Independence of circadian timing from cell division in cyanobacteria

In the cyanobacterium Synechococcus elongatus, cell division is regulated by a circadian clock. Deletion of the circadian clock gene, kaiC, abolishes rhythms of gene expression and cell division timing. Overexpression of the ftsZ gene halted cell division but not growth, causing cells to grow as filaments without dividing. The nondividing filamentous cells still exhibited robust circadian rhythms of gene expression. This result indicates that the circadian timing system is independent of rhythmic cell division and, together with other results, suggests that the cyanobacterial circadian system is stable and well sustained under a wide range of intracellular conditions.     

A kaiC-interacting sensory histidine kinase, SasA, necessary to sustain robust circadian oscillation in cyanobacteria

Both regulated expression of the clock genes kaiA, kaiB, and kaiC and interactions among the Kai proteins are proposed to be important for circadian function in the cyanobacterium Synechococcus sp. strain PCC 7942. We have identified the histidine kinase SasA as a KaiC-interacting protein. SasA contains a KaiB-like sensory domain, which appears sufficient for interaction with KaiC. Disruption of the sasA gene lowered kaiBC expression and dramatically reduced amplitude of the kai expression rhythms while shortening the period. Accordingly, sasA disruption attenuated circadian expression patterns of all tested genes, some of which became arrhythmic. Continuous sasA overexpression eliminated circadian rhythms, whereas temporal overexpression changed the phase of kaiBC expression rhythm. Thus, SasA is a close associate of the cyanobacterial clock that is necessary to sustain robust circadian rhythms.     

Overexpression of COL9, a CONSTANS-LIKE gene, delays flowering by reducing expression of CO and FT in Arabidopsis thaliana

CONSTANS (CO) is an important floral regulator in the photoperiod pathway, integrating the circadian clock and light signal into a control for flowering time. It is known that CO promotes flowering in Arabidopsis under long-day conditions. CONSTANS-LIKE 9 (COL9) is a member of the CONSTANS-LIKE gene family, encoding a nuclear protein. The expression of COL9 is regulated by the circadian clock in the photoperiod pathway and is detected in various organs. Unexpectedly, overexpression of COL9 in transgenic Arabidopsis resulted in delayed flowering, while co-suppression lines and a transferred DNA (T-DNA) knockout line showed earlier flowering under long-day conditions. Overexpression of COL9 did not enhance the late-flowering phenotype in a co mutant background. Double overexpressors produced by overexpression of CO in COL9 transgenic lines showed an early flowering phenotype similar to single CO overexpressors. The pattern of oscillation of a number of circadian-associated genes remained unchanged in the COL9 transgenic lines. Compared with wild-type plants, the abundance of CO and FLOWERING LOCUS T (FT) mRNA was reduced in the COL9 overexpression lines. Our results indicate that COL9 is involved in regulation of flowering time by repressing the expression of CO, concomitantly reducing the expression of FT and delaying floral transition.     

A general and islet cell-enriched overexpression of IGF-I results in normal islet cell growth, hypoglycemia, and significant resistance to experimental diabetes

Insulin-like growth factor I (IGF-I) is normally produced from hepatocytes and various other cells and tissues, including the pancreas, and is known to stimulate islet cell replication in vitro, prevent Fas-mediated beta-cell destruction and delay the onset of diabetes in nonobese diabetic mice. Recently, however, the notion that IGF-I stimulates islet cell growth has been challenged by the results of IGF-I and receptor gene targeting. To test the effects of a general, more profound increase in circulating IGF-I on islet cell growth and glucose homeostasis, we have characterized MT-IGF mice, which overexpress the IGF-I gene under the metallothionein I promoter. In early reports, a 1.5-fold-elevated serum IGF-I level caused accelerated somatic growth and pancreatic enlargement. We demonstrated that the transgene expression, although widespread, was highly concentrated in the beta-cells of the pancreatic islets. Yet, islet cell percent and pancreatic morphology were unaffected. IGF-I overexpression resulted in significant hypoglycemia, hypoinsulinemia, and improved glucose tolerance but normal insulin secretion and sensitivity. Pyruvate tolerance test indicated significantly suppressed hepatic gluconeogenesis, which might explain the severe hypoglycemia after fasting. Finally, due to a partial prevention of beta-cell death against onset of diabetes and/or the insulin-like effects of IGF-I overexpression, MT-IGF mice (which overexpress the IGF-I gene under the metallothionein I promoter) were significantly resistant to streptozotocin-induced diabetes, with diminished hyperglycemia and prevention of weight loss and death. Although IGF-I might not promote islet cell growth, its overexpression is clearly antidiabetic by improving islet cell survival and/or providing insulin-like effects.     

Chronic shift-lag alters the circadian clock of NK cells and promotes lung cancer growth in rats

Prolonged subjection to unstable work or lighting schedules, particularly in rotating shift-workers, is associated with an increased risk of immune-related diseases, including several cancers. Consequences of chronic circadian disruption may also extend to the innate immune system to promote cancer growth, as NK cell function is modulated by circadian mechanisms and plays a key role in lysis of tumor cells. To determine if NK cell function is disrupted by a model of human shift-work and jet-lag, Fischer (344) rats were exposed to either a standard 12:12 light-dark cycle or a chronic shift-lag paradigm consisting of 10 repeated 6-h photic advances occurring every 2 d, followed by 5-7 d of constant darkness. This model resulted in considerable circadian disruption, as assessed by circadian running-wheel activity. NK cells were enriched from control and shifted animals, and gene, protein, and cytolytic activity assays were performed. Chronic shift-lag altered the circadian expression of clock genes, Per2 and Bmal1, and cytolytic factors, perforin and granzyme B, as well as the cytokine, IFN-γ. These alterations were correlated with suppressed circadian expression of NK cytolytic activity. Further, chronic shift-lag attenuated NK cell cytolytic activity under stimulated in vivo conditions, and promoted lung tumor growth following i.v. injection of MADB106 tumor cells. Together, these findings suggest chronic circadian disruption promotes tumor growth by altering the circadian rhythms of NK cell function.     

The circadian gene per1 plays an important role in cell growth and DNA damage control in human cancer cells

The Per1 gene is a core clock factor that plays an essential role in generating circadian rhythms. Recent data reveal that major biological pathways, including those critical to cell division, are under circadian control. We report here that Per1 provides an important link between the circadian system and the cell cycle system. Overexpression of Per1 sensitized human cancer cells to DNA damage-induced apoptosis; in contrast, inhibition of Per1 in similarly treated cells blunted apoptosis. The apoptotic phenotype was associated with altered expression of key cell cycle regulators. In addition, Per1 interacted with the checkpoint proteins ATM and Chk2. Ectopic expression of Per1 in human cancer cell lines led to significant growth reduction. Finally, Per1 levels were reduced in human cancer patient samples. Our results highlight the importance of circadian regulation to fundamental cellular functions and support the hypothesis that disruption of core clock genes may lead to cancer development.     

Extracellular production of cloned alpha-amylase by Escherichia coli

Overexpression of Bacillus stearothermophilus gene coding for thermostable alpha-amylase in Escherichia coli was shown to cause outer-membrane damage leading to extracellular location of periplasmic proteins. Prolonged high expression of the alpha-amylase gene under lacZpo control eventually also lysed cells. Surprisingly, expression controlled by the pL promoter of phage lambda allowed specific release of periplasmic proteins into the growth medium without total cell lysis. Accumulation of alpha-amylase in the growth medium continued for at least 24 h under lambda pL control, whereas beta-lactamase activity ceased to increase beyond the exponential growth phase. The extent of outer membrane damage caused by alpha-amylase expression was monitored by following growth kinetics in the presence of lysozyme and by electron microscopy of the cells. Supplementing growth medium with Mg2+ restored the normal growth kinetics. It is suggested that periplasmic protein release caused by alpha-amylase overexpression is a stress response of the cell. A role for induced autolytic activity of the cell as a final effector of protein release is also proposed.     

Calpain inhibition decreases the growth rate of mammalian cell colonies

The calpains, a family of calcium-requiring intracellular proteases, are proposed regulators of cell proliferation. However, ablation of the calpain small subunit gene necessary for function of the conventional calpains did not result in decreased rate of proliferative growth of mouse stem cells under routine culture conditions. To address the reasons for this discrepancy, Chinese hamster ovary cell lines were established that overexpress the calpain inhibitor protein, calpastatin, under control of the ecdysone congener, ponasterone A. Overexpression of calpastatin in these cell lines resulted in a decreased growth of isolated colonies adhering to tissue culture plates. However, when cells were plated at higher density, calpastatin overexpression had no influence on proliferative growth rate. Growth of colonies in soft agar was not inhibited by calpastatin overexpression. Cell adhesion, cell de-adhesion, and cell motility all appeared to be normal after calpastatin overexpression. Differential display analysis was initiated to detect possible alteration of gene expression upon calpastatin overexpression. Analysis of approximately 3000 differential display PCR signals resulted in identification of one band that was underexpressed. Northern blot analysis confirmed a decreased amount of approximately 1 kb mRNA in cells overexpressing calpastatin. Sequence analysis identified a putative protein, Csr, containing a region homologous to two ubiquitin transferases and a putative cation channel protein.     

光周期和温度对生物钟基因cwo在棉铃虫幼虫节律表达的影响

【目的】生物钟普遍存在于生物体的生长发育、行为及生理等各个方面。本研究旨在探究光周期和温度对生物钟基因cwo在棉铃虫Helicoverpa armigera幼虫昼夜节律表达的影响。【方法】通过转录组测序获得了生物钟基因cwo的c DNA开放阅读框全长,利用实时荧光定量PCR技术分析了生物钟基因cwo在棉铃虫幼虫中的时空表达,以及其在不同光周期和不同温度条件下的昼夜表达节律。【结果】序列分析结果显示,棉铃虫生物钟基因cwo的c DNA序列开放阅读框为1 335 bp,编码444个氨基酸残基,预测蛋白的分子质量为49.95 ku。时空表达分析表明,cwo基因在棉铃虫幼虫组织中以脑和腹神经索表达量较高,在卵孵化期、蜕皮期、蛹变态期及羽化前1 d高峰度表达。头部昼夜节律表达分析表明,正常光周期条件下生物钟基因cwo呈昼夜节律表达,在暗期高表达;短时间持续暗期/光期处理使cwo表达的时间相位提前,长时间持续暗期/光期处理使cwo表达的节律性显著降低;38℃处理使cwo表达短期内显著上调,并改变了其节律性,18℃处理使cwo表达在暗期显著下调,但是节律性不变。【结论】生物钟基因cwo在棉铃虫幼虫头部的昼夜节律表达受到光周期和温度的影响。     

Cloning,expression, characterization,and role in autocrine cell growth of cell surface retention sequence binding protein-1

Cell surface retention sequence binding protein-1 (CRSBP-1) is a cell surface binding protein for the cell surface retention sequence (CRS) motif of the v-sis gene product (platelet-derived growth factor-BB). It has been shown to be responsible for cell surface retention of the v-sis gene product in v-sis-transformed cells (fibroblasts) and has been hypothesized to play a role in autocrine growth and transformation of these cells. Here we demonstrate that the CRSBP-1 cDNA cloned from bovine liver libraries encodes a 322-residue type I membrane protein containing a 23-residue signal peptide, a 215-residue cell surface domain, a 21-residue transmembrane domain, and a 63-residue cytoplasmic domain. CRSBP-1 expressed in transfected cells is an approximately 120-kDa disulfide-linked homodimeric glycoprotein and exhibits dual ligand (CRS-containing growth regulators (v-sis gene product and insulin-like growth factor binding protein-3, IGFBP-3) and hyaluronic acid) binding activity. CRSBP-1 overexpression (by stable transfection of cells with CRSBP-1 cDNA) enhances autocrine loop signaling, cell growth, and tumorigenicity (in mice) of v-sis-transformed cells. CRSBP-1 expression also enhances autocrine cell growth mediated by IGFBP-3 in human lung carcinoma cells (H1299 cells), which express very little, if any, endogenous CRSBP-1 and exhibits a mitogenic response to exogenous IGFBP-3, stably transfected with IGFBP-3 cDNA. However, CRSBP-1 overexpression does not affect growth of normal and transformed cells that do not produce these CRS-containing growth regulators. These results suggest that CRSBP-1 plays a role in autocrine regulation of cell growth mediated by growth regulators containing CRS.     

Clock genes in cell clocks: roles, actions, and mysteries

Cellular events must be organized in the time dimension as well as in the space dimension for many proteins to perform their cellular functions effectively. The intracellular molecular oscillating loops that compose the cell's circadian clock coordinate the timing of the expression of a variety of genes with basic or specific cellular functions. In mammals, the temporal pattern of clock gene expression generated in each SCN neuron is coupled to those of other cells and, amplified, spreads its signals through the brain and then, via feeding behavior, glucocorticoids, and sympathetic nerves, to peripheral organs. These peripheral organs have their own circadian clocks. In some tissues, such as liver, there is also a clock-regulating cell cycle, which interacts strongly with the components and temporal organization of the circadian clock. Some tissues, however, such as testis, express clock genes whose function, if any, remains unclear. Furthermore, circadian clock function may be suspended in differentiating tissue. Thus, the prominence of circadian organization may not apply equally to all tissues under all conditions.