Browsing by Subject "Circadian clock"
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Item Cellular Function and Localization of Circadian Clock Proteins in Cyanobacteria(2011-08-08) Dong, GuogangThe cyanobacterium Synechococcus elongatus builds a circadian clock on an oscillator comprised of three proteins, KaiA, KaiB, and KaiC, which can recapitulate a circadian rhythm of KaiC phosphorylation in vitro. The molecular structures of all three proteins are known, and the phosphorylation steps of KaiC, the interaction dynamics among the three Kai proteins, and a weak ATPase activity of KaiC have all been characterized. A mutant of a clock gene in the input pathway, cikA, has a cell division defect, and the circadian clock inhibits the cell cycle for a short period of time during each cycle. However, the interaction between the circadian cycle and the cell cycle and the molecular mechanisms underlying it have been poorly understood. In addition, the subcellular localization of clock proteins and possible localization dynamics, which are critical in the timing circuit of eukaryotic clock systems and might also shed light on the interaction between circadian cycle and cell cycle, have remained largely unknown. A combination of genetics, cell biology, and microscopy techniques has been employed to investigate both questions. This work showed that the cell division defect of a cikA mutant is a function of the circadian clock. High ATPase activity of KaiC coincides with the inhibition of cytokinesis by the circadian clock. CikA likely represses KaiC's ATPase activity through an unknown protein, which in cikA's absence stimulates both the ATPase and autokinase activities independently of KaiA or KaiB. SasA-RpaA acts as an output in the control of cell division, and the localization of FtsZ is the target, although it still remains to be seen how RpaA, directly or indirectly, inhibits FtsZ localization. The project also showed that clock proteins are localized to the cell poles. KaiC is targeted to the cell pole in a phosphorylation-dependent manner. KaiB and CikA are also found at the poles independently of KaiC. KaiA likely only localizes to the cell pole during the dephosphorylation phase, which is dependent on both KaiB and KaiC, specifically on the phosphorylation of KaiC at S431. Overall, significant progress was made in both areas and this project sheds light on how the circadian oscillator operates in cyanobacterial cells and interacts with another fundamental cellular function.Item Circadian clock gene expression and growth vigor in arabidopsis hybrids and mRNA stability in arabidopsis allotetraploids(2010-12) Kim, Eun Deok; Chen, Z. JeffreyHybrids and polyploids are very common in plants and some animals. Although hybrid vigor or heterosis has been widely adopted in agricultural practices, the underlying mechanisms are poorly understood partly because of their multigenic nature and the lack of a good model system for the study. Allotetraploidy is an emerging model system for investigating molecular mechanisms of hybrid vigor. An allotetraploid is formed by interspecific hybridization followed by chromosome doubling or hybridization between two autotetraploid parents and is genetically stable. A previous study showed nonadditive expression (different from the mid-parent value) of over 5% of genes in the allotetraploids, suggesting altered transcriptional and post-transcriptional regulation. Here oligo-gene microarray analysis of mRNA stability in allotetraploids was carried out to investigate how nonadditive gene regulation upon allopolyploidization is achieved at the posttranscriptional level. Approximately 1% of annotated genes were identified as unstable transcripts, and their estimated half-life is less than 60 minutes. The unstable transcripts in Arabidopsis allotetraploids are associated with nonadditive gene expression and with stress and environmental responses. The nonadditively expressed genes identified in the previous study include those encoding proteins involved in energy and metabolic pathways, which are putative targets of circadian clock regulators. To test how circadian clock genes affect downstream genes and pathways, expression of CIRCADIAN CLOCK ASSOCIATED1 (CCA1) was up- or down-regulated by overexpressing CCA1 or cca1(RNAi) driven by the promoter of TIMING OF CAB EXPRESSION 1 (TOC1). Upregulation of CCA1 was associated with repression of downstream genes in chlorophyll biosynthesis and starch metabolism, whereas down-regulation of CCA1 correlated with upregulation of these downstream genes. As a result, chlorophyll and starch content was ~10% higher in the TOC1::cca1(RNAi) transgenic plants than the controls, while the growth vigor is lower in the TOC1::CCA1 transgenic plants. To further test the effects of clock genes in growth vigor, CCA1 expression was examined in reciprocal hybrids of A. thaliana ecotypes. The maternal effect on starch content was observed in several combinations of hybrids, which was correlated with preferential expression of maternal CCA1 during early stages of seed development. Although the cause of parent-of-origin effects is still unclear, the data have clearly documented parent-of-origin effects on circadian clock gene expression and starch metabolism in hybrids.Item The day/night switch of the circadian clock of synechococcus elongatus and hydrogen bonds of dna and rna(2009-05-15) Kim, Yong-IckThe circadian oscillator of the cyanobacterium Synechococcus elongatus is composed of only three proteins, KaiA, KaiB, and KaiC, which together with ATP can generate a self-sustained ~24 hour oscillation of KaiC phosphorylation for several days. KaiA induces KaiC to autophosphorylate whereas KaiB blocks the stimulation of KaiC by KaiA, which allows KaiC to autodephosphorylate. We propose and support a model in which the C-terminal loops of KaiC, the ?A-loops?, are the master switch that determines overall KaiC activity. When the A-loops are in their buried state, KaiC is an autophosphatase. When the A-loops are exposed, however, KaiC is an autokinase. The data suggest that KaiA stabilizes the exposed state of the A-loops through direct binding. We also show evidence that if KaiA cannot stabilize the exposed state KaiC remains hypophosphorylated. We propose that KaiB inactivates KaiA by preventing it from stabilizing the exposed state of the A-loops. Thus, KaiA and KaiB likely act by shifting the dynamic equilibrium of the A-loops between exposed and buried states, which shifts the balance of autokinase and autophosphatase activities of KaiC. A-loop exposure likely moves the ATP closer to the sites of phosphorylation and we show evidence in support of how this movement may be accomplished. Density functional theory calculations of isolated Watson?Crick A:U and A:T base pairs predict that adenine 13C2 trans-hydrogen bond deuterium isotope shifts due to isotopic substitution at the pyrimidine H3, 2h?13C2, are sensitive to the hydrogen-bond distance between the N1 of adenine and the N3 of uracil or thymine, which supports the notion that 2h?13C2 is sensitive to hydrogen-bond strength. Calculated 2h?13C2 values at a given N1?N3 distance are the same for isolated A:U and A:T base pairs. Replacing uridine residues in RNA with 5-methyl uridine and substituting deoxythymidines in DNA with deoxyuridines do not statistically shift empirical 2h?13C2 values. Thus, we show experimentally and computationally that the C7 methyl group of thymine has no measurable affect on 2h?13C2 values. Furthermore, 2h?13C2 values of modified and unmodified RNA are more negative than those of modified and unmodified DNA, which supports our hypothesis that RNA hydrogen bonds are stronger than those of DNA. It is also shown here that 2h?13C2 is context dependent and that this dependence is similar for RNA and DNA.