were classified as "copy number" mutants; and the remaining 81% showed altered fluorescence without a change in the reporter gene itself and were classified as "trans-acting" mutants. As a group, coding mutants had the strongest effect on reporter gene activity and always decreased it. By contrast, 50%-95% of the mutants in each of the other three classes increased gene activity, with mutants affecting copy number and cis-regulatory sequences having larger median effects on gene activity than trans-acting mutants. When made heterozygous in diploid cells, coding, cis-regulatory, and copy number mutant genotypes all had significant effects on gene activity, whereas 88% of the trans-acting mutants appeared to be recessive. These differences in the frequency, effects, and dominance among functional classes of mutations might help explain why some types of mutations are found to be segregating within or fixed between species more often than others.

Figure 1. EMS treatment increased the frequency of cells with extreme YFP fluorescence.

(A) The relationship between “forward scatter” (“FSC”, a proxy for cell size) and YFP fluorescence is shown for a population of control cells. FSC and YFP fluorescence are both reported in arbitrary units on a log scale. A similar linear relationship was observed for other genotypes. Approximately 2% of flow cytometry “events” had YFP fluorescence less than the range plotted and are not shown. The fluorescence phenotype of each sample in the secondary screen was calculated as the median YFP/FSC ratio for FACS events with FSC values between 5.30 and 5.55 (indicated with dotted lines). (B) The distribution of YFP fluorescence phenotypes is shown for EMS-treated (dashed curve) and control cells (solid curve) from one of the nine replicate populations of EMS-treated and control cells analyzed in the primary screen. Locations of the 1st, 5th, 95th, and 99th percentiles of the control sub-population are indicated, and vertical dashed lines show the average thresholds used for cell sorting (see also Table S1). (C) The difference between the number of EMS-treated and control cells in the population is plotted for a range of fluorescence levels (grey line). The black curve shows a spline fit to these data. Positive values indicate fluorescence phenotypes that were more abundant in the EMS-treated sample, whereas negative values indicate fluorescence phenotypes that were more abundant in the control sample. The spline crosses zero at approximately the 17th and 84th percentiles of the control population. The percentage of the EMS-treated population that is either over or under represented is shown for the following percentile ranges: 1–17, 17–84, 84–99. The X-axis representing YFP fluorescence levels has the same scale as in panel B.

Figure 2. Mutations in the coding and promoter sequences of P_TDH3-YFP as well as changes in its copy number were found among the 231 mutant genotypes.

(A) The schematic depicts the P_TDH3-YFP reporter gene and is drawn to scale. Annotations indicate the location of each mutation relative to the transcription start site (+1) of TDH3 , the nucleotide substitution observed, and the change in amino acid sequence if applicable. Asterisks indicate mutations observed in two mutant genotypes. Mutations at positions −255, −240 and −140 are located within the promoter, and the mutation at position 348 is a synonymous change. (B) In 221 of the 231 mutants, the relative copy number of P_TDH3-YFP and P_TDH3-CFP was determined by pyrosequencing. Clusters of points representing genotypes with one copy of P_TDH3-YFP (CFP and YFP≈1/2) and two copies of P_TDH3-YFP (CFP≈1/3 and YFP≈2/3) are indicated with arrows. (C) Median YFP fluorescence is plotted for each of the replicate populations analyzed for eight distinct genotypes: unmutagenized cells containing the “wildtype” P_TDH3-YFP sequence (diamonds); each of the four regulatory mutant genotypes (8Q1D4, 4Q4E4, 3Q3C11, and 1Q4D11 in Table S3) that contained a promoter mutation (filled circles); and genotypes in which one of the promoter mutations (−255, −240, or −140) was introduced into same genetic background as the wildtype P_TDH3-YFP gene (open circles). Populations of cells containing any one of the promoter mutations showed a significant change in YFP fluorescence relative to the wildtype genotype (P₋₂₅₅ = 0.002, P₋₂₄₀ = 0.002, P₋₁₄₀ = 0.015, MWW test). The mutation at −255 had an effect on YFP fluorescence equivalent to that of mutant genotype 8Q1D4 (P = 0.261, MWW), but not to that of mutant genotype 4Q4E4 (P = 0.0002, MWW). The effects on YFP fluorescence of mutations at −240 and −140 were equivalent to those of the mutant genotypes (3Q3C11 and 1Q4D11, respectively) that harbored them (P₋₂₄₀ = 0.262 and P₋₁₄₀ = 0.262, MWW).

Figure 3. Effects on YFP fluorescence in haploid cells differ among mutational classes.

The effects on YFP fluorescence of the 4 cis-regulatory (black), 16 coding (green), 22 CNV (red), and 179 trans-acting (blue) mutants are summarized in histograms. For each mutational class, the height of each bar indicates the number of mutants with the corresponding effect (as measured by Z-score) on YFP fluorescence in haploid cells. Positive Z-scores indicate increases in YFP fluorescence relative to control cells and negative Z-scores indicate decreases in YFP fluorescence relative to control cells. The relative frequency of mutants in each of the four mutational classes is also shown in the inset pie chart.

Figure 4. Effects of heterozygous mutant alleles on YFP and CFP fluorescence differ among mutational classes in diploid cells.

(A) The effect of each mutant genotype on YFP fluorescence in haploid cells (X-axis) is compared to the effect of the heterozygous mutant genotype on YFP fluorescence in diploid cells (Y-axis). These diploid cells were heterozygous for the mutant P_TDH3-YFP haploid genome and a reference haploid genome containing P_TDH3-CFP. Black squares indicate cis-regulatory mutants, blue circles indicate trans-acting mutants, green triangles indicate coding mutants, and red crosses indicate CNV mutants. (B) Using the same symbols to represent the four mutational classes as in (A), the effect of each mutant on YFP (X-axis) and CFP (Y-axis) fluorescence in diploid cells is shown. Insets in (A) and (B) show only coding mutants and cover the larger ranges of Z-scores needed to plot all of the mutants in this class.