Journal: G3: Genes|Genomes|Genetics

Article Title: Reconstructed evolutionary history of the yeast septins Cdc11 and Shs1

doi: 10.1093/g3journal/jkaa006

Figure Lengend Snippet: Reconstructed ancestral septins localize to the yeast bud neck congruent with endogenous septins. A CDC11 shs1Δ strain (GFY-6) expressing Cdc10-mCherry from the chromosomal CDC10 locus to mark the location of the septin collar at the bud neck was transformed with plasmids expressing either S. cerevisiae Shs1-GFP (pGF-preIVL-59) (top panels), Anc.11-S-GFP (pGF-IVL-159) (middle panels), or Anc.S-GFP (pGF-IVL-168) (bottom panels). The cultures were incubated overnight in SD-LEU at 30°C, back-diluted into YPD, grown for an additional 4.5 h at 30°C, washed with water, and visualized under white light by Nomarski optics (Differential Interference Contrast (DIC), leftmost images) and by fluorescence microscopy with appropriate cutoff filters to detect mCherry (middle images) and GFP (rightmost images), respectively. Representative images, adjusted using ImageJ, are shown. Faint dotted white lines demarcate the cell periphery. Scale bar, 3 μm.

Article Snippet: Cultures were grown overnight at 30°C, back diluted into rich medium for 4.0 or 4.5 h at 30°C, harvested, and examined within 30 min at room temperature under a fluorescence microscope (Leica, model DMI6000; Leica Microsystems, Buffalo Grove, IL, USA), equipped with a 100× lens and appropriate cutoff filters for visualization of GFP and mCherry (monomeric red fluorescent protein derivative) fluorescence (Semrock), and images were acquired using a Leica DFC340 FX camera.

Techniques: Expressing, Transformation Assay, Incubation, Fluorescence, Microscopy