Project description:CWO binding sites were genome-widely searched with Drosophila genome tiling array. Abstract: The Drosophila circadian clock consists of integrated autoregulatory feedback loops, making the clock difficult to elucidate without comprehensively identifying the network components in vivo. Previous studies have adopted genome-wide screening for clock-controlled genes using high-density oligonucleotide arrays that identified hundreds of clock-controlled genes. In an attempt to identify the core clock genes among these candidates, we applied genome-wide functional screening using an RNAi system in vivo. Here we report the identification of novel clock gene candidates including clockwork orange (cwo), a transcriptional repressor belonging to the basic helix-loop-helix-ORANGE family. cwo is rhythmically expressed and directly regulated by CLK-CYC through canonical E-box sequences. A genome-wide search for its target genes using the Drosophila genome tilling array revealed that cwo forms its own negative feedback loop and directly suppresses the expression of other clock genes through the E-box sequence. Furthermore, this negative transcriptional feedback loop contributes to sustaining a high-amplitude circadian oscillation in vivo. Based on these results, we propose that the competition between cyclic CLK-CYC activity and the adjustable threshold imposed by CWO keeps E-box-mediated transcription within the controllable range of its activity, thereby rendering a Drosophila circadian clock capable of generating high-amplitude oscillation. Keywords: ChIP-chip
Project description:Squalene synthase (SQS) is a bifunctional enzyme that catalyzes the condensation of two molecules of farnesyl diphosphate (FPP) to give presqualene diphosphate (PSPP) and the subsequent rearrangement of PSPP to squalene. These reactions constitute the first pathway-specific steps in hopane biosynthesis in Bacteria and sterol biosynthesis in Eukarya. The genes encoding SQS were isolated from the hopane-producing bacteria Thermosynechococcus elongatus BP-1, Bradyrhizobium japonicum, and Zymomonas mobilis and cloned into an Escherichia coli expression system. The expressed proteins with a His(6) tag were found exclusively in inclusion bodies when no additives were used in the buffer. After extensive optimization, soluble recombinant T. elongatus BP-1 SQS was obtained when cells were disrupted and purified in buffers containing glycerol. The recombinant B. japonicum and Z. mobilis SQSs could not be solubilized under any of the expression and purification conditions used. Purified T. elongatus His(6)-SQS gave a single band at 42 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and molecular ion at m/z 41886 by electrospray mass spectrometry. Incubation with FPP and NADPH gave squalene as the sole product. Incubation of the enzyme with [(14)C]FPP in the absence of NADPH gave PSPP. The enzyme requires Mg(2+) for activity, has an optimum pH of 7.6, and is strongly stimulated by detergent. Under optimal conditions, the K(m) of FPP is 0.97 +/- 0.10 microM and the k(cat) is 1.74 +/- 0.04 s(-1). Zaragozic acid A, a potent inhibitor of mammalian, fungal, and Saccharomyces cerevisiae SQSs, also inhibited recombinant T. elongatus BP-1 SQS, with a 50% inhibitory concentration of 95.5 +/- 13.6 nM.
Project description:To investigate the temperature response in Thermosynechococcus elongatus (BP-1) and look for coordinated control in the cell, transcriptomes of BP-1 were measured using RNAseq following exposure to low and high temperature stress. The amount of temperature increase (53 to 61 °C) and decrease (53 to 45 °C) was based on the allowable range of continuous growth. The cells were growth in three separate culture tubes under LED light in a temperature-controlled submersible chamber under batch growth conditions. The temperature shift was conducted once the cells reached their target concentration of 1.5E7 cells/ml. A control experiment was conducted, in which the temperature remained at 53 °C, ensuring that the cellular physiology and light attenuation were comparable and that the only difference between the hot and cold treatments relative to the control treatment was temperature.
Project description:In the present study we show that the master myogenic regulatory factor, MYOD1, is a positive modulator of molecular clock amplitude and functions with the core clock factors for expression of clock-controlled genes in skeletal muscle. We demonstrate that MYOD1 directly regulates the expression and circadian amplitude of the positive core clock factor Bmal1. We identify a non-canonical E-box element in Bmal1 and demonstrate that is required for full MYOD1-responsiveness. Bimolecular fluorescence complementation assays demonstrate that MYOD1 colocalizes with both BMAL1 and CLOCK throughout myonuclei. We demonstrate that MYOD1 and BMAL1:CLOCK work in a synergistic fashion through a tandem Ebox to regulate the expression and amplitude of the muscle specific clock-controlled gene, Titin-cap (Tcap). In conclusion, these findings reveal mechanistic roles for the muscle specific transcription factor MYOD1 in the regulation of amplitude of the molecular clock as well as synergistic regulation of clock-controlled genes in skeletal muscle.
Project description:NDH (NADH-quinone oxidoreductase)-1 complexes in cyanobacteria have specific functions in respiration and cyclic electron flow as well as in active CO2 uptake. In order to isolate NDH-1 complexes and to study complex-complex interactions, several strains of Thermosynechococcus elongatus were constructed by adding a His-tag (histidine tag) to different subunits of NDH-1. Two strains with His-tag on CupA and NdhL were successfully used to isolate NDH-1 complexes by one-step Ni2+ column chromatography. BN (blue-native)/SDS/PAGE analysis of the proteins eluted from the Ni2+ column revealed the presence of three complexes with molecular masses of about 450, 300 and 190 kDa, which were identified by MS to be NDH-1L, NDH-1M and NDH-1S respectively, previously found in Synechocystis sp. PCC 6803. A larger complex of about 490 kDa was also isolated from the NdhL-His strain. This complex, designated 'NDH-1MS', was composed of NDH-1M and NDH-1S. NDH-1L complex was recovered from WT (wild-type) cells of T. elongatus by Ni2+ column chromatography. NdhF1 subunit present only in NDH-1L has a sequence of -HHDHHSHH- internally, which appears to have an affinity for the Ni2+ column. NDH-1S or NDH-1M was not recovered from WT cells by chromatography of this kind. The BN/SDS/PAGE analysis of membranes solubilized by a low concentration of detergent indicated the presence of abundant NDH-1MS, but not NDH-1M or NDH-1S. These results clearly demonstrated that NDH-1S is associated with NDH-1M in vivo.
Project description:A comparative genomics analysis among all forty whole genome sequences available for cyanobacteria (3 thermophiles- Thermosynechococcus elongatus BP-1, Synechococcus sp. JA-2-3B'a (2-13), Synechococcus sp. JA-3-3Ab and 37 mesophiles) was performed to identify genomic and proteomic factors responsible for the behaviour of T. elongatus BP-1, a thermophilic unicellular cyanobacterium with optimum growth temperature [OGT] of 55°C. Majority of genomic and proteomic characteristics for this cyanobacterium indicated contrasting features indicating its mesophilic behaviour while the role of mutational biasness and selection pressure is thought to be responsible for high OGT. Contradictory results were obtained for T. elongatus for synonymous codon usage, CvP-bias and amino acid composition with respect to thermophilic behaviour. Calculated J2 index is lowest among all cyanobacterial genomes. Except for proline and termination codons, T. elongatus showed synonymous codon usage pattern which is expected for mesophiles. Results indicated that among cyanobacterial genomes, majority of genomic and proteomic determinants put T. elongatus very close to mesophiles and the whole genome of this organism represents continuous gain of mesophilic rather than thermophilic behavior.