Circadian Rhythm and cDNA Cloning of the Clock Gene period in the Honeybee Apis cerana japonica

Isolated individual foragers of Apis cerana japonica could be entrained under a light-dark cycle, and the predominant activity was concentrated to the later part of the photophase. Foragers showed circadian rhythm under conditions of constant light and constant dark with free-running periods of more and less than 24 hr, respectively. These observations indicated that A. cerana possesses a circadian clock controlling locomotor activity. To investigate the molecular mechanism underlying the circadian system we cloned cDNA for a homolog of the clock gene period (per) from the honeybee by a PCR-strategy. The cloned percDNAs consisted of two types, α and β, encoding a putative protein of 1124 amino acids and 1116 amino acids, respectively. The sequences of types α and β were identical except that the former possessed an additional 24 bp stretch corresponding to 8 amino acids in the conserved C2 block. These two types were assumed to be differentially spliced variants and found also in per cDNA of A. mellifera. In support of this idea, Southern blotting experiments showed that per of A. cerana is a single copy gene. RT-PCR analysis and subcloning of the products revealed that the both types α and β are expressed in the brain of the forager. A quantitative RT-PCR assay by which the level of per mRNA in one single brain can be detected was established. Per mRNA level showed daily oscillation under a light-dark cycle with a change of the ratio of type α to β

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

Complete chloroplast DNA sequence of the moss Physcomitrella patens: evidence for the loss and relocation of rpoA from the chloroplast to the nucleus

The complete chloroplast DNA sequence (122 890 bp) of the moss Physcomitrella patens has been determined. The genome contains 83 protein, 31 tRNA and four rRNA genes, and a pseudogene. Four protein genes (rpoA, cysA, cysT and ccsA) found in the liverwort Marchantia polymorpha and the hornwort Anthoceros formosae are absent from P.patens. The overall structure of P.patens chloroplast DNA (cpDNA) differs substantially from that of liverwort and hornwort. Compared with its close relatives, a 71 kb region from petD to rpoB of P.patens is inverted. To investigate whether this large inversion and the loss of rpoA usually occur in moss plants, we analyzed amplified cpDNA fragments from four moss species. Our data indicate that the large inversion occurs only in P.patens, whereas the loss of the rpoA gene occurs in all mosses. Moreover, we have isolated and characterized the nuclear rpoA gene encoding the α subunit of RNA polymerase (RNAP) from P.patens and examined its subcellular localization. When fused to green fluorescent protein, RpoA was observed in the chloroplasts of live moss protonemata cells. This indicates that chloroplast RNAP is encoded separately by chloroplast and nuclear genomes in the moss. These data provide new insights into the regulation and evolution of chloroplast transcription

Differential Expression on a Daily Basis of Plastid Sigma Factor Genes from the Moss Physcomitrella patens. Regulatory Interactions among PpSig5, the Circadian Clock, and Blue Light Signaling Mediated by Cryptochromes

The nuclear-encoded plastid sigma factors are supposed to be a regulatory subunit of the multisubunit bacteria-type plastid RNA polymerase. We studied here whether or not three genes, PpSig1, PpSig2, and PpSig5 encoding plastid sigma factors, are controlled by the circadian clock and/or by blue light signaling in the moss Physcomitrella patens. Among the three PpSig genes, only PpSig5 was clearly controlled by the circadian clock. In contrast to the differential regulation on a daily timescale, a pulse of blue light induced the expression of all the three PpSig genes. This induction was significantly reduced in a knockout mutant that lacked the blue light photoreceptor cryptochromes PpCRY1a and PpCRY1b, indicating that PpCRY1a and/or PpCRY1b mediate the blue light signal that induces the expression of the PpSig genes. In a daily cycle of 12-h blue light/12-h dark, the timing of peak expression of PpSig5 and a chloroplast gene psbD, encoding the D2 subunit of photosystem II, advanced in the cryptochrome mutant relative to those in the wild type, suggesting the presence of regulatory interactions among the expression of PpSig5 and psbD, the circadian clock, and the blue light signaling mediated by the cryptochrome(s)