Protein Sequence Alignment of Selected Gnathostome RARs

<p>RARs are represented from lamprey (LampRAR, Petromyzon marinus), amphioxus (AmphiRAR, Branchiostoma floridae), tunicate (RAR_POLM1, Polyandrocarpa misakiensis), and the synthetic predicted ancestral RAR (Ancestor). The position of the 12 helices is indicated above the alignment (H1–H12). Residues implicated in direct contacts with the ligand are numbered from 1 to 25 below the alignment. The three divergent residues within the LBP between vertebrate RARs are within vertical rectangles in helices 3, 5, and 11. Gnathostome and <i>Polyandrocarpa</i> sequences are named with the nomenclature code used in the nuclear receptor database NUREBASE (<a href="http://www.ens-lyon.fr/LBMC/laudet/nurebase/nurebase.html" target="_blank">http://www.ens-lyon.fr/LBMC/laudet/nurebase/nurebase.html</a>) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020102#pgen-0020102-b039" target="_blank">39</a>].</p

Transcriptional Activity and Binding Selectivity of Vertebrate RARs

<p>Transcriptional activity is shown in (A–C), (G–I), (M), and (N), and corresponding binding selectivity in (D–F), (J–L), (O), and (P). Identities of the vertebrate RARs for each activity-selectivity pair are indicated above each bar graph. In each case, a chimera comprising the RAR LBD fused to the GAL4 DNA-binding domain (GAL-RAR(LBD)) has been used. The analysis of transcriptional activity in (A–C), (G–I), (M), and (N) shows transient transactivation assays in Cos1 cells with the indicated GAL-RAR(LBD) expression vector and the cognate (17m)5x-G-luc reporter plasmid, in the presence of increasing concentrations (10⁻¹⁰ to 10⁻⁶ M) of ATRA (red bars), BMS753 (yellow bars), BMS641 (light green bars), and BMS961 (dark green bars) respectively. The black bars indicate transactivation in the absence of hormone. Partial proteolysis maps of different in vitro-translated RARs are shown in (D–F), (J–L), (O), and (P). For each proteolysis gel lane 1 represents the undigested protein, lane 2 shows digestion of the receptor in the absence of ligand, lanes 3 and 4 show digestion of the receptor in the presence of ATRA (10⁻⁴ to 10⁻⁵ M), lanes 5 and 6 show digestion in the presence of BMS753 (10⁻⁴ to 10⁻⁵ M), lanes 7 and 8 show digestion in the presence of BMS641 (10⁻⁴ to 10⁻⁵ M), and lanes 9 and 10 show digestion in the presence of BMS961 (10⁻⁴ to 10⁻⁵ M). Protected bands in the presence of BMS641 are indicated by an asterisk, and slightly protected bands are indicated by arrowheads.</p

Ancient and present-day sampling of goats in Corsica.

<p>The medieval samples are all located in the archeological site of Rostino, in the northern part of the island. The present-day samples come from 5 different localities identified by numbers on the map.</p

Mitochondrial diversity of Corsican goats compared to world-wide goats diversity.

<p>Ancient (circles) and current (stars) Corsican sequences have been used with 20 sequences of reference corresponding to all haplogroups described up to now <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030272#pone.0030272-Naderi2" target="_blank">[14]</a> to reconstruct a Bayesian phylogenetic tree using a GTR+I+G model of evolution (see text). Only a single sequence by haplotype has been used, so haplotypes in common between medieval and today periods are indicated by both a circle and star. Only posterior probabilities higher than 0.6 are indicated.</p

Schematic Representation of the Expression Territories of RARs

<p>Staining of embryos indicates expression of mRARα (A), mRARβ (B), and mRARγ (C) in mouse embryos at E9; of xRARα (G), xRARβ (H), and xRARγ (I) in stage 30 <i>Xenopus</i> embryos, and of AmphiRAR (M) in 20 h old amphioxus larvae. Schematic representations are shown of the expression territories of mRARs (D–F), xRARs (J–L), and AmphiRAR (N) in mouse, <i>Xenopus,</i> and amphioxus embryos, respectively. Regions with high levels of expression are red and those with lower levels of expression are pink. Arrowheads indicate regions in mouse and <i>Xenopus</i> embryos where the RAR expression cannot be correlated with AmphiRAR expression and can be described as “new expression territories.”</p

Transcriptional Activity and Binding Selectivity of Chordate RARs

<p>Transcriptional activity is shown in (A–D) and (I–L), and corresponding binding selectivity in (E–H) and (M–P). Identities of the chordate RARs for each activity-selectivity pair are indicated above each bar graph. Transcriptional activity is shown in (A–D) for LampRAR, AmphiRAR, PmRAR, and AncRAR, and that of AmphiRAR mutants is shown in (I–L). Partial proteolysis maps of the different in vitro-translated RARs are shown in (E–H) and (M–P). Chimeric GAL-RAR(LBD) transactivation methods, colour code of the transactivation figures, and contents of each proteolysis gel are as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020102#pgen-0020102-g003" target="_blank">Figure 3</a>. Protected bands in the presence of BMS641 are indicated by an asterisk, and slightly protected bands are indicated by arrowheads.</p

Representation of the Transactivation and Binding Capacities of the RARs Used in the Present Study

<p>The three synthetic retinoids are shown as α, BMS753; β, BMS641; and γ, BMS961. The phylogenetic relationships between the RARs have been schematized by a phylogenetic tree (the tunicate and amphioxus RARs have been polytomised, LampRAR is also polytomised with the vertebrate RARs). The putative position in the tree of the ancestral sequence is indicated by a dashed branch in red.</p

Genetic diversity parameters.

<p>The different parameters for all datasets (see text) have been computed on a 130 bp fragment of the HVI region of the mitochondrial control region (CR). The values are given with their confidence interval (±) and tests with their p-value (p).</p

Networks generated with CR sequences of Corsican goats from A) medieval samples only or B) combined with present-day samples.

<p>The circles are proportional to the number of sequences obtained and colours indicated the period: yellow corresponds to XII^th century, red to XIV^th century, orange to all medieval and blue to present-day sequences. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030272#pone-0030272-t001" target="_blank">Table 1</a> for details about haplotypes and archeological samples. Haplotypes indicated in italics are those only observed in Corsica up to now. Haplotype Ha 26 is the only one from the haplogroup C and differs from the closer haplotype by 17 positions that is materialized by a square with this number.</p

Allelic states of 21 ancient individuals with dog-morphotype and 2 ancient individuals with wolf-morphotype at the R301C, R306ter and K locus.

*<p>Hypothetical coat color can be deduced considering allelic state of R306ter and K locus. <i>In italic</i>: individuals with a wolf-morphotype (CH1075 and CH1244). ND: not determined, no positive amplification; ?: Hypothetical coat color could not be deduced;−/−: <i>CBD103</i>: ΔG23 mutation.</p>1<p>Date obtained directly on dog bones (cal BP);</p>2<p>Date derived from other remains (BP);</p>3<p>Chronological periods derived from cultural attributions.</p