Identification of ovine <i>Vegfa</i> transcripts by standard PCR using primers O14I/O15I: No evidence for <i>Vegfaxxxb</i> isoforms.

<p><u>A</u>/ Picture obtained after agarose gel electrophoresis of <i>Vegfa</i> PCR products. PCR was performed on a cDNA mix obtained from MBH of ewes sampled in May, August and November. The primers were designed to amplify both Vegfaxxx and Vegfaxxxb products. A typical “3-bands pattern” was obtained for all 4 primer combinations. The 3 bands, labeled b1, b2 and b3 (arrows), were individually extracted on gel for the primer combination O14I/O15I. <u>B</u>/ Schematics summarizing the results of Sanger sequencing after cloning of PCR products contained in b1, b2 and b3 (location of primers O14I/O15I is provided). Five distinct amplicons were identified: 3 for b1 (labeled b1a, b1b and b1c) and a single product for both b2 and b3. The number of clones sequenced for each amplicon is indicated on the right. An annotated sequence of ovine exon 6 is provided and the alternative splice sites used to generate the transcripts b1b and b1c are identified. Note that 0/84 clones included the putative exon 8b.</p

RNA-seq identifies intron-spanning, uniquely mapping reads at the <i>Vegfa</i> gene.

<p>RNA-seq identifies intron-spanning, uniquely mapping reads at the <i>Vegfa</i> gene.</p

Seasonal expression of <i>Vegfa</i> and <i>VegfR2</i> in the ovine MBH and pituitary.

<p>All values on the y-axis are normalized relative levels of expression (qPCR) or optical density (ISH). <u>A</u>/ 1^st Experiment: qRT-PCR for <i>Vegfa</i> and <i>VegfR2</i> on MBH cDNA samples from OVX+E2 ewes maintained under natural conditions and culled in May, August and November. Post-hoc Tukey test: **p<0.01, ***p<0.005 & ****p<0.001. <u>B</u>/ 2^nd Experiment: qRT-PCR for <i>Vegfa</i> and <i>VegfR2</i> on MBH (left column) and PD (right column) cDNA samples from OVX+E2 ewes, which had been kept indoors under prolonged SP or exposed to 3 weeks of LP. A group of LP-exposed ewes had been THX months before photoperiodic transfer (LP-THX). <u>C</u>/ 3^rd Experiment: ISH for <i>Vegfa</i> on coronal brain sections at the level of the caudal PT / infundibulum region. Sham-operated and THX ewes (OVX+E2 model) were sampled in May and November and quantification was performed in the PT. <u>D</u>/ 4^th Experiment: ISH for <i>Vegfa</i> on coronal brain sections at the level of the caudal PT / infundibulum region. Intact ewes were sampled in May and November and quantification was performed in the PT. Representative autoradiograms are shown (scale bar = 2mm).</p

Exon 8 and putative exon 8b share a common 4 bases motif at their 5’ end.

<p><u>A</u>/ Nucleotide sequence of ovine exon 8, splice sites are indicated. Both exon 8 and exon 8b would start with the same 4 bases motif ATGT, in green and bold. <u>B</u>/ Standard PCR / agarose gel electrophoresis was performed using MBH extracts from ewes culled across seasons. The use of downstream primers, which all contain the 3’end with the 11 bases complementary to exon 7 and a 5’end that includes these 4 bases or less (O73I-O76I), did not yield any PCR products. However downstream primers, which all contain the 3’end with the 11 bases complementary to exon 7, along with a 5’end that includes these 4 bases preceeded by a random stretch of 5 bases (O77I-O79I), all yielded robust amplification. <u>C</u>/ Single or double point mutation were made to the ATGT motif in the backbone of “exon 8b-specific” O63I primer. Note that a single point mutation (ATCT, O81I) is enough to blunt PCR (see text for details).</p

<i>Vegfaxxxb</i> transcripts as PCR artefacts: Extending findings to the human-derived HEK293 cell line.

<p><u>A</u>/ Proposed sequence for the prototypical “<i>Vegfaxxxb</i>-specific” PCR primer. Note that beyond the 11 bases complementary to exon 7 and a minimum of 2–3 bases complementary to exon 8, the sequence at the 5’end of the primer does not confer specificity. However, this 5’ tail is mandatory as it increases the Tm of the primer. <u>B</u>/ Sequence alignments of the ovine “<i>Vegfaxxxb</i>-specific” PCR primer O63I, and the human “<i>Vegfaxxxb</i>-specific” PCR primer O81I as defined by Bates and coll. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197123#pone.0197123.ref019" target="_blank">19</a>]. Note that the two sequences differ only by a single nucleotide, located in the 4 bases motif. The sequence of the proximal part of human <i>Vegfa</i> exon 8 is provided. <u>C</u>/ PCR on cDNA samples obtained from HEK293 cells. The sequences of the 9 bases stretches of the 5’end of the downstream primers are provided above the gel picture to facilitate interpretation. The human “<i>Vegfaxxxb</i>-specific” primer O81I yielded a faint signal compared to O63I and O80I, which are either single or double point mutations of O81I - but respectively spare all 4 bases and 3 bases of the ATGT motif for exon 8. Note that even a primer bearing an unrelated 7 bases stretch at its 5’ end (O90I) could outperform O81I.</p

Searching for <i>Vegfaxxxb</i> transcripts with isoform-specific PCR primers.

<p><u>A</u>/ Standard PCR / agarose gel electrophoresis was performed using MBH extracts from ewes culled across seasons (same samples used for qPCR data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197123#pone.0197123.g001" target="_blank">Fig 1</a>). O21I and O22I are Vegfaxxxb-specific primers while O23I spans the putative splice site for exon 8b (and is therefore a Vegfaxxx-specific primer). <u>B</u>/ Standard PCR / agarose gel electrophoresis was performed using MBH extracts from ewes culled under SP or after a photoperiodic transfer to LP (intact and THX ewes; same samples used for qPCR data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197123#pone.0197123.g001" target="_blank">Fig 1</a>).</p

List of primers.

<p>List of primers.</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

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

<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