quantitative analyses of circadian gene expression in mammalian cell cultures生理基因表达的定量分析在哺乳动物细胞培养.pdfVIP

Quantitative Analyses of Circadian Gene Expression in Mammalian Cell Cultures 1¤ 2 3 1* Mariko Izumo , Takashi R. Sato , Martin Straume , Carl Hirschie Johnson 1 Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee, United States of America, 2 Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, United States of America, 3 Customized Online Biomathematical Research Applications, Charlottesville, Virginia, United States of America The central circadian pacemaker is located in the hypothalamus of mammals, but essentially the same oscillating system operates in peripheral tissues and even in immortalized cell lines. Using luciferase reporters that allow automated monitoring of circadian gene expression in mammalian fibroblasts, we report the collection and analysis of precise rhythmic data from these cells. We use these methods to analyze signaling pathways of peripheral tissues by studying the responses of Rat-1 fibroblasts to ten different compounds. To quantify these rhythms, which show significant variation and large non-stationarities (damping and baseline drifting), we developed a new fast Fourier transform–nonlinear least squares analysis procedure that specifically optimizes the quantification of amplitude for circadian rhythm data. This enhanced analysis method successfully distinguishes among the ten signaling compounds for their rhythm-inducing properties. We pursued detailed analyses of the responses to two of these compounds that induced the highest amplitude rhythms in fibroblasts, forskolin (an activator of adenylyl cyclase), and dexamethasone (an agonist of glucocorticoid receptors). Our quantitative analyses clearly indicate that the synchronization mechanisms by