A single circular chromosome yeast

Characterization of the single circular chromosome yeast SY15. a Construction of the SY15 strain. Ligation of two chromosome ends via both CRISPR-Cas9 induced DSBs and homologous recombination. The red arrowheads indicated the cutting sites of Cas9. DR: direct repeat. The URA3 selection marker was further deleted via homologous recombination of two DR regions by negative selection. b The myc-tagged telomere binding protein Sir2 was detected with polyclonal anti-myc antibody and Cy3-conjugated (red) secondary antibody. Nop1, a nucleolar protein, was detected with a monoclonal anti-Nop1 antibody and Alexa 488-conjugated (green) secondary antibody. DNA was stained by DAPI (blue).1 c 3D conformation of the SY15 genome in comparison to that of SY14. d Classification of differentially expressed genes, defined as those with log2 (fold change) ≥1 and P<0.05 in SY15 compared to SY14. Data were collected from three biological replicates. e Scanning electron microscopy pictures of SY14 and SY15 cells. Representative images from three independent experiments. f Heatmap of the Phenotype Microarray profiles of SY14 and SY15 cells. Low to high metabolic activities are depicted by a color spectrum from light blue to yellow. Data were collected from two biological replicates. g Cell cycle analysis. The yeast cells were synchronized with hydroxyurea and the progression of the cell cycle was analyzed by flow cytometry. Data are representative of two independent experiments. h Fitness analysis of SY15 cells under various growth conditions. Representative results of three independent experiments. i Senescence assay in liquid medium. The growth of wild type BY4742 (dark brown), SY14 (dark green), SY15 (dark blue), BY4742 tlc1Δ (light brown), SY14 tlc1Δ (light green), SY15 tlc1Δ (light blue) strains were monitored for 16 days. Every 24？h, the growth of the strains was measured in the value of OD600. The diluted cultures were started from OD600？=？0.01. For each strain, two clones were examine