Fig. 3: esa-1 knockdown causes a long period.
From: A crucial role for dynamic expression of components encoding the negative arm of the circadian clock
a Bioluminescent analysis of strains that knock down esa-1 (middle) or overexpress esa-1E395Q (right). qa-2-driven double-stranded RNA against esa-1, or esa-1E395Q targeting the csr locus, was transformed to a WT strain. Circadian periods of WT and ∆brd-8 at 25 °C were determined by the luciferase assay in the absence or presence of 10-2 M QA as indicated. Race tube images on the top indicate the viability of the qa-2-driven double-stranded RNA against esa-1 strain grown with 10-2 M QA despite a slow and unstable growth rate compared with the no QA control. b qa-2:dsRNA against esa-1 was transformed to esa-1V5; strains were cultured in constant light, and 10-2 M QA was added to the cultures and immediately transferred to the dark for 24, 47, and 72 hrs; Western blotting against V5 was performed and a non-specific band was shown for equal loadings. The assay was performed three times, and similar results were observed. c In vitro HAT assays of recombinant histone H4 (upper left) or H2A (upper right) with affinity-purified ESA-1V5. Details for this assay are described in Methods. Bottom, a HAT assay of ESA-1V5 in vitro. ESA-1V5 in the background of WT, ∆wc-1, or ∆brd-8 was immunoprecipitated by V5 antibody and assayed in vitro with recombinant histone H4 to test its histone acetylation activity. Western blotting using antibodies against V5, acetyl histone H2A (K9), and acetyl histone H4 (K5, K8, K12, and K16) respectively were followed to show ESA-1V5 and acetylated histone H2A and H4 levels. The assay was done three times with similar results. Source data were deposited in the Source Data file.
