The RFLP mapping of the calmodulin gene of Neurospora crassa

The map position of the calmodulin gene (cmd) was determined by RFLP (Restriction Fragment Length Polymorphism) mapping in Neurospora crassa. The cmd gene was mapped on chromosome V, between al-3 and inl

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

Real-Time Monitoring of Chloroplast Gene Expression by a Luciferase Reporter: Evidence for Nuclear Regulation of Chloroplast Circadian Period

Chloroplast-encoded genes, like nucleus-encoded genes, exhibit circadian expression. How the circadian clock exerts its control over chloroplast gene expression, however, is poorly understood. To facilitate the study of chloroplast circadian gene expression, we developed a codon-optimized firefly luciferase gene for the chloroplast of Chlamydomonas reinhardtii as a real-time bioluminescence reporter and introduced it into the chloroplast genome. The bioluminescence of the reporter strain correlated well with the circadian expression pattern of the introduced gene and satisfied all three criteria for circadian rhythms. Moreover, the period of the rhythm was lengthened in per mutants, which are phototactic rhythm mutants carrying a long-period gene in their nuclear genome. These results demonstrate that chloroplast gene expression rhythm is a bona fide circadian rhythm and that the nucleus-encoded circadian oscillator determines the period length of the chloroplast rhythm. Our reporter strains can serve as a powerful tool not only for analysis of the circadian regulation mechanisms of chloroplast gene expression but also for a genetic approach to the molecular oscillator of the algal circadian clock

A systematic forward genetic analysis identified components of the Chlamydomonas circadian system

The molecular bases of circadian clocks have been studied in animals, fungi, bacteria, and plants, but not in eukaryotic algae. To establish a new model for molecular analysis of the circadian clock, here we identified a large number of components of the circadian system in the eukaryotic unicellular alga Chlamydomonas reinhardtii by a systematic forward genetic approach. We isolated 105 insertional mutants that exhibited defects in period, phase angle, and/or amplitude of circadian rhythms in bioluminescence derived from a luciferase reporter gene in their chloroplast genome. Simultaneous measurement of circadian rhythms in bioluminescence and growth rate revealed that some of these mutants had defects in the circadian clock itself, whereas one mutant had a defect in a specific process for the chloroplast bioluminescence rhythm. We identified 30 genes (or gene loci) that would be responsible for rhythm defects in 37 mutants. Classification of these genes revealed that various biological processes are involved in regulation of the chloroplast rhythmicity. Amino acid sequences of six genes that would have crucial roles in the circadian clock revealed features of the Chlamydomonas clock that have both partially plant-like and original components

The ATP-Mediated Regulation of KaiB-KaiC Interaction in the Cyanobacterial Circadian Clock

<div><p>The cyanobacterial circadian clock oscillator is composed of three clock proteins—KaiA, KaiB, and KaiC, and interactions among the three Kai proteins generate clock oscillation <i>in vitro</i>. However, the regulation of these interactions remains to be solved. Here, we demonstrated that ATP regulates formation of the KaiB-KaiC complex. In the absence of ATP, KaiC was monomeric (KaiC^1mer) and formed a complex with KaiB. The addition of ATP plus Mg²⁺ (Mg-ATP), but not that of ATP only, to the KaiB-KaiC^1mer complex induced the hexamerization of KaiC and the concomitant release of KaiB from the KaiB-KaiC^1mer complex, indicating that Mg-ATP and KaiB compete each other for KaiC. In the presence of ATP and Mg²⁺ (Mg-ATP), KaiC became a homohexameric ATPase (KaiC^6mer) with bound Mg-ATP and formed a complex with KaiB, but KaiC hexamerized by unhydrolyzable substrates such as ATP and Mg-ATP analogs, did not. A KaiC N-terminal domain protein, but not its C-terminal one, formed a complex with KaiB, indicating that KaiC associates with KaiB <i>via</i> its N-terminal domain. A mutant KaiC^6mer lacking N-terminal ATPase activity did not form a complex with KaiB whereas a mutant lacking C-terminal ATPase activity did. Thus, the N-terminal domain of KaiC is responsible for formation of the KaiB-KaiC complex, and the hydrolysis of the ATP bound to N-terminal ATPase motifs on KaiC^6mer is required for formation of the KaiB-KaiC^6mer complex. KaiC^6mer that had been hexamerized with ADP plus aluminum fluoride, which are considered to mimic ADP-Pi state, formed a complex with KaiB, suggesting that KaiB is able to associate with KaiC^6mer with bound ADP-Pi. </p> </div

Native PAGE gels, gel filtration chromatography elution profiles, and the immunoblots of the KaiB_1-94-KaiC complexes.

<p>A, B. Native PAGE gels. Reaction mixtures containing 15 μM KaiB_1-94 and 5 μM KaiC_N^1mer (A) or KaiC_C/DD^1mer (B) were incubated at 4 °C for the periods indicated (hereafter, unless otherwise stated, 0 h shows data for samples taken at the onset of incubation) and subjected to native PAGE. Other conditions were the same as for Figure 1C legend. C. Gel filtration chromatography elution profiles and an immunoblot. Reaction mixtures containing 7.5 μM KaiB_1-94, 2.5 μM KaiC_N^6mer, and 1 mM ATP plus 5 mM MgCl₂ (Mg-ATP) were incubated at 4 °C for 6 h and then subjected to gel filtration chromatography on a Superdex 200/HR 10/30 column equilibrated with reaction buffer containing 0.5 mM ATP and 5 mM MgCl₂ at 4 °C. We also separately analyzed KaiB_1-94 and KaiC_N^6mer by gel filtration chromatography as controls. The peak fraction samples were subjected to SDS-PAGE, blotted to PVDF membranes, and reacted with an anti-KaiB antiserum. Other conditions were the same as for Figure 1C legend. Black solid line, KaiB_1-94 + KaiC_N^6mer; gray solid line, KaiC_N^6mer; black broken line, KaiB_1-94. </p

The sequence motifs of KaiC and KaiB-KaiC complex formation.

<p>A. A diagram of the sequence motifs of KaiC. B. Time courses of KaiB-KaiC_DD^6mer complex formation. KaiB_WT (1 μM) and KaiB_1-94 (2 μM) were separately incubated with 1 μM KaiC_DD^6mer in reaction buffer containing Mg-ATP at 4 °C or 40 °C for the various periods indicated, and then aliquots of the reaction mixtures were subjected to native PAGE on 10 % gels. After staining the gels with CBB to visualize proteins, we estimated the amounts of KaiBs-KaiC_DD^6mer complex by densitometry. We used the maximum value obtained at a time of 6 h as the maximum value. KaiBs added and temperature conditions: open circles, KaiB_WT (40 °C); closed circles, KaiB_1-94 (40 °C); open squares, KaiB_1-94 (4 °C). C. A native PAGE gel of the reaction products of KaiC_DD^1mer with KaiB_1-94. Reaction mixtures containing 15 μM KaiB_1-94 and 5 μM KaiC_DD^1mer in reaction buffer were incubated at 4 °C for 6 h and then subjected to native PAGE. Proteins were visualized by CBB staining of the gel. Because KaiB_1-94 has an isoelectric point of 9.7, it moved in the opposite direction and could not detect by native PAGE. D. A native PAGE gel of the reaction products of KaiC_DD^1mer with KaiB_WT. Reaction mixtures containing 7.5 μM KaiB_WT and 5 μM KaiC_DD^1mer in reaction buffer were incubated at 4 °C for 6 h and then subjected to native-PAGE. E. Time course of KaiB_1-94-KaiC_DD^1mer complex formation. Reaction mixtures containing 5 μM KaiB_1-94 and 5 μM KaiC_DD^1mer in reaction buffer were incubated at 4 °C for the periods indicated and then subjected to native PAGE. We used the value obtained at a time of 9 h as the maximum value. A typical experimental data is shown. F. KaiB_1-94 concentration-dependence of the KaiB_1-94-KaiC_DD^1mer complex formation. Reaction mixtures containing 1 μM KaiC_DD^1mer and various amounts (0.5, 0.75, 1.0, 1.25, 1.5, 2.0, 2.5, 3.0, and 4.0 μM) of KaiB_1-94 in reaction buffer were incubated at 4 °C for 6 h and then subjected to native PAGE. We used the value obtained at a KaiB_1-94 concentration of 4 μM as the maximum value. A typical experimental data is shown.</p