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

<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

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

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

List of primers.

<p>List of primers.</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

[Hosta sp.]

原著和名: [記載なし]科名: ユリ科 = Liliaceae採集地: 宮崎県 東臼杵郡 大崩山 (日向 東臼杵郡 大崩山)採集日: 1973/8/1採集者: 萩庭丈壽整理番号: JH003752国立科学博物館整理番号: TNS-VS-953752備考: DB作成協力会による補足あ

Additional file 8: of Deep landscape update of dispersed and tandem repeats in the genome model of the red jungle fowl, Gallus gallus, using a series of de novo investigating tools

Diversity of CR1 within galGal4. The clustering of CR1 copies into subfamilies was re-investigated using SiLiX. Eight subfamilies were found, 7 of them matching with the Repbase sub-families CR1-C, CR1-D, CR1_F, CR1-G, CR1_GG, CR1-H, and CR1-Y. Their respective abundance in galGal4 was summarized in Table S2. (ODT 30 kb

<i>Mariner</i> Transposons Contain a Silencer: Possible Role of the Polycomb Repressive Complex 2 - Fig 11

<p><b>Counting of YY1 (a) and NFAT-5 (b) binding sites along the sequence of genomic Δ7 <i>Hsmar2</i> segments.</b> Areas filled in grey located the Δ8 segment within the Δ7 <i>Hsmar2</i> segment.</p