, 2000). The ‘additional’ KaiC proteins from Cyanothece and Crocosphaera lack both DXXG motifs and display a proline or arginine aligning to Q115 of S. elongatus-KaiC following the DXXG2 motif. In S. elongatus-KaiC mutation of Q115 to arginine abolishes kaiBC expression ( Nishiwaki et al., 2000). Hence it is very unlikely that these additional KaiC proteins drive kaiBC expression rhythms. Cyanobacteria form a highly diverse group of photoautotrophic prokaryotes, which

PF-562271 cell line is reflected not only by their various habitats, morphologies, metabolic needs and behavior but also by the complex diversity of their KaiC-based timing systems. When we analyzed the conservation of clock-related genes in a subset of marine cyanobacterial genomes (Table 1), we detected a large genetic diversity. There are strains lacking some or even all Kai components, others encode multiple copies of kaiC and/or kaiB. For other known clock-related components a similar complex pattern appeared. In summary, the diversity in cyanobacterial Kai-based timing systems appears to be evident primarily regarding the core oscillator and the input pathways. The phosphorylation Selleckchem AZD2281 status of KaiC differs in Cyanobacteria, which possess KaiA and in those, where KaiA is absent. In the first case, KaiC exhibits periodic oscillations of phosphorylation like in S. elongatus. In the other case however, KaiC remains hyperphosphorylated as shown for MED4 in vitro.

Thus, KaiA might be required to turn a timing system into a self-sustained oscillation ( Simons, 2009). Accordingly, diurnal cycles observed in MED4 and other Cyanobacteria lacking KaiA are very likely under the control of an hourglass

instead of a true circadian clock. The KaiABC core clock is not universal when we look at diverse marine genomes of Cyanobacteria. Only KaiC homologs can be found almost always, and even in Proteobacteria, Chloroflexi and Archaea (Aoki and Onai, 2009 and Dvornyk et al., 2003). Thus, a minimal timing system simply based on KaiC might exist that could Adenosine represent a general prokaryotic mechanism. Here, UCYN-A presents a fascinating and unprecedented example. Although KaiA and KaiB homologs are absent, output components are present as well as some input components. The exploration of such a minimal KaiC-based system will be an exciting future challenge. Some of the species listed in Table 1 produce cyanotoxins and other secondary metabolites. The most common toxin-producing Cyanobacteria also include the genera Nodularia and Trichodesmium ( Paerl and Otten, 2013). Both of them are also potent formers of the highly visible and harmful cyanobacterial blooms mentioned above and they inhabit brackish water as well as marine ecosystems ( Huisman et al., 2005 and Paerl and Otten, 2013). The circadian clock was shown to improve reproductive fitness in Cyanobacteria living in rhythmic environments ( Gonze et al., 2002, Mori and Johnson, 2001, Ouyang et al., 1998 and Woelfle et al., 2004).

The blot was washed twice again for 5 min with T-TBS and twice for 5 min with TBS. The blot was then developed using a chemiluminescence ECL kit. Immunoblots were quantified by scanning the films with a Hewlett-Packard Scanjet 6100C scanner and determining optical densities with an OptiQuant version 02.00 software (Packard Instrument Company). Optical density values were obtained for the studied proteins. RNA

was isolated from striatum OSI-744 research buy using the TRIzol Reagent (Invitrogen). Approximately 2 μg of total RNA were added to each cDNA synthesis reaction using the SuperScript-II RT pre-amplication system. Reactions were performed at 42 °C for 1 h using the primer T23 V (5′ TTT TTT TTT TTT TTT TTT TTTTTV). Quantitative

PCR amplification was carried out using specific primer pairs designed with Oligo Calculator version 3.02 (http://www.basic.nwu.edu/biotools/oligocalc.html) and synthesized by IDT (MG, Brazil). The sequences of the primers used are listed in Table 1. Quantitative PCRs were carried out in an Applied-Biosystem StepOne Plus real-time cycler and done in quadruplicate. Reaction settings were composed of an initial denaturation step of 5 min at 95 °C, followed by 40 cycles of 10 s at 95 °C, 10 s at 60 °C, 10 s at 72 °C; samples were kept for 1 min at 60 °C for annealing and then heated from 55 to 99 °C with a ramp of 0.3 °C/s to acquire data to produce the denaturing curve of the amplified products. Quantitative PCRs were made in a 20 μl final volume composed of 10 μl of each reverse transcription sample diluted 50–100 LDK378 supplier times, 2 μl of 10 times PCR buffer, 1.2 μl of 50 mM MgCl2, 0.4 μl of 5 mM dNTPs, 0.8 μl of 5 μM primer pairs, 3.55 μl of water, 2.0 μl of SYBRgreen (1:10,000 Molecular Probe), and 0.05 μl of Platinum Taq mafosfamide DNA polymerase

(5 U/μl). All results were analyzed by the 2 − DDCT method (Livak and Schmittgen, 2001). TBP (TATA box binding protein) was used as the internal control gene for all relative expression calculations. Twelve pups (six per group) were anesthetized using ketamine/xylazine (75 and 10 mg/kg, respectively, i.p.) and were perfused through the left cardiac ventricle with 40 ml of 0,9% saline solution, followed by 40 ml of 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS), pH 7.4, and the descendent aorta was clamped. After the perfusion the brains were removed, post-fixed in the same fixative solution for 4 h at room temperature and cryoprotected by immersing in 15% and after in 30% sucrose solution in PBS at 4 °C. The brains were then frozen by immersion in isopentane cooled with CO2 and stored in a freezer (− 80 °C) for later analyses. Serial coronal sections (40 μm) of striatum were obtained using a cryostat at − 20 °C (Leica).