Phosphorylation of RNA Polymerase II by CDKC;2 Maintains the Arabidopsis Circadian Clock Period.

The circadian clock is an internal timekeeping system that governs about 24 h biological rhythms of a broad range of developmental and metabolic activities. The clocks in eukaryotes are thought to rely on lineage-specific transcriptional-translational feedback loops. However, the mechanisms underlying the basic transcriptional regulation events for clock function have not yet been fully explored. Here, through a combination of chemical biology and genetic approaches, we demonstrate that phosphorylation of RNA polymerase II by CYCLIN DEPENDENT KINASE C; 2 (CDKC;2) is required for maintaining the circadian period in Arabidopsis. Chemical screening identified BML-259, the inhibitor of mammalian CDK2/CDK5, as a compound lengthening the circadian period of Arabidopsis. Short-term BML-259 treatment resulted in decreased expression of most clock-associated genes. Development of a chemical probe followed by affinity proteomics revealed that BML-259 binds to CDKC;2. Loss-of-function mutations of cdkc;2 caused a long period phenotype. In vitro experiments demonstrated that the CDKC;2 immunocomplex phosphorylates the C-terminal domain of RNA polymerase II, and BML-259 inhibits this phosphorylation. Collectively, this study suggests that transcriptional activity maintained by CDKC;2 is required for proper period length, which is an essential feature of the circadian clock in Arabidopsis.

Functional hypophysectomy simply by neck-strangulation in the rat: applications for ACTH and LH assays.

The classical methods for hypophysectomy require a long training-period and at the end of each experiment, it is required to check any undue remnant of the hypophyseal tissue in the sella turcica, and there must be exclusion of such datum with the remnant from the experimental group. The present method for functional hypophysectomy by neck-strangulation in the rat is very simple to perform so that even an experimenter with little experience can become easily accustomed to the technique and obtain a number of functionally hypophysectomized preparations at any time he intends, with no need to check the remnant. The cerebral blood circulation of vertebrally dislocated rats was immediately intercepted by means of neck-strangulation and artificially respired. Fifteen minutes after intravenous injection of ACTH (0.1 - 3.2 mU/rat) or LH (312.5 - 2500 ng/rat), blood was sampled from the vena cava, and sera were examined for corticosterone or testosterone by radioimmunoassay. A linear dose-response relationship was obtained within a dose range of 0.2 - 1.6 mU/rat of ACTH and 312.5 - 1250 ng/rat of LH. In the ACTH assay, parapharyngeally hypophysectomized rats were compared. It was found that the sensitivity of functionally hypophysectomized preparations was 2.72 times higher than that of the parapharyngeally hypophysectomized rats.