Functional transcriptional effects of Fbxl3 and Fbxl21.

<p>A. oFbxl3 and oFbxl21 dose-dependently reverse oCRY1 inhibition of oCLK/oBM1 induced transactivation of an <i>oRev-erb α</i> reporter construct. Transfection conditions <i>per</i> well: 100 ng of oClock, 100 ng of oBmal1, 100 ng of oCry1, 100 or 200 ng of each Fbxl or ev, 50 ng of <i>oRev-erb alpha</i> promoter reporter, 50 ng of b-Gal reporter and/or necessary amount of empty vector to make a final amount of 600 ng. B. mFbxl21 dose-dependently reverses mCRY1 inhibition of mCLK/mBM1 induced transactivation of an <i>mPer1</i> reporter construct. Transfection conditions <i>per</i> well: 100 ng of mClock, 100 ng of mBmal1, 20 ng of oCry1, 100 or 200 ng of mFbxl21 or ev, 50 ng of <i>Per1</i> promoter reporter, 50 ng of b-Gal reporter and/or necessary amount of empty vector to make a final amount of 600 ng. Data in A/B are mean±SEM of one triplicate representative from 3 replicate experiments. Different letters above bars indicate significantly different groups (p<0.05).</p

Cloning and mRNA distribution of ovine Fbxl3 and Fbxl21.

<p>A/D. Schematics depicting the cds of mRNA of Fbxl3 and Fbxl21, respectively. Primers used for RT-PCR are indicated. Note that Fbxl3sv is an in-frame splice-variant while Fbxl21sv is an out-of-frame splice-variant (fs for frame-shift) with an early stop-codon (TGA, nt 489). B/E. Schematics depicting the proteins encoded by the Fbxl3 and Fbxl21 transcripts, respectively. Note that part of F-Box motif of Fbxl21sv is disrupted due to the splicing event while the CBD is spared. Note also that Fbxl21sv is a short protein product of 171 amino acids with the last 8 amino acids (indicated in green) differing from Fbxl21fl due to the AS event. The F-Box motif is left intact but Fbxl21sv does not have a CBD. C. RT-PCR for Fbxl3 on central and peripheral tissues sampled at ZT4. Co-amplification of a CKIδ fragment was used as an internal positive control (oCKIδ, GenBank EF643522), absence of cDNA in the PCR mix as a negative control. Upper panel: screening with O47C/O48C primers. Lower panel: screening with O51C/O52C primers clarifies the tissue distribution of Fbxl3sv. F. RT-PCR for Fbxl21 on central and peripheral tissues sampled at ZT4. G. Sheep - mouse amino acid conservation within the CBD of Fbxl3 and Fbxl21. Residues associated with the <i>Afterhours</i> (<i>Afh</i>) and <i>Overtime</i> (<i>Ovtm</i>) Fbxl3 mutants are highlighted in red. Blue shading indicates residues that differ in at least one of the four sequences. Star, two dots and one dot indicate identical, conservative and semi-conservative substitutions, respectively. Pfam scores for F-box motifs: Fbxl3fl 3.9e⁻⁰⁹, Fbxl3sv 2.1e⁻⁰⁵, Fbxl21fl and Fbxl2sv 6.5e⁻¹¹.</p

mFbxl21 is a clock-controlled gene.

<p>A. Schematics depicting the promoter region of the mouse <i>Fbxl21</i> gene, E' indicate non-canonical E-Box motifs, D-Box indicates a putative response element for the PAR-bZIP proteins. B. The mouse <i>Fbxl21</i> gene is responsive to both CLK/BM1 and the PAR-bZIP transcription factors. Transfection conditions per well: 50 ng of <i>Fbxl21</i> promoter reporter, 50 ng of b-Gal reporter and either 100 ng of mClock+100 ng of mBmal1 or 100 ng of each of the PAR-bZIP transcription factors and/or necessary amount of empty vector to make a final amount of 300 ng. Data in A/B are mean±SEM of one triplicate representative from 3 replicate experiments. Different letters above bars indicate significantly different groups (p<0.05).</p

Fbxl21 and Fbxl3 promote degradation of ovine CRY1 and BM1.

<p>A. COS7 cells were transfected with oCry1, oBm1 and oClk, and an oFbxl expression vector as indicated above each lane. Extracts were made at the start of cycloheximide (CHX) treatment (t = 0) or 2 or 8 h later as indicated and protein levels assessed by WB. Transfection conditions <i>per</i> well: 1 µg of oClock, 600 ng of oBmal1, 600 ng of each oFbxl/ev and 400 ng of oCry1. B. Quantitation of baseline levels (t = 0) of oCRY1, oBM1 or oCLK proteins. Data are normalised to values observed in cells not transfected with any oFbxl (ev). **: significantly lower density than for ev, p<0.01, One-way ANOVA followed by Student-Newman-Keuls post-hoc test. C. Quantitation of temporal decline in protein levels following the start of CHX treatment. Data are expressed relative to t = 0 values for each vector combination. p values for the interaction (treatment×time, two-way ANOVA) are given on the right. Data in B/C are mean±SEM of observations from 3 replicate experiments.</p

<i>In-situ</i> hybridisation for Fbxl3 and Fbxl21 within the SCN of the sheep and mouse.

<p>A. Representative images for expression of Fbxl21 (top), Fbxl3 and the neuropeptides, Avp and Vip (bottom) in the hypothalamus of sheep sampled at ZT4-6. Sense controls for Fbxl riboprobes are also shown (top and middle right panels). B. Fbxl21 undergoes circadian expression in the SCN of the mouse. Top: mRNA profiles in the SCN of mice sampled at 6 different time-points (n = 5 per time-point) under either a 12/12 light-dark cycle (LD) or under constant darkness (DD). Data for ZT/CT0 are double-plotted. Bottom: representative <i>in-situ</i> autoradiograms. Note the SCN-restricted pattern of expression. Two-way ANOVA: time-effect, P<0.001, light condition×time interaction, p = 0.39. C. Fbxl3 does not show circadian expression in the SCN of the mouse. Representative images for expression of Fbxl3 in the brain of mouse sampled at ZT4 and ZT16. Note the ubiquitous pattern of labelling including diffuse expression in the SCN.</p

Ovine and murine Fbxl3 and Fbxl21 physically associate with oCry1.

<p>A. Extracts from COS7 cells co-transfected with Myc-Fbxl and Flag-Cry1 expression vectors were submitted to immunoprecipitation (IP) with α-Myc beads followed by western-blotting (WB). The oCRY1 protein was readily co-purified with oFbxl3fl, oFbxl3sv and oFbxl21fl but not with oFbxl21sv. Note the low CRY1 levels in the input when Fbxl3fl was present. Similar detection levels of IgG (from Myc antibodies) across all conditions demonstrate loading of comparable amounts of supernatant. ev = empty expression vector. B. The use of similar methods demonstrates that the mouse Fbxl21 also interacts with oCRY1.</p

Neuropeptide gene expression in the sheep SCN.

<p>The left panel shows daily mRNA expression profiles for the neuropeptides <i>Avp</i> and <i>Vip</i>. <i>Avp</i> shows a pronounced daily rhythm of expression, but this was unaffected by the photoperiod manipulation. <i>Vip</i> expression shows no significant changes during the daily cycle, or in response to photoperiod manipulation. Data presentation in the graphs is as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159201#pone.0159201.g001" target="_blank">Fig 1</a>. The right panels show representative images for each of the 3 neuropeptide genes studied, with phases from which images were taken. Note the absence of <i>Grp</i> expression in the SCN despite strong labelling in the neighbouring supraoptic and paraventricular nuclei. Scale bar = 4 mm.</p

Processed 454 reads for olfactory epithelium

Processed 454 reads for olfactory epithelium from Salmo salar at the parr stage. Trimmed for quality and adaptors (see paper for detail

Python script for identifying duplicate pairs in Salmo salar

Python script for identifying duplicate pairs in Salmo sala

Processed 454 reads for brain

454 reads for brain from Salmo salar in the parr stage. Trimmed for quality and adaptors (see paper for detail