*Department of Biological Sciences, Graduate School of Science, University of Tokyo;
Laboratory of Evolutionary Genetics, National Institute of Genetics, Mishima 411-8540, Japan
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Abstract |
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Introduction |
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In view of the evolutionary hypervariability among hominoids and the unusually high polymorphism of the C gene hinge region among Old World monkeys, there is a possibility that this gene has experienced positive selection during primate evolution. With this aim, we obtained 31 new C
hinge sequences from Old World and New World monkeys and evaluated their dN and dS values by using a tree-based comparison method.
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Materials and Methods |
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PCR Amplification and Fragment Analysis
The following PCR primer pair was used to amplify an approximately 400-bp DNA fragment involving the intron, hinge, and CH2 regions: 5'-CCA AGC TTC TAC ACG AAT CCC AGC CAG GAT GTG-3' and 5'-CCG AAT TCT CCC ACT TGA GGG CGT CCA GGT GAA-3'. The PCR program was 3540 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 2 min. A PCR-SSCP analysis was performed to check whether the PCR products contain single or multiple sequences, where PCR products were run on nondenaturing gel containing 5% acrylamide, 5% glycerol, and 0.5x TBE buffer at 16°C constant temperature, as described before (Sumiyama et al. 1998
). In the PCR-SSCP analysis, the following internal primers were used to obtain better resolution in gel: 5'-TCT GAC CAG TTC AGG CCA TCT C-3' and 5'-GGC CGG CGC AGC GAC AGT-3'.
Sequence Determination
Nucleotide sequences of both strands were determined using the ABI Taq DyeDeoxy Terminator Cycle sequencing kit, according to the manufacturer's instructions, using an ABI PRISM 377 DNA sequencer. In some heterozygous cases, their nucleotide sequences were determined by using allele-specific sequencing primers.
Sequence Analyses
Hominoid sequences were taken from Kawamura et al. (1991)
. The Neighbor-joining (NJ) method (Saitou and Nei 1987
) was used for tree reconstruction of the hinge-flanking region by using CLUSTAL W (Thompson, Higgins, and Gibson 1994
), with "exclude gap" option. Maximum likelihood analysis was done by using the NucML program of the MOLPHY package (Adachi and Hasegawa 1996
) with the HKY85 model in order to evaluate the reliability of tree topology. We used the PAUP 3.1.1. program (Illinois Natural History Survey) for the construction of the maximum parsimony (MP) tree for the hinge region. For the purpose of estimating the ancestral sequences on the tree internal nodes, we used the baseml program of the PAML package (Yang 1999
) with the HKY85 model. Values of dN and dS were calculated for each phylogenetic tree branch between the given sequences using the computer program package ODEN (Ina 1994
) that employs Nei and Gojobori's (1986)
method. Window analysis was conducted using the wina program of Endo, Ikeo, and Gojobori (1996)
. Statistical tests for positive selection were conducted by using Fisher's exact probability test, where we set the null hypothesis that nonsynonymous substitutions per site and synonymous substitutions per site are equal (strictly neutral condition). Therefore, the ratio of unchanged nonsynonymous sites to unchanged synonymous sites was expected to be equal to that of the nonsynonymous changes to synonymous changes (e.g., Zhang, Kumar, and Nei 1997
).
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Results and Discussion |
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We calculated the total distance (DT) of the phylogenetic tree among species for dS and dN, according to the established tree topology(((human, chimpanzee), gorilla), orangutan). Ancestral nucleotide sequences of all interior nodes were reconstructed by using the PAML program, and then the number of nucleotide substitutions on each branch was calculated. DT is defined as the sum of all branches. DT of each window was plotted against the first nucleotide position of each window along the entire coding sequence (fig. 1 ). The DT of the nonsynonymous substitutions clearly exceeds that of the synonymous substitutions at the hinge region. This observation supports the idea that the hominoid hinge region has been evolving under some kind of positive selection. Besides the hinge region, both synonymous and nonsynonymous rates were raised at the end of the CH2 domain. We do not know the reason for this observation, however, it is possible that recombinations or gene conversions occurred and raised overall variation in this region.
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In contrast to the flanking region, the hinge region is unusually hypervariable. Because of the complexity associated with the hinge region (different length, many substitutions, and partially reiterated structure; see table 1 ), a multiple alignment of all sequences could not be performed at once. Instead of making an overall multiple alignment in the hinge region, we show the alignment within the hinge to be the largest collection of sequences for which homology can be clearly assessed within Old World monkeys, New World monkeys, and hominoids (fig. 2 ). We use these unambiguously aligned sequences for subsequent analysis of the hinge region.
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We previously reported a very high extent of heterozygosity in the hinge region in macaques (Sumiyama et al. 1998
), which is another indication of overdominant selection. Considering these results together, existence of overdominant selection is quite probable in the C
gene hinge region for Old World monkeys.
When hominoids are considered, special attention is necessary because they have two C loci (Kawamura, Saitou, and Ueda 1992
). C
1 and C
2 are positioned close to each other in the phylogenetic tree in every hominoid species. This is the result of increased similarity caused by a gene conversion event between the two duplicated C
loci (Kawamura, Saitou, and Ueda 1992
). In other primates, there is only one locus for the C
gene; thus, they are free from this gene conversion problem.
Phylogeny and Substitution Pattern of the Hypervariable Hinge Sequences
Because the hinge region itself is small and hypervariable, it was necessary to use methods different from those used for the hinge-flanking regions. First, we constructed MP trees for those nucleotide sequences corresponding to 10 Old World monkey hinge regions shown in figure 2a.
Ten equally parsimonious trees were obtained (fig. 4a
). Nine of the ten MP trees contained only nonsynonymous substitutions. Only one MP tree (MPTREE_5) contained one synonymous substitution, as shown with a diamond in figure 4a.
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To further examine our observations, we computed Fisher's exact tests (table 2
). Note that the commonly used t-test is not suitable for very low expected values of substitutions. The results are listed in table 2
. Note that the nonsynonymous and synonymous substitution reconstruction may be biased by the codon usage and the GC%, it is observed that GC3 = 0.795 and ENc (effective number of codon) = 39.75 for the entire crab-eating macaque C-coding sequence (X53705). Nevertheless, the null hypothesis of equal expectation of nonsynonymous and synonymous substitutions was rejected for all MP trees. We also conducted the same analysis on hominoids and New World monkeys. They also show high nonsynonymous to synonymous ratios, but these were not statistically significant (see table 2
).
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Other Supports for Positive Selection: IgA Sequence Variation in Other Mammals and Vast IgA Protease Variation in Pathogenic Bacteria
We found that the IgA hinge region showed evidence of adaptive evolution in primates. Furthermore, some other mammalian species also show hypervariability in their IgA hinge regions, although nonsynonymous-synonymous substitution ratio tests have not been conducted because of alignment difficulties (Osborne et al. 1988
; Burnett et al. 1989
; Brown and Butler 1994
). Our results suggest that adaptive evolution of the IgA hinge is likely to have occurred in many mammalian species. IgA is the most prevalent immunoglobulin in secretions, and it plays a crucial role for the prevention of pathogenic bacterial penetration of the mucosal surface. Because IgA is an indispensable factor for mammalian species, the bacterial IgA protease could be a strong selective pressure on the IgA gene.
There is a possibility of coevolution between host IgA and proteases of the pathogenic bacteria. IgA proteases of pathogenic bacteria consist of several different classes. They are found in a wide variety of pathogenic bacteria, including Haemophilus, Neisseria, and Streptococcus (Plaut et al. 1975
; Kilian, Mestecky, and Schrohenloher 1979
; Male 1979
). These proteases are highly specific enzymes that cleave certain peptide bonds of the IgA hinge region. Interestingly, their catalytic mechanisms and gene structures are completely different among bacteria (Mortensen and Kilian 1984
; Bachovchin et al. 1990
; Gilbert, Plaut, and Wright 1991
; Lomholt, Poulsen, and Kilian 1995
), such as thiol-activated protease from Bacteroides melaninogenicus, serine-type proteases from Haemophilus influenzae and Neisseria gonorrhoeae, and metalloprotease from Streptococcus sanguis. Such wide variations among IgA proteases from different origins with the same substrate specificity strongly suggest functional convergence in IgA protease evolution. This convergent evolution of the IgA proteases of pathogenic bacteria indicates the importance of inactivation of the host IgA to retain their pathogenicity. This fact indicates strong host-parasite interaction between host IgA and parasite protease, which results in positive selection pressure on both sides.
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Conclusions |
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Supplementary Materials |
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Acknowledgements |
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Footnotes |
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Keywords: immunoglobulin C
IgA
hinge
positive selection
Address for correspondence and reprints: Kenta Sumiyama, Department of Molecular, Cellular and Developmental Biology, Yale University, 266 Whitney Avenue, New Haven, Connecticut 06520. kenta.sumiyama{at}yale.edu
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