Regulation of Cell Division Cycles by Circadian Oscillators:Role of the cAMP Pathway

在已经提出的控制微生物细胞分裂周期的其他机制中，自主生物振荡器提供了一种可能的方法来整合细胞周期、退出和转运的不同概念。拟议的研究将关注振荡器和细胞分裂周期之间的这种耦合是如何实现的。在微藻Euglena gracilis中表现出双峰昼夜节律性的环状AMP，在许多细胞类型中具有控制细胞分裂周期进程中某些限速步骤的能力，可能起到这样的耦合作用，参与将细胞分裂周期事件门控到昼夜周期的特定阶段。两个问题将被解决：第一，时钟如何赋予腺苷环化酶/cAMP-磷酸二酯酶系统节律性，第二，腺苷环化酶/cAMP-磷酸二酯酶如何与细胞分裂周期相互作用和调节。腺苷环化酶、磷酸二酯酶和其他第二信使(如cGMP和钙)对cAMP水平和节律性的调节将在无绿叶突变体的同步分裂和不分裂培养中进行研究，该突变体保持在恒定的黑暗中，通过检测已知能激活或抑制这些生化物种的各种药物和化学物质的脉冲能力，来改变cAMP水平、腺苷环化酶和磷酸二酯酶活性以及细胞分裂的节律。初步结果表明，cAMP是一种依赖于cAMP的激酶，它的细胞受体将被鉴定，并将寻找cAMP或cAMP类似物对细胞分裂周期的影响与激酶刺激之间的关系。最后，将寻找p34(分裂酵母的CDC 2蛋白激酶基因产物)的Euglena同源物，如果鉴定出它，将在受到cAMP脉冲干扰的同步培养中，在细胞分裂周期的不同阶段测量其组蛋白H1激酶活性。从单细胞真核生物到高等动植物的生物体都存在昼夜节律，并涉及植物的叶片运动、昆虫和哺乳动物的运动活动以及单细胞藻类的光合作用等各种活动。许多藻类、真菌和原生动物的细胞分裂周期也表现出昼夜节律；细胞分裂只发生在昼夜周期的有限间隔期间，通常是在与昼夜明暗周期中的暗间隔相对应的时间。这种现象被认为反映了生物钟和细胞周期振荡器之间的相互作用。这项研究的长期目标是确定生物钟(S)的性质，推测它与细胞分裂周期有关，并产生分裂的周期性，以及总体上的昼夜节律性。

Among other mechanisms that have been proposed to control microbial cell division cycles, autonomous biological oscillators provide a possible means of integrating the disparate concepts of cell cycle, exit, and transit. The proposed research will be concerned with the means by which this coupling between oscillator and cell division cycle is effected. Cyclic AMP, which exhibits bimodal circadian rhythmicity in the algal flagellate Euglena gracilis, and which is known to have the capacity to control certain rate- limiting steps in the progression of the cell division cycle in many cell types, may play such a coupling role, participating in the gating of cell division cycle events to specific phases of the circadian cycle. Two problems will be addressed: First, how the clock confers rhythmicity upon the adenyl cyclase/cAMP- phosphodiesterase system, and second, how the adenyl cyclase/cAMP- phosphodiesterase interacts with and modulates the cell division cycle. The regulation of the level and rhythmicity of cAMP by adenyl cyclase, phosphodiesterase, and other second messengers (such as cGMP and calcium) will be studied in synchronously dividing and nondividing cultures of the achlorophyllous ZC mutant of Euglena, maintained in constant darkness, by examining the ability of pulses of various drugs and chemicals that are known to activate or inhibit these biochemical species to phase-shift the rhythms of cAMP level, adenyl cyclase and phosphodiesterase activities, and cell division. The cellular receptor for cAMP, which preliminary results suggest is a cAMP-dependent kinase, will be characterized, and a correlation between kinase stimulation and effects of cAMP or cAMP analogs on the cell division cycle will be sought. Finally, a Euglena homolog of p34 (the cdc 2 protein- kinase gene product of fission yeast) will be sought, and if it is identified, its histone H1 kinase activity will be measured at different phases of the cell division cycle in synchronous cultures perturbed by a cAMP pulse. Circadian rhythms have been documented in organisms ranging from unicellular eukaryotes to higher plants and animals and concern activities as varied as leaf movements in plants, locomotor activity in insects and mammals, and photosynthesis in unicellular algae. The cell division cycle of many algae, fungi, and protozoa also exhibit circadian rhythms; cell division occurs only during restricted intervals of the circadian cycle, often at the times corresponding to the dark intervals in a diurnal light-dark cycle. This phenomenon is thought to reflect an interaction between the circadian clock and the cell cycle oscillator. The long range goal of this research is to determine the nature of the circadian clock(s) presumed to couple to the cell division cycle and to generate periodicity of division, as well as to underlie circadian rhythmicity in general.