Analysis of intron positions shared with <i>S. frugiperda fut8</i> (i<i>number</i>l) in arthropoda and chordata orthologs <i>fut8</i> genes.

<p>IS: intronic sequence, Nb: number. The intron phase was defined as follows: phase 0 introns are inserted between two codons; phase 1 introns are inserted after the first nucleotide of the codon, and phase 2 introns are inserted between the second and the third nucleotide of the codon.</p><p>(*) The gene is truncated and the 5′ exon is missing.</p><p>Analysis of intron positions shared with <i>S. frugiperda fut8</i> (i<i>number</i>l) in arthropoda and chordata orthologs <i>fut8</i> genes.</p

Conserved aa and motifs found in all the α1,6-fucosyltransferases sequences.

<p>Schematic representations of the FUT8 protein showing the cytoplasmic (C), transmembrane (T) and stem region (S) characteristic of α1,6-fucosyltransferases. The catalytic domain is in white and motifs I, II and III in grey. In addition, a region found only in α1,6-fucosyltransferase with conserved cysteine residues is indicated by dashed lines and was named “Cys-rich” domain. Conserved aa and those implicated in the enzymatic activity are highlighted with orange stars. The conserved peptide sequences used to generate the motif I, motif II and motif III sequence logos were extracted from multiple alignments of 96 α1,6-fucosyltransferase sequences identified in the databases (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110422#pone.0110422.s004" target="_blank">Table S2</a>) and visualized at the Weblogos site at Berkeley, as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110422#pone.0110422-Felgner1" target="_blank">[63]</a>. In the logos, aa are colored according to their chemical properties: polar aa (G, C, S, T, Y) are green, basic (K, R, H) are blue, acidic (D, E) are red, hydrophobic (A, V, L, I, P, W, F, M) are black and neutral polar aa (N, Q) are pink. The overall height of the stacks indicates the sequence conservation at a given position, while the height of the symbol within the stack indicates the relative frequency of each aa at that position. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110422#pone.0110422-Nei1" target="_blank">[69]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110422#pone.0110422-Crooks1" target="_blank">[70]</a>.</p

Molecular phylogenetic analysis using the Maximum Likelihood method.

<p>Evolutionary analyses were conducted in MEGA5 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110422#pone.0110422-Tamura1" target="_blank">[67]</a> and the evolutionary history was inferred using the Maximum Likelihood method based on the Whelan and Goldman model <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110422#pone.0110422-Whelan1" target="_blank">[72]</a>. This analysis involved 92 FUT8 aa sequences and the final dataset contained 336 positions (42% of 787). The bootstrap consensus tree inferred from 1050 replicates is taken to represent the evolutionary history of the analyzed taxa. The percentage of trees (only those >75%) in which the associated taxa clustered together is shown next to the branches <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110422#pone.0110422-Felsenstein1" target="_blank">[73]</a>. Initial tree(s) for the heuristic search were obtained automatically as follows. When the number of common sites was <100 or less than one fourth of the total number of sites, the maximum parsimony method was used; otherwise the BIONJ method with the MCL distance matrix was used.</p

Schematic illustration of the correlation between animal <i>fut8</i> gene phylogeny, intron gain/loss and intron position (gene organization).

<p>This analysis was carried out by considering only the intron insertion sites in the <i>fut8</i> gene sequences encoding the conserved region of FUT8 proteins, between i<i>3</i>l and the stop codon. On the right, column 1 shows the total number of intron insertion sites (IS) identified in the different <i>fut8</i> genes, and column 2 shows the number of order- or family-specific intron insertion sites. Intron gains and losses are highlighted (grey and white boxes, respectively) as well as putative intron sliding in “near-intron-pairs” (light grey boxes). Putative ancestral introns are in a dark grey box.</p

Analysis of the sequences immediately flanking the three potential start codons (ATG1, ATG2 and ATG3 in Figure 1) identified in the <i>fut8</i> cDNA.

1<p>Consensus sequence determined in this work (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110422#pone-0110422-t001" target="_blank">Table 1</a>).</p>2<p>Consensus sequence determined by Cavener and Ray (1991) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110422#pone.0110422-CavenerDRRay1" target="_blank">[41]</a> for invertebrates.</p><p>Analysis of the sequences immediately flanking the three potential start codons (ATG1, ATG2 and ATG3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110422#pone-0110422-g001" target="_blank">Figure 1</a>) identified in the <i>fut8</i> cDNA.</p

Intron positions shared by <i>S. frugiperda fut8</i> (i<i>number</i>l) and with <i>fut8</i> genes identified in chordata (i<i>number</i>c).

<p>IS: Intronic Sequence.</p><p>(X): Presence of lepidopteran intron insertion site.</p><p>(<b>+</b>): Presence of chordata intron insertion site.</p><p>(*) The gene is truncated and the 5′ exon is missing.</p><p>Intron positions shared by <i>S. frugiperda fut8</i> (i<i>number</i>l) and with <i>fut8</i> genes identified in chordata (i<i>number</i>c).</p

Southern and Northern blot analyses.

<p><b>A.</b> Ten µg/lane of EcoRI-digested genomic DNA from <i>Sf</i>9 cells was hybridized with a mono-exonic (exon 3, {E<i>3</i>l}) digoxigenin-labeled Fut8 probe prepared by PCR using the Forint10 and B24 primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110422#pone.0110422.s003" target="_blank">Table S1</a>), as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110422#s4" target="_blank">Materials and Methods</a>. <b>B.</b> One µg/lane of total RNA was analyzed by Northern blotting. Hybridization was performed with a <i>fut8</i> anti-sense RNA probe (pos. 825 to pos. 1686 on the cDNA, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110422#pone-0110422-g001" target="_blank">Figure 1</a>). Molecular weight markers in bp are indicated on the left of each panel.</p

Analysis of intron positions in <i>fut8</i> orthologs in hymenoptera (i<i>number</i>h).

<p>The intron phase was defined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110422#pone-0110422-t004" target="_blank">Table 4</a>. IS: intronic sequence, Nb: number.</p><p>Analysis of intron positions in <i>fut8</i> orthologs in hymenoptera (i<i>number</i>h).</p

Nucleotide and deduced aa sequences of the <i>Sf</i>9 α1,6-fucosyltransferase cDNA.

<p>The three potential start codons are presented in boxes. ATG3 was considered as the start codon (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110422#pone-0110422-t002" target="_blank">Table 2</a>). A potential transmembrane domain (residues 9 to 25) is highlighted in grey. The conserved motifs I, II and III in the active site are underlined. The putative SH3 domain is in bold and underlined with a dashed line. The two potential <i>N</i>-glycosylation sites are underlined with a double line. Solid arrowheads indicate the position of introns. The numbers between brackets represent the position of critical aa conserved in human FUT8.</p

Schematic illustration of the genomic organization of the <i>fut8</i> genes identified in animal genomes.

<p>Coding exons are represented by rectangles with their relative size in codons (in bold). Italic numbers represent the number of nucleotides belonging to codons that flank the intron insertion site and determine the intron insertion phase. Dashed lines represent conserved intron insertion sites. Conserved exons are in the same color. <i>S. frugiperda</i> and <i>H. sapiens</i> FUT8 proteins are in blue, specific domains are represented by rectangles with their relative size in aa (Cys-rich domain, in black and motifs I, II and III, in grey).</p