Table 1.
Characterized full-length spider silk gene sequences.
Figure 1.
Complete amino acid sequence for A. ventricosus minor ampullate spidroin
(MiSp). The sequence is read from left to right and top to bottom. The first methionine (Met) marks the start position and the asterisk denotes the stop position. The protein is dominated by poly-A (red), GGX (blue and fuchsia), GGGX (fuchsia) and GX (red and cyan) motifs. The protein sequence can be divided into nonrepetitive N- and C-terminal regions and a predominantly repetitive region. The repetitive region is interrupted by two nonrepetitive spacer regions which have 100% identity. Gaps (–) have been inserted in order to align repeat units within a type. Non-repetitive N- and C-terminal regions are in black, spacers are in green and linker regions are in gray. The green arrow marks the intron insertion position.
Table 2.
Primers used for intron identification.
Figure 2.
Alignment of genomic DNA upstream sequences.
Aligned DNA ranges approximately 210 nucleotides upstream of the predicted transcription start (indicated by a box and a black triangle). The red triangle indicates A. ventricosus MiSp upstream sequence. The translation start ATG codons, and the conserved motifs CACG and TATA are boxed. The sequences shown are the following: NimFlag, N. inaurata madagascariensis flagelliform (Flag) spidroin gene, upstream (GenBank accession no. AF218623S1); NimMaSp2, N. inaurata madagascariensis major ampullate spidroin 2-like gene (GenBank accession no. DQ059135); LhMaSp1, L. hesperus major ampullate spidroin 1 (GenBank accession no. EF595246) and 2 (GenBank accession no. EF595245) genes; LgMaSp1, L. geometricus major ampullate spidroin 1-like gene (GenBank accession no. DQ059133S1); AtMaSp2, A. trifasciata major ampullate spidroin 2 gene (GenBank accession no. DQ059136S1); AvMiSp, A. ventricosus minor ampullate spidroin gene (GenBank accession no. JX513956).
Figure 3.
Alignment of predicted 3′ UTR sequences.
TGA/TAA stop codons and the polyadenylation signals, AATAAA, are boxed. The UTRs are determined from genomic DNA and mRNA. Nim-UTR was determined by alignment of N. inaurata madagascariensis flagelliform spidroin gene (GenBank accession no. AF218623S2) and N. clavipes flagelliform (Flag) mRNA (GenBank accession no. AF027973); Lh-UTR 1 was determined by alignment of L. hesperus major ampullate spidroin 1 (MaSp1) gene (GenBank accession no. EF595246) and mRNA (GenBank accession no. DQ409057); Lh-UTR 2 was determined by alignment of L. hesperus major ampullate spidroin 2 (MaSp2) gene (GenBank accession no. EF595245) and mRNA (GenBank accession no. DQ409058); Av-UTR was determined by alignment of A. ventricosus minor ampullate spidroin gene (GenBank accession no. JX513956) and A. ventricosus major ampullate gland dragline silk protein-2 (F2) mRNA (GenBank accession no. AY177203).
Figure 4.
Sequence alignment of spacer regions.
The spacer sequences are aligned using ClustalW2 and amino acid residues are shown in different colors. X indicates missing amino acid residues and Gaps (–) have been inserted in order to align the amino acid residues. Positions with identical residues in a majority of the proteins are boxed. α-Helices and strands predicted by PSI-Pred v3.0 are shaded in red and yellow, respectively. (A) The sequences shown are the following: MiSp-Spacer (Ds), D. spinosa minor ampullate spidroin (GenBank accession no. ABD61589); MiSp-Spacer (Na), Nephila antipodiana minor ampullate fibroin 1 (GenBank accession no. ABC72645); MiSp-Spacer (Nc), N. clavipes minor ampullate silk protein MiSp1 (GenBank accession no. AAC14589); MiSp-Spacer (Ncr), Nephilengys cruentata minor ampullate spidroin-like protein (GenBank accession no. ABR37278); MiSp-Spacer (Aar), Argiope argentata minor ampullate spidroin (GenBank accession no. AFM29835); ADF1-Spacer (Ad), A. diadematus fibroin-1 (ADF-1) (GenBank accession no. AAC47008); MiSp (Av), A. ventricosus minor ampullate spidroin (GenBank accession no. JX513956). (B) The Nephila Flag-spacer sequences shown are the following: Flag-Spacer1–4 (Nc), N. clavipes flagelliform silk protein (Flag) (GenBank accession no. AAF36090); Flag-Spacer1–4 (Nc-U), N. clavipes flagelliform silk protein (Flag) (GenBank accession no. AAF36090); Flag-Spacer (Nc-D), N. clavipes flagelliform silk protein (Flag), downstream (GenBank accession no. AAF36089); FlagA-Spacer (Nc), N. clavipes flagelliform silk protein (Flag) (GenBank accession no. AAC38846); FlagB-Spacer1–2 (Nc), N. clavipes flagelliform silk protein (Flag) (GenBank accession no. AAC38847); Flag-Spacer1–4 (Nim), N. inaurata madagascariensis flagelliform silk protein (Flag) (GenBank accession no. AAF36091); Flag-Spacer1–4 (Nim-U), N. inaurata madagascariensis flagelliform silk protein (Flag) (GenBank accession no. AAF36091); Flag-Spacer 1–2(Nim-D), N. inaurata madagascariensis flagelliform silk protein (Flag), downstream (GenBank accession no. AAF36092); Flag-Spacer1–3 (Ncr), N. cruentata flagelliform spidroin-like protein (GenBank accession no. ABR37273). (C) The Argiope and Araneus Flag-spacer sequences shown are the following: FlagA-Spacer1–2 (At), A. trifasciata flagelliform silk protein (Flag) (GenBank accession no. AF350265_1); FlagB-Spacer1–3 (At), A. trifasciata flagelliform silk protein (Flag) (GenBank accession no. AF350264_1); Flag1-Spacer1–2 (Av), A. ventricosus flagelliform silk protein-1 (Ff1) (GenBank accession no. AAT36347); Flag-Spacer (Av), A.ventricosus flagelliform fibroin (GenBank accession no. ABK00016).
Figure 5.
Kyte and Doolittle hydropathicity plots for A. ventricosus MiSp.
Positive scores indicate hydrophobicity. NT = N-terminal region, RT = repetitive region, S = spacer and CT = C-terminal region.
Figure 6.
Architectures of A. ventricosus MiSp, A. trifasciata MaSp2, Nephila Flag, L. hesperus MaSp1 and MaSp2 genes.
Arrangement of exons (red) interrupted by introns (grey light green) in the spidroin genes. Due to the huge variability in size the lengths of the different segments are not drawn to scale.