Figure 1.
Nucleotide and deduced aa sequences of the Sf9 α1,6-fucosyltransferase cDNA.
The three potential start codons are presented in boxes. ATG3 was considered as the start codon (see Table 2). 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 N-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.
Table 1.
Determination of the optimal sequence for initiation of translation in Sf9 cells.
Table 2.
Analysis of the sequences immediately flanking the three potential start codons (ATG1, ATG2 and ATG3 in Figure 1) identified in the fut8 cDNA.
Table 3.
Determination of α1–6-fucosyltransferase activity.
Figure 2.
Southern and Northern blot analyses.
A. Ten µg/lane of EcoRI-digested genomic DNA from Sf9 cells was hybridized with a mono-exonic (exon 3, {E3l}) digoxigenin-labeled Fut8 probe prepared by PCR using the Forint10 and B24 primers (Table S1), as described in Materials and Methods. B. One µg/lane of total RNA was analyzed by Northern blotting. Hybridization was performed with a fut8 anti-sense RNA probe (pos. 825 to pos. 1686 on the cDNA, Figure 1). Molecular weight markers in bp are indicated on the left of each panel.
Figure 3.
Conserved aa and motifs found in all the α1,6-fucosyltransferases sequences.
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 (Table S2) and visualized at the Weblogos site at Berkeley, as described previously [63]. 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. [69], [70].
Figure 4.
Molecular phylogenetic analysis using the Maximum Likelihood method.
Evolutionary analyses were conducted in MEGA5 [67] and the evolutionary history was inferred using the Maximum Likelihood method based on the Whelan and Goldman model [72]. 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 [73]. 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.
Figure 5.
Schematic illustration of the genomic organization of the fut8 genes identified in animal genomes.
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. S. frugiperda and H. sapiens 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).
Table 4.
Analysis of intron positions shared with S. frugiperda fut8 (inumberl) in arthropoda and chordata orthologs fut8 genes.
Table 5.
Analysis of intron positions in fut8 orthologs in hymenoptera (inumberh).
Table 6.
Intron positions shared by S. frugiperda fut8 (inumberl) and with fut8 genes identified in chordata (inumberc).
Figure 6.
Schematic illustration of the correlation between animal fut8 gene phylogeny, intron gain/loss and intron position (gene organization).
This analysis was carried out by considering only the intron insertion sites in the fut8 gene sequences encoding the conserved region of FUT8 proteins, between i3l and the stop codon. On the right, column 1 shows the total number of intron insertion sites (IS) identified in the different fut8 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.