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Fig. 4. Comparisons of the Siglec gene cluster in human, chimpanzee, baboon, rat, and mouse. Combinatorial pair-wise alignments. Using the generated genomic sequence, dot plots were constructed using the BLASTZ-generated alignments for each species pair, as indicated on the x and y axes. The relative positions of the kallikrein (KLK) genes are shown in green, Siglec genes in blue, and other genes in yellow. Note that the human and mouse sequences used in this study was generated elsewhere (1, 2). An interactive version of this figure can be found at www.nisc.nih.gov/data/Angata_etal_2004_Siglec (requires that JAVA be installed).

1. International Human Genome Sequencing Consortium (2001) Nature 409, 860-921.

2. International Mouse Genome Sequencing Consortium (2002) Nature 420, 520-562.

Fig. 5. Molecular phylogenetic trees of exons encoding the (A) first, (B) second, and (C) third Ig-like domains of CD33rSiglecs. Trees were reconstructed by the neighbor-joining method, based on a distance matrix (uncorrected P distance) calculated from the nucleotide sequences encoding each domain. Due to the unusual structure of Siglec-12/XII (which has two V-set domains, whereas all other Siglecs have only one V-set domain), "Ig2" and "Ig3" of SIGLEC12/XII used in this analysis were exon sequences encoding the first two C2-set domains (Ig3 and Ig4 of this molecule), respectively. Primate CD33/SIGLEC3, rodent Cd33/Siglec3, and rodent SiglecH are absent from C, because they lack such a domain. The "primate SIGLEC10 and 11 + rodent SiglecG" clade was used as an internal outgroup, because they are considered ancestral to the CD33rSiglecs and are consistently monophyletic, whereas SIGLEC4 is not similar enough to CD33rSiglecs at nucleotide level to give reliable alignment with them.

Fig. 6. Primate SIGLEC6, a possible example of hybrid gene. (A) A schematic representation of human SIGLEC3, 5, and 6. A closed box represents an exon, whereas an open box represents a remnant of exon. (B) Nucleotide sequence identity between SIGLEC6 and SIGLEC3 or SIGLEC5, calculated for individual exon and intron. The number in each box or above each line between boxes stands for the nucleotide length of each exon or intron, respectively. The percent identity between the equivalent segments in two Siglec genes is shown in italics. (C) Possible scenarios leading to the current configuration of primate SIGLEC6. In Scenario 1, a gene duplication generating SIGLEC5 and proto-SIGLEC6 from their ancestor, and one generating a SIGLEC3-like Siglec gene, should have preceded the gene arrangement shown on top. The subsequent recombination and loss of a part of SIGLEC3-like Siglec gene and proto-SIGLEC6 may have led to the current configuration of SIGLEC6. In Scenario 2, gene duplication happened only once, generating SIGLEC5 and proto-SIGLEC6 from their ancestor, which was followed by a partial gene conversion of a part of proto-SIGLEC6 by SIGLEC3 or a gene similar to it. Note that these scenarios are grossly simplified and may not represent all events that have happened in this region.

Fig. 7. Amino acid differences between human and chimpanzee CD33rSiglec orthologs, overlaid onto the structure of Siglec-7. (A) Amino acid differences between human and chimpanzee ortholog pairs were projected onto the equivalent residues of human Siglec-7 and then binned for each secondary structural element (b -strand and loop). The fractional number beside each strand/loop represents the number of differences per the total number of amino acids in each strand/loop. (B) Birds-eye view of the Ig1 of human Siglec-7, with amino acid differences between all human–chimpanzee CD33rSiglec ortholog pairs marked in yellow. The attached N-glycan is represented by a ball-and-stick model. The dotted white line represents the division between two "faces" of the molecule (which does not exactly correspond to the two b -sheets).

Data Set 1. Nonsynonymous and synonymous differences between human and chimpanzee CD33rSiglec orthologs, mapped onto the 3D structure of Siglec-7. Amino acid residues showing differences between human and chimpanzee CD33rSiglec orthologs were mapped onto 3D structure of human Siglec-7 (PDB ID code 1O7V; Molecular Modeling Database no. 22472) and highlighted. The file provided should be visualized using CN3D (www.ncbi.nlm.nih.gov/Structure/CN3D/cn3d.shtml). To see the annotation (nonsynonymous differences), choose the Structure window, select "Style: Annotate" from the menu, choose "NonSyn" from the available annotation panel, and click "Show," then the "Done" button. Synonymous changes (synonymous nucleotide substitutions) are also mapped on the structure for comparison (to see synonymous changes, follow the procedure described above, except choosing "Syn" instead of "Nonsyn" from the available annotation panel).