Journal: Nucleic Acids Research
Article Title: Chromatin assembly factor-1 (CAF-1) chaperone regulates Cse4 deposition into chromatin in budding yeast
Figure Lengend Snippet: When Scm3 is absent and Cse4 levels are high, CAF-1 may help target Cse4 to the centromere. ( A ) Cac1 localization to centromeres is cell-cycle independent. ChIP was performed for Cac1 using a FLAG-tagged strain using asynchronously growing cells and also G1, S and G2/M arrested cells. qPCR was used to detect Cac1 levels at CEN3. The y -axis indicates arbitrary units representing the Cac1 enrichment at CEN3 from ChIP performed with and without antibody, with respect to the signal for total chromatin for each sample. The error bars represent the standard deviation for ChIP performed in triplicate. ( B ) Rescue of Scm3 off strain by overexpression of Cse4 is less efficient when CAC2 is deleted. Strains containing a single copy of Scm3 under the control of the gal promoter, allowing the expression of Scm3 to be controlled with glucose/galactose, were used in the spotting assay. Cse4 was under the control of a copper-inducible promoter on a plasmid. ‘EV’ indicates empty vector. When Scm3 is expressed (Scm3 on ), no growth differences are observed in 10-fold serial dilutions. Induction of Cse4 does not efficiently rescue growth of the Scm3 off strain when CAC2 is deleted as compared to WT. Three independent isolates of the cac2Δ strain (1, 2 and 3) were tested.
Article Snippet: Antibodies used for ChIP experiments are 2 μl of α-Cse4 antibody (polyclonal rabbit antibody against recombinant Cse4) and anti-FLAG (Sigma-F3165).
Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Standard Deviation, Over Expression, Expressing, Spotting Assay, Plasmid Preparation