Chlamydomonas reinhardtii as a new model system for studying the molecular basis of the circadian clock

AbstractThe genome of the unicellular green alga Chlamydomonas reinhardtii has both plant-like and animal-like genes. It is of interest to know which types of clock genes this alga has. Recent forward and reverse genetic studies have revealed that its clock has both plant-like and algal clock components. In addition, since C. reinhardtii is a useful model organism also called “green yeast”, the identification of clock genes will make C. reinhardtii a powerful model for studying the molecular basis of the eukaryotic circadian clock. In this review, we describe our forward genetic approach in C. reinhardtii and discuss some recent findings about its circadian clock

The role of actin in temperature-dependent gel-sol transformation of extracts of Ehrlich ascites tumor cells

Ehrlich aseites tumor cell extracts form a gel when warmed to 25 ~ at pH 7.0 in sucrose solution, and the gel rapidly becomes a sol when cooled to 0*C. This gelsol transformation was studied quantitatively by determining the volume or the total protein of pellets of gel obtained by low-speed centrifugation. The gelation depended on nucleotide triphosphates, Mg ~+, KC1, and a reducing agent. Gelation was inhibited reversibly by 0.5 /~M free Ca 2+, and 25-50 ng/ml of either cytochalasin B or D, but it was not affected by 10 mM colchicine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the gel was composed of six major proteins with mol wt>300,000, 270,000, 89,000, 51,000, 48,000, and 42,000 daltons. The last component was identified as cell actin because it had the same molecular weight as muscle actin and bound with muscle myosin and tropomyosin. The role of actin in gelation was studied by use of actin-inhibitors. Getation was inhibited by a chemically modified subfragment-1 of myosin, which binds with F-actin even in the presence of ATP, and by bovine pancreatic DNase I, which tightly binds with G-actin. Muscle G-actin neutralized the inhibitory effect of DNase I when added at an equimolar ratio to the latter, and it also restored gelation after its inhibition by DNase I. These findings suggest that gelation depends on actin. However, the extracts showed temperature-dependent, cytochalasin-sensitive, and Ca~+-regulated gelation as did the original extracts when the cell actin in the extracts was replaced by muscle actin, suggesting that components other than cell actin might be responsible for these characteristics of the gelation. KEY WORDS Ehrlich aseites tumor cells actin 9 gelation Ca ~+ regulation cytochalasins Recently, it has become possible to study the assembly and disassembly of actin-containing filaments (microfilaments) in ~ritro. Kane first found that actin filaments form a gel when extracts of sea urchin eggs are warmed in the presence of ATP, ethyleneglycol-bis (fl-aminoethylether)-N,N'-tetraacetate 0EGTA), and KC1 (26, 27). Similar temperature- and ATP-dependent gelatio

Circadian rhythm of the cyanobacterium Synechocystis sp

The cyanobacterium Synechocystis sp. strain PCC 6803 exhibited circadian rhythms in complete darkness. To monitor a circadian rhythm of the Synechocystis cells in darkness, we introduced a P dnaK1 ::luxAB gene fusion (S. Aoki, T. Kondo, and M. Ishiura, J. Bacteriol. 177:5606-5611, 1995), which was composed of a promoter region of the Synechocystis dnaK1 gene and a promoterless bacterial luciferase luxAB gene set, as a reporter into the chromosome of a dark-adapted Synechocystis strain. The resulting dnaK1-reporting strain showed bioluminescence rhythms with a period of 25 h (on agar medium supplemented with 5 mM glucose) for at least 7 days in darkness. The rhythms were reset by 12-h-light-12-h-dark cycles, and the period of the rhythms was temperature compensated for between 24 and 31°C. These results indicate that light is not necessary for the oscillation of the circadian clock in Synechocystis. Circadian rhythms are biological oscillations with a period of about 1 day and are found ubiquitously in organisms from cyanobacteria to humans. Since circadian rhythms in diverse organisms persist under constant conditions (i.e., constant light or darkness at a constant temperature), an endogenous mechanism called "circadian clock" that generates the rhythms is postulated. Light resets the phases of circadian rhythms in all organisms examined so far. In green plants, a phase-resetting light signal or signals are supposed to be mediated by phytochrome (8, 23) or a blue-light receptor (21). Light is also required for sustaining circadian rhythms in plants and algae Previously, by using a bacterial luciferase luxAB gene set as a reporter, we demonstrated that a circadian clock controls the expression of the dnaK1 gene, which encodes a heat shock protein, DnaK, in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 (2). The dnaK1-reporting strain CFC2 cultured under continuous illumination (LL) exhibited circadian rhythms of bioluminescence, which reflect the rhythmic activation of the dnaK1 promoter (2). However, the bioluminescence declined completely within 1 or 2 days when the cells were maintained in DD. In cyanobacteria, it remains to be determined whether a light signal is necessary for the oscillation of the circadian clock. We took advantage of the fact that Synechocystis can grow heterotrophically on glucose in darkness when the cells are exposed to a daily, brief light pulse (light-activated heterotrophic growth [LAHG] [1]). The Synechocystis cells grown under LAHG conditions (dark-adapted cells) can continue to grow for 6 to 8 days even in DD (1). We constructed a dark-adapted dnaK1-reporting strain and found that this strain showed a persistent circadian rhythm of bioluminescence even in DD, indicating that the oscillation of the clock does not need any light signal in Synechocystis. MATERIALS AND METHODS Bacterial strains, media, and cultures. Wild-type cells of Synechocystis sp. strain PCC 6803 were maintained in BG-11 liquid medium (19) or on BG-11 agar that contained 1 mM sodium thiosulfate, 10 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES [pH 8.2]), and 1.5% (wt/vol) Bacto-Agar (Difco Laboratories, Detroit, Mich.) under LL (intensity of about 35 mol m Ϫ2 s Ϫ1 from white fluorescent lamps), as described previously (2). A bioluminescent dnaK1-reporting strain of Synechocystis, CFC2 (2), was maintained in BG-11 medium (or agar) supplemented with 40 g of spectinomycin sulfate per ml. Dark-adapted cells of Synechocystis were maintained under LAHG conditions, in which cells were grown in the presence of 5 mM glucose in darkness except for a 15-min light pulse of 40 mol m Ϫ2 s Ϫ1 every 24 h (1). (Although Anderson et al. [1] used daily 5-min light pulses, we used 15-min light pulses for technical convenience.) A dnaK1-reporting strain of the dark-adapted Synechocystis constructed in this study, CFC4, was maintained under the LAHG conditions in the presence of 40 g of spectinomycin sulfate per ml. Unless otherwise stated, cells were cultured at 27°C. Construction of a dnaK1-reporting strain of dark-adapted Synechocystis. We first adapted wild-type cells of Synechocystis and CFC2 cells gradually to LAHG conditions as follows (23a). Cells were inoculated into 100 ml of BG-11 liquid medium containing 5 mM glucose and cultured with vigorous shaking (rotation speed of about 180 rpm) under constant illumination (LL) of 35 mol m Ϫ2 s Ϫ1 on the first day. The light period was then decreased stepwise by 2 h a day. On the 13th day, the cells were transferred to and then maintained under LAHG conditions and used as dark-adapted cells. The dark-adapted cells grew continuously under LAHG conditions, both in liquid medium and on agar medium

CSL encodes a leucine-rich-repeat protein implicated in red/violet light signaling to the circadian clock in Chlamydomonas

緑藻の体内時計 : 赤や紫の光情報を体内時計に伝える因子を発見. 京都大学プレスリリース. 2017-03-24.The green alga Chlamydomonas reinhardtii shows various light responses in behavior and physiology. One such photoresponse is the circadian clock, which can be reset by external light signals to entrain its oscillation to daily environmental cycles. In a previous report, we suggested that a light-induced degradation of the clock protein ROC15 is a trigger to reset the circadian clock in Chlamydomonas. However, light signaling pathways of this process remained unclear. Here, we screened for mutants that show abnormal ROC15 diurnal rhythms, including the light-induced protein degradation at dawn, using a luciferase fusion reporter. In one mutant, ROC15 degradation and phase resetting of the circadian clock by light were impaired. Interestingly, the impairments were observed in response to red and violet light, but not to blue light. We revealed that an uncharacterized gene encoding a protein similar to RAS-signaling-related leucine-rich repeat (LRR) proteins is responsible for the mutant phenotypes. Our results indicate that a previously uncharacterized red/violet light signaling pathway is involved in the phase resetting of circadian clock in Chlamydomonas