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. 2008 Apr 1;22(7):918-30.
doi: 10.1101/gad.1650408. Epub 2008 Mar 11.

A Systematic Forward Genetic Analysis Identified Components of the Chlamydomonas Circadian System

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Free PMC article

A Systematic Forward Genetic Analysis Identified Components of the Chlamydomonas Circadian System

Takuya Matsuo et al. Genes Dev. .
Free PMC article

Abstract

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.

Figures

Figure 1.
Figure 1.
Screening of a genetic background suitable for the high-throughput assay of bioluminescence rhythms. All samples were prepared according to the high-throughput assay protocol (see Materials and Methods). Traces of bioluminescence from the 96 samples on agar media are shown on the left, and numbers of samples (per total samples examined) that exhibited a significant circadian rhythmicity (error value [calculated by the RAP] ≤ 0.1) are indicated on the right of the traces. Histograms of period length, peak phase angle, bottom phase angle, and amplitude of the rhythmic samples are shown. Strains or pairs of the crosses are shown on the right.
Figure 2.
Figure 2.
Screening and genetic analysis of circadian rhythm mutants. (A) Representative bioluminescence traces of several types of roc mutants. (SP) roc55; (LP) roc66; (APA) roc83; (DPA) roc25; (LA I) roc77; (LA IIa) roc39; (LA IIb) roc109; (LA III) roc76. The maximum values are expressed as 100. (B) Pie chart showing the distribution of all mutants by phenotype. (C) Representative results of the cosegregation analysis. Histograms of hygromycin-resistant (red) and hygromycin-sensitive (blue) progenies are shown. (D) Pie chart showing the distribution of all mutants by segregation pattern. (E) Southern blot analysis. The 3′ region of the aph7″ coding sequence was used as a probe. The positions of size markers (2 and 12 kb) are also shown on the left.
Figure 3.
Figure 3.
Growth rhythms of isolated mutants. (A) Growth rhythms of mutants. Growth rhythms were monitored by a continuous culture system (see Materials and Methods) in LL. Two-hour moving averages of growth rates (added fresh medium per culture volume per day) are shown. (B) Simultaneous measurement of growth and bioluminescence rhythms. Growth and bioluminescence rhythms were monitored simultaneously from an identical liquid culture by the continuous culture system.
Figure 4.
Figure 4.
Schematic representation of the integration sites of roc15/roc74 (A), roc40 (B), roc11/roc55 (C), roc66 (D), roc75 (E), and roc108/roc114 (F). Boxes indicate exons. Black boxes indicate gene models of JGI Chlamydomonas version 3.0. Predicted (first) initiation codons and stop codons are shown. Red arrows indicate integration sites of the marker in each mutant. The phenotype of the mutants is indicated. Gray bars indicate the predicted genome structures of the roc55 and roc114 mutants. Blue bars indicate the regions used for probes in the Northern blot analysis. The asterisk in B indicates an exon/intron boundary that does not follow the GT–AG rule. Scaffold numbers and directions (arrows) are indicated on the left.
Figure 5.
Figure 5.
Complementation of the mutant phenotypes. Transformants with the genomic DNA fragments were subjected to the high-throughput bioluminescence assay. (A) Representative traces of complemented (red) and control (blue) strains on agar. For roc55 and roc114, traces of complemented strains with roc55HS and roc114KK genomic fragments, respectively, are shown. (B–G) Histograms of the complementation analysis of roc15 (B), roc40 (C), roc55 (D), roc66 (E), roc75 (F), and roc114 (G). For period length mutants (roc15, roc55, and roc66), transformants with period lengths in the range of 24.0–26.0 h were judged to be complemented. For roc40, transformants in the range of 24.0- to 27.0-h periods in LL were judged to be complemented. For low-amplitude mutants (roc75 and roc114), transformants with amplitudes (calculated by the RAP) >0.4 and in the range of 0.2–0.4 were judged to be complemented and partially complemented, respectively. Numbers of complemented (roc15, roc40, roc55, roc66, and roc75) and partially complemented (roc114) transformants among the transformants examined are shown in the graphs.
Figure 6.
Figure 6.
Northern blot analysis of ROC15 (A), ROC40 (B), ROC55 (C), ROC66 (D), ROC75 (E), and ROC114 (F). Total RNA (10 μg; A,B,D–F) and polyadenylated RNA (from 100 μg of total RNA; C) from asynchronous cultures of the wild-type, mutant (roc15, roc74, roc40, roc55, roc11, roc66, roc75, roc114, and roc108), complemented (roc15C, roc40C, roc55C, roc66C, and roc75C), and partially complemented (roc114PC) strains were analyzed. The roc55C and roc114PC strains are complemented and partially complemented strains obtained by gene transfer of the genomic DNA fragments roc55HS and roc114KK, respectively. Arrows indicate the band sizes of wild-type mRNA. Probes are indicated in Figure 4.
Figure 7.
Figure 7.
Predicted amino acid sequences of ROC proteins. (A) Schematic view of primary structures of ROC proteins. (B–F) Alignment of amino acid sequences. The amino acid sequences of ROC15, ROC40, ROC66, and ROC75 were aligned to the Arabidopsis proteins using CLUSTAL W (http://clustalw.ddbj.nig.ac.jp). (B) GARP motif of ROC15, PCL1(LUX), and ARR1. (C) Single MYB repeat and following homologous region of ROC40, CCA1, and LHY. (D) B-box zinc-finger domain of ROC66 and CO. The consensus residues of CO-type B-box are denoted in yellow. B-box2 region of ROC66 does not match the consensus of B-box. (E) CCT domain of ROC66, CO, and TOC1. (F) GARP motif of ROC75, PCL1(LUX), and ARR1. Identities of amino acid sequences are indicated.
Figure 8.
Figure 8.
Circadian expression patterns of ROC mRNAs. (A) Northern blot analysis of ROC15, ROC40, ROC66, ROC75, and ROC114 mRNAs. Total RNA (5 μg) from a synchronous culture of the wild-type strain in LL (10 μmol m−2 s−1) at 17°C was analyzed. The ROC55 mRNA was undetectable under the experimental conditions. Probes are the same as those used in Figure 6. Equal RNA loading was confirmed by reprobing the blot for rbcL mRNA. (B) Temporal expression profiles of the ROC mRNAs. The maximum values are expressed as 100.

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