Fig 4. Reconstructing individual evolved mutations increases the fitness of the Rec8-expressing ancestor.

(A) The effect of single evolved mutations and deletion of genes encoding subunits of the Cdk8 complex on fitness of the Rec8-expressing ancestor. (B) The effect of deleting CLN2 and MBP1 on fitness of the Rec8-expressing ancestor. (C) The effect of single evolved mutations in genes that encode other cohesin components or separase and an extra copy of SCC3 on fitness of the Rec8-expressing ancestor. In 4A-4C, each single evolved mutation was reconstructed in the Rec8 ancestral strain used in the evolution experiment. The relative fitness of reconstructed strains to the ancestor was measured by competing it against a fluorescently labeled Rec8 ancestor. The darker gray points represent the values of three biological replicates, and the thinner gray bar represents one standard deviation on each side of the mean of these measurements. The fitness of the wild-type strain, labeled as SCC1, is shown in each panel. The statistical significance between data from the Rec8-expressing strain and each mutation-reconstructed strain was calculated by two-tailed Student t test, **p < 0.01. (D) esp1 evolved mutations (esp1-P8 and esp1-P15) are hypomorphic. A CEN plasmid carrying ESP1 was transformed into a wild-type strain, two ancestors (Anc), and two evolved populations that had acquired esp1 mutations (P8 and P15) at generation 375. Cells were subjected to 10-fold serial dilutions and spotted on YPD plates to assay growth. Cells transformed with an empty plasmid (pRS413) served as control. (E) The effect of deleting one copy of SCC3 on fitness of the evolved populations with disomic Chromosome 9 at generation 1,750. Data associated with Fig 4A, 4B and 4C, and 4E can be found in S1 Data. Cdk8, cyclin-dependent kinase 8; CEN, centromere; CLN2, cyclin 2; esp1, extra spindle pole bodies 1; MBP1, mlul-binding protein; Rec8, recombination 8; SCC, sister chromosome cohesion; YPD, yeast extract, peptone, and dextrose.