Degeneration and Domestication of a Selfish Gene in Yeast: Molecular Evolution Versus Site-Directed Mutagenesis
http://www.100md.com
分子生物学进展 2005年第7期
Department of Biological Sciences, Imperial College London, Silwood Park, Ascot, Berks, United Kingdom
Correspondence: E-mail: v.koufopanou@imperial.ac.uk.
Abstract
VDE is a homing endonuclease gene in yeasts with an unusual evolutionary history including horizontal transmission, degeneration, and domestication into the mating-type switching locus HO. We investigate here the effects of these features on its molecular evolution. In addition, we correlate rates of evolution with results from site-directed mutagenesis studies. Functional elements have lower rates of evolution than degenerate ones and higher conservation at functionally important sites. However, functionally important and unimportant sites are equally likely to have been involved in the evolution of new function during the domestication of VDE into HO. The domestication event also indicates that VDE has been lost in some species and that VDE has been present in yeasts for more than 50 Myr.
Key Words: VDE ? HEG ? site-directed mutagenesis ? molecular evolution ? Saccharomycetaceae
VDE (vacuolar membrane–derived endonuclease) is one of the best-studied selfish genetic elements (Gimble and Thorner 1992; recent reviews include Chevalier and Stoddard 2001; Gogarten et al. 2002). It encodes a bifunctional protein with two domains, a homing endonuclease and a self-splicing intein, and is situated in the middle of the nuclear VMA1 locus of yeasts. The intein domain allows the VDE protein to splice out of the VMA1 host protein after they have been translated together in frame, thus reconstituting a functional VMA1 protein. The endonuclease domain, a member of the LAGLIDADG family of homing endonuclease genes (HEGs), recognizes, binds, and cuts a 31-bp recognition sequence in a VMA1 allele that does not contain VDE. Recent comparative studies in yeasts have revealed an unusual evolutionary history including regular horizontal transmission and degeneration (Koufopanou, Goddard, and Burt 2002; Okuda et al. 2003; Posey et al. 2004). This supports a model of cyclical evolution in which HEGs degenerate after becoming fixed within a species, due to the absence of purifying selection, with regular horizontal transmission being necessary for long-term persistence (Goddard and Burt 1999; Burt and Koufopanou 2004). In addition, the wild VDE has been domesticated in some yeasts, evolving into HO, a gene that improves the efficiency of mating-type switching (Dalgaard et al. 1997; Butler et al. 2004). Similar features are found in other selfish genetic elements, such as transposable elements (Hurst and Werren 2001; Pinsker et al. 2001; Kazazian 2004).
We analyze here a number of VDE and HO sequences from saccharomycete yeasts to study the effect of these unusual events on its sequence evolution. Furthermore, numerous site-directed mutagenesis studies available on VDE from Saccharomyces cerevisiae enable us to compare rates of evolution for sites that are found experimentally to be critical, semicritical, or neutral for the splicing and endonuclease functions and assess their contribution to the evolution of new function in HO. It is commonly assumed that functionally important sites evolve more slowly than functionally unimportant sites, and indeed, degree of conservation is often used to infer the importance of particular sites, but there are few studies directly correlating rates of evolution with results from site-directed mutagenesis (Li 1997).
The cyclical model of HEG evolution predicts that the two domains of VDE are under different selection regimes. Evolution in the splicing domain should be simple, splicing being critical for survival in all species. Indeed, maximum likelihood analysis gives a dN/dS ratio of 0.1 ± 0.01, well below the neutral expectation of 1, and no heterogeneity among lineages, indicating strong purifying selection to preserve function. There is also complete conservation at all six amino acid sites identified as critical for self-splicing, showing a strict amino acid requirement.
Evolution in the endonuclease domain might be more complex, as this domain can degenerate after becoming fixed. Indeed, only three out of the 14 VDEs studied here are active (S. cerevisiae, Saccharomyces cariocanus, and Zygosaccharomyces bailii; Posey et al. 2004). Maximum likelihood analysis gives a significantly lower dN/dS ratio in functional than degenerate endonuclease domains, indicating slower evolution in active domains and faster following degeneration (0.07 ± 0.01 vs. 0.17 ± 0.02 for functional vs. degenerate lineages, respectively, P < 0.001; fig. 1; see Supplementary Material online for full details). Note that divergence in degenerate endonuclease domains is considerably underestimated here due to a large number of insertions or deletions, treated as ambiguities in the analysis. Nevertheless, purifying selection is still indicated even in degenerate domains, not only by dN/dS < 1 but also by 10 invariant sites, five of which are in the LAGLIDADG-signature motifs forming the two -helices of the catalytic center (supplementary fig. 1). Perhaps these sites are structurally important in folding the molecule and enhancing self-splicing. This could explain why we do not observe deletions of the entire endonuclease domain, as are frequently found in intron-associated HEGs in yeasts and in bacterial inteins (Goddard and Burt 1999; Gogarten et al. 2002). Other intron-associated HEGs have evolved RNA maturase function and are thus less likely to be deleted (Chatterjee et al. 2003). Testing the effects of substitutions at these sites on self-splicing activity should clarify this point. Finally, for the three functional elements (all of which recognize the same sequence), sites important for endonuclease function are more conserved than neutral sites (table 1). No such difference is found among degenerate elements. Nevertheless, the functional Z. bailii VDE differs from the S. cerevisiae VDE at eight sites, which the mutagenesis studies indicate are important. Either the substitutions in Z. bailii are less drastic than alanine substitutions or there have been compensating changes at other sites.
FIG. 1.— Phylogram showing the evolution of VDE and HO (endonuclease domain only; maximum parsimony tree, numbers indicating >70% bootstrap support). Branch lengths are given by dN estimates from a free-ratio model in PAML, with the dN/dS ratio estimated separately for each branch. In bold are the three functional VDEs, connected by thick lines; the ancestor of the Saccharomyces castelli clade is also assumed to be functional, horizontal transfer events having been inferred within that clade (Koufopanou, Goddard, and Burt 2002). When Kluyveromyces lactis is removed, there is 91% bootstrap support for a sister clade relationship between the HO clade and the Saccharomyces cerevisiae clade of VDE, indicating that HO originated within the saccharomycete VDEs. The two alleles of HO from Saccharomyces pastorianus reflect its hybrid origin from S. cerevisiae and Saccharomyces bayanus.
Table 1 Conservation of Amino Acids at Sites Critical, Semicritical, and Neutral for Endonuclease Activity, as Determined by Site-Directed Mutagenesis (Alanine Substitutions Only), in Different Functional Classes of Elements
HO is a free-standing gene, homologous to the full length of VDE, including the self-splicing domain (even though HO does not splice), with 66% amino acid divergence between S. cerevisiae HO and VDE. HO has also acquired a C-terminal zinc finger domain (Bakhrat et al. 2004) and has evolved a new specificity toward an 18-bp sequence in the MAT locus, to which it binds and cleaves but does not insert. In an analysis of a large number of LAGLIDADG HEGs encompassing the entire range of their diversification, Dalgaard et al. (1997) found VDE and HO from S. cerevisiae to be closest relatives, suggesting that HO represents a domesticated form of VDE. Our analysis, on a much larger sample of VDE and HO sequences, confirms this result, with the HOs forming a clade nested within the VDEs, indicating a single origin of HO from VDE (fig. 1). Unlike VDE, and consistent with its domesticated status, HO shows no evidence for horizontal transmission (no difference between HO and host genealogy; data not shown) nor is there evidence for loss of HO in any species (fig. 2) or degeneration. The evolution of HO provides the clearest example to date for the domestication of a selfish element, and its study allows some insight into the evolution of novelty.
FIG. 2.— The distribution of VDE among species of yeasts where HO is known to be present or absent. Phylogenetic relationships are from Kurtzman and Robnett (2003). Numbers indicate >80% bootstrap support.
Maximum likelihood branch lengths calculated from the rates of nonsynonymous substitutions, dN, show the origin of HO as the longest internal branch in the phylogeny (fig. 1). Reliable estimates of dN/dS for this branch could not be obtained, perhaps due to changes in codon usage; within the HO cluster, dN/dS was 0.04 ± 0.002, approximately half the estimate for functional VDEs. Correlating rates of evolution with results from site-directed mutagenesis, we find no significant tendency for functionally important sites to be more (or less) involved in the evolution of the novel function. Sites important or unimportant in VDE are equally likely to be divergent in S. cerevisiae HO or conserved among the 10 HOs (table 1). Thus, the change in function from VDE to HO (which recognizes a sequence different and unrecognizable by VDE; Gimble and Wang 1996) has apparently entailed a change in critical sites. This is contrary to the diversification of functional VDEs, where important sites are more conserved. Nevertheless, six HO sites homologous to important VDE sites were also found important for mating-type switching, and amino acids in four of these are identical to those in VDE (including the two Mg2+-binding amino acids at the active site; Bakhrat et al. 2004; supplementary fig. 1), highlighting the similarity of action in the two molecules. It will be interesting to test how general these observations are for other types of genes, for example, during the evolution of a new function following gene duplication (Gaucher et al. 2002).
The domestication event, which is followed by normal vertical transmission, also provides evidence relevant to the cyclical model of VDE evolution. The presence of HO in several species that do not contain VDE (fig. 2) supports the idea that VDE was present in their ancestors and was subsequently lost. The mechanism of precise loss remains unknown. The domestication event also provides an anchor point to the endless cycling of VDE and allows us to obtain a minimum estimate for the length of residence of VDE in the Saccharomycetaceae. The phylogeny in figure 2 suggests that HO originated after the divergence of S. cerevisiae and Kluyveromyces lactis, estimated at approximately 70 Myr (Berbee and Taylor 1993), and is therefore younger than 70 Myr, perhaps 50 Myr old. VDE, which is ancestral to HO, must therefore be much older and must have been in the Saccharomycetaceae for at least that long.
Methods
VDE sequences were previously recovered by screening the VMA1 locus of 24 species of Saccharomycetaceae yeasts (Koufopanou, Goddard, and Burt 2002) and homologues from Chlamydomonas eugametos and Methanococcus jannaschii used as out-group in phylogenetic analyses (Dalgaard et al. 1997). Information on sites critical for splicing or endonuclease function was compiled from the literature (Cooper et al. 1993; Gimble and Stephens 1995; Kawasaki et al. 1997; Gimble et al. 1998; He et al. 1998; Hu et al. 1999; Wende et al. 2000). Sites were categorized as critical when mutants had <1% of wild-type activity, semicritical when <25%, and neutral when 25% or higher. "Neutral" does not necessarily imply the absence of selective effects. For consistency, only the effects of alanine substitutions are considered here; this is thought to effectively remove any important functional group that is normally present (He et al. 1998). Of a total of 454 amino acid sites in the S. cerevisiae VDE, 83 have been tested for endonucleolytic function, and of these, 40 were found critical or semicritical for function, distributed in both endonuclease and self-splicing domains. Only six sites have been tested for self-splicing activity, all found critical. Twelve sequences for HO were available in the database; sequences for Zygosaccharomyces rouxii and Saccharomyces exiguus were only partial and were not included in the main analysis, but results were qualitatively unchanged if they were included. Sequences were aligned using ClustalX (Thompson, Higgins, and Gibson 1994), and phylogenetic relationships were analyzed with PAUP* (Swofford 1999) and rates of evolution with PAML (Yang 1997).
Supplementary Material
A Logo alignment of VDEs (supplementary fig. 1), illustrating amino acid conservation at functionally important and unimportant sites, is available at Molecular Biology and Evolution online (www.mbe.oupjournals.org). Also given are plots of pairwise amino acid divergence between the two domains and between important and unimportant sites (supplementary fig. 2), full details for the maximum likelihood analysis (supplementary table 1), and the full alignment of VDE and HO sequences.
Acknowledgements
This work was supported by the Daphne Jackson Trust, Natural Environment Research Council, and the Wellcome Trust.
References
Bakhrat, A., M. S. Jurica, B. L. Stoddard, and D. Raveh. 2004. Homology modeling and mutational analysis of HO endonuclease of yeast. Genetics 166:721–728.
Berbee, M. L., and J. W. Taylor. 1993. Dating the evolutionary radiations of the true fungi. Can. J. Bot. 71:1114–1127.
Burt, A., and V. Koufopanou. 2004. Homing endonuclease genes: the rise and fall and rise again of a selfish element. Curr. Opin. Genet. Dev. 14:609–615.
Butler, G., C. Kenny, A. Fagan, C. Kurischko, C. Gaillardin, and K. H. Wolfe. 2004. Evolution of the MAT locus and its HO endonuclease in yeast species. Proc. Natl. Acad. Sci. USA 101:1632–1637.
Chatterjee, P., K. L. Brady, A. Solem, Y. Ho, and M. G. Caprara. 2003. Functionally distinct nucleic acid binding sites for a group I encoded RNA maturase/DNA homing endonuclease. J. Mol. Biol. 329:239–251.
Chevalier, B., and B. L. Stoddard. 2001. Homing endonucleases: structural and functional insight into the catalysts of intron/intein mobility. Nucleic Acids Res. 29:3757–3774.
Cooper, A. A., Y.-J. Chen, M. A. Lindorfer, and T. H. Stevens. 1993. Protein splicing of the yeast TFP1 intervening protein sequence: a model for self-excision. EMBO J. 12:2575–2583.
Dalgaard, J. Z., A. J. Klar, M. J. Moser, W. R. Holley, A. Chatterjee, and I. S. Mian. 1997. Statistical modeling and analysis of the LAGLIDADG family of site-specific endonucleases and identification of an intein that encodes a site-specific endonuclease of the HNH family. Nucleic Acids Res. 25:4626–4638.
Gaucher, E. A., X. Gu, M. M. Miyamoto, and S. A. Benner. 2002. Predicting functional divergence in protein evolution by site-specific rate shifts. Trends Biochem. Sci. 27:315–321.
Gimble, F. S., X. Duan, D. Hu, and F. A. Quiocho. 1998. Identification of Lys-403 in the PI-SceI homing endonuclease as part of a symmetric catalytic center. J. Biol. Chem. 273:30524–30529.
Gimble, F. S., and B. W. Stephens. 1995. Substitutions in conserved dodecapeptide motifs that uncouple the DNA binding and DNA cleavage activities of PI-SceI endonuclease. J. Biol. Chem. 270:5849–5856.
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Goddard, M. R., and A. Burt. 1999. Recurrent invasion and extinction of a selfish gene. Proc. Natl. Acad. Sci. USA 96:1380–1385.
Gogarten, J. P., A. G. Senejani, O. Zhaxybayeva, L. Olendzenski, and E. Hilario. 2002. Inteins: structure, function and evolution. Annu. Rev. Microbiol. 56:263–287.
He, Z., M. Crist, H. Yen, X. Duan, F. A. Quiocho, and F. S. Gimble. 1998. Amino acid residues in both the protein splicing and endonuclease domains of the PI-SceI intein mediate DNA binding. J. Biol. Chem. 273:4607–4615.
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Correspondence: E-mail: v.koufopanou@imperial.ac.uk.
Abstract
VDE is a homing endonuclease gene in yeasts with an unusual evolutionary history including horizontal transmission, degeneration, and domestication into the mating-type switching locus HO. We investigate here the effects of these features on its molecular evolution. In addition, we correlate rates of evolution with results from site-directed mutagenesis studies. Functional elements have lower rates of evolution than degenerate ones and higher conservation at functionally important sites. However, functionally important and unimportant sites are equally likely to have been involved in the evolution of new function during the domestication of VDE into HO. The domestication event also indicates that VDE has been lost in some species and that VDE has been present in yeasts for more than 50 Myr.
Key Words: VDE ? HEG ? site-directed mutagenesis ? molecular evolution ? Saccharomycetaceae
VDE (vacuolar membrane–derived endonuclease) is one of the best-studied selfish genetic elements (Gimble and Thorner 1992; recent reviews include Chevalier and Stoddard 2001; Gogarten et al. 2002). It encodes a bifunctional protein with two domains, a homing endonuclease and a self-splicing intein, and is situated in the middle of the nuclear VMA1 locus of yeasts. The intein domain allows the VDE protein to splice out of the VMA1 host protein after they have been translated together in frame, thus reconstituting a functional VMA1 protein. The endonuclease domain, a member of the LAGLIDADG family of homing endonuclease genes (HEGs), recognizes, binds, and cuts a 31-bp recognition sequence in a VMA1 allele that does not contain VDE. Recent comparative studies in yeasts have revealed an unusual evolutionary history including regular horizontal transmission and degeneration (Koufopanou, Goddard, and Burt 2002; Okuda et al. 2003; Posey et al. 2004). This supports a model of cyclical evolution in which HEGs degenerate after becoming fixed within a species, due to the absence of purifying selection, with regular horizontal transmission being necessary for long-term persistence (Goddard and Burt 1999; Burt and Koufopanou 2004). In addition, the wild VDE has been domesticated in some yeasts, evolving into HO, a gene that improves the efficiency of mating-type switching (Dalgaard et al. 1997; Butler et al. 2004). Similar features are found in other selfish genetic elements, such as transposable elements (Hurst and Werren 2001; Pinsker et al. 2001; Kazazian 2004).
We analyze here a number of VDE and HO sequences from saccharomycete yeasts to study the effect of these unusual events on its sequence evolution. Furthermore, numerous site-directed mutagenesis studies available on VDE from Saccharomyces cerevisiae enable us to compare rates of evolution for sites that are found experimentally to be critical, semicritical, or neutral for the splicing and endonuclease functions and assess their contribution to the evolution of new function in HO. It is commonly assumed that functionally important sites evolve more slowly than functionally unimportant sites, and indeed, degree of conservation is often used to infer the importance of particular sites, but there are few studies directly correlating rates of evolution with results from site-directed mutagenesis (Li 1997).
The cyclical model of HEG evolution predicts that the two domains of VDE are under different selection regimes. Evolution in the splicing domain should be simple, splicing being critical for survival in all species. Indeed, maximum likelihood analysis gives a dN/dS ratio of 0.1 ± 0.01, well below the neutral expectation of 1, and no heterogeneity among lineages, indicating strong purifying selection to preserve function. There is also complete conservation at all six amino acid sites identified as critical for self-splicing, showing a strict amino acid requirement.
Evolution in the endonuclease domain might be more complex, as this domain can degenerate after becoming fixed. Indeed, only three out of the 14 VDEs studied here are active (S. cerevisiae, Saccharomyces cariocanus, and Zygosaccharomyces bailii; Posey et al. 2004). Maximum likelihood analysis gives a significantly lower dN/dS ratio in functional than degenerate endonuclease domains, indicating slower evolution in active domains and faster following degeneration (0.07 ± 0.01 vs. 0.17 ± 0.02 for functional vs. degenerate lineages, respectively, P < 0.001; fig. 1; see Supplementary Material online for full details). Note that divergence in degenerate endonuclease domains is considerably underestimated here due to a large number of insertions or deletions, treated as ambiguities in the analysis. Nevertheless, purifying selection is still indicated even in degenerate domains, not only by dN/dS < 1 but also by 10 invariant sites, five of which are in the LAGLIDADG-signature motifs forming the two -helices of the catalytic center (supplementary fig. 1). Perhaps these sites are structurally important in folding the molecule and enhancing self-splicing. This could explain why we do not observe deletions of the entire endonuclease domain, as are frequently found in intron-associated HEGs in yeasts and in bacterial inteins (Goddard and Burt 1999; Gogarten et al. 2002). Other intron-associated HEGs have evolved RNA maturase function and are thus less likely to be deleted (Chatterjee et al. 2003). Testing the effects of substitutions at these sites on self-splicing activity should clarify this point. Finally, for the three functional elements (all of which recognize the same sequence), sites important for endonuclease function are more conserved than neutral sites (table 1). No such difference is found among degenerate elements. Nevertheless, the functional Z. bailii VDE differs from the S. cerevisiae VDE at eight sites, which the mutagenesis studies indicate are important. Either the substitutions in Z. bailii are less drastic than alanine substitutions or there have been compensating changes at other sites.
FIG. 1.— Phylogram showing the evolution of VDE and HO (endonuclease domain only; maximum parsimony tree, numbers indicating >70% bootstrap support). Branch lengths are given by dN estimates from a free-ratio model in PAML, with the dN/dS ratio estimated separately for each branch. In bold are the three functional VDEs, connected by thick lines; the ancestor of the Saccharomyces castelli clade is also assumed to be functional, horizontal transfer events having been inferred within that clade (Koufopanou, Goddard, and Burt 2002). When Kluyveromyces lactis is removed, there is 91% bootstrap support for a sister clade relationship between the HO clade and the Saccharomyces cerevisiae clade of VDE, indicating that HO originated within the saccharomycete VDEs. The two alleles of HO from Saccharomyces pastorianus reflect its hybrid origin from S. cerevisiae and Saccharomyces bayanus.
Table 1 Conservation of Amino Acids at Sites Critical, Semicritical, and Neutral for Endonuclease Activity, as Determined by Site-Directed Mutagenesis (Alanine Substitutions Only), in Different Functional Classes of Elements
HO is a free-standing gene, homologous to the full length of VDE, including the self-splicing domain (even though HO does not splice), with 66% amino acid divergence between S. cerevisiae HO and VDE. HO has also acquired a C-terminal zinc finger domain (Bakhrat et al. 2004) and has evolved a new specificity toward an 18-bp sequence in the MAT locus, to which it binds and cleaves but does not insert. In an analysis of a large number of LAGLIDADG HEGs encompassing the entire range of their diversification, Dalgaard et al. (1997) found VDE and HO from S. cerevisiae to be closest relatives, suggesting that HO represents a domesticated form of VDE. Our analysis, on a much larger sample of VDE and HO sequences, confirms this result, with the HOs forming a clade nested within the VDEs, indicating a single origin of HO from VDE (fig. 1). Unlike VDE, and consistent with its domesticated status, HO shows no evidence for horizontal transmission (no difference between HO and host genealogy; data not shown) nor is there evidence for loss of HO in any species (fig. 2) or degeneration. The evolution of HO provides the clearest example to date for the domestication of a selfish element, and its study allows some insight into the evolution of novelty.
FIG. 2.— The distribution of VDE among species of yeasts where HO is known to be present or absent. Phylogenetic relationships are from Kurtzman and Robnett (2003). Numbers indicate >80% bootstrap support.
Maximum likelihood branch lengths calculated from the rates of nonsynonymous substitutions, dN, show the origin of HO as the longest internal branch in the phylogeny (fig. 1). Reliable estimates of dN/dS for this branch could not be obtained, perhaps due to changes in codon usage; within the HO cluster, dN/dS was 0.04 ± 0.002, approximately half the estimate for functional VDEs. Correlating rates of evolution with results from site-directed mutagenesis, we find no significant tendency for functionally important sites to be more (or less) involved in the evolution of the novel function. Sites important or unimportant in VDE are equally likely to be divergent in S. cerevisiae HO or conserved among the 10 HOs (table 1). Thus, the change in function from VDE to HO (which recognizes a sequence different and unrecognizable by VDE; Gimble and Wang 1996) has apparently entailed a change in critical sites. This is contrary to the diversification of functional VDEs, where important sites are more conserved. Nevertheless, six HO sites homologous to important VDE sites were also found important for mating-type switching, and amino acids in four of these are identical to those in VDE (including the two Mg2+-binding amino acids at the active site; Bakhrat et al. 2004; supplementary fig. 1), highlighting the similarity of action in the two molecules. It will be interesting to test how general these observations are for other types of genes, for example, during the evolution of a new function following gene duplication (Gaucher et al. 2002).
The domestication event, which is followed by normal vertical transmission, also provides evidence relevant to the cyclical model of VDE evolution. The presence of HO in several species that do not contain VDE (fig. 2) supports the idea that VDE was present in their ancestors and was subsequently lost. The mechanism of precise loss remains unknown. The domestication event also provides an anchor point to the endless cycling of VDE and allows us to obtain a minimum estimate for the length of residence of VDE in the Saccharomycetaceae. The phylogeny in figure 2 suggests that HO originated after the divergence of S. cerevisiae and Kluyveromyces lactis, estimated at approximately 70 Myr (Berbee and Taylor 1993), and is therefore younger than 70 Myr, perhaps 50 Myr old. VDE, which is ancestral to HO, must therefore be much older and must have been in the Saccharomycetaceae for at least that long.
Methods
VDE sequences were previously recovered by screening the VMA1 locus of 24 species of Saccharomycetaceae yeasts (Koufopanou, Goddard, and Burt 2002) and homologues from Chlamydomonas eugametos and Methanococcus jannaschii used as out-group in phylogenetic analyses (Dalgaard et al. 1997). Information on sites critical for splicing or endonuclease function was compiled from the literature (Cooper et al. 1993; Gimble and Stephens 1995; Kawasaki et al. 1997; Gimble et al. 1998; He et al. 1998; Hu et al. 1999; Wende et al. 2000). Sites were categorized as critical when mutants had <1% of wild-type activity, semicritical when <25%, and neutral when 25% or higher. "Neutral" does not necessarily imply the absence of selective effects. For consistency, only the effects of alanine substitutions are considered here; this is thought to effectively remove any important functional group that is normally present (He et al. 1998). Of a total of 454 amino acid sites in the S. cerevisiae VDE, 83 have been tested for endonucleolytic function, and of these, 40 were found critical or semicritical for function, distributed in both endonuclease and self-splicing domains. Only six sites have been tested for self-splicing activity, all found critical. Twelve sequences for HO were available in the database; sequences for Zygosaccharomyces rouxii and Saccharomyces exiguus were only partial and were not included in the main analysis, but results were qualitatively unchanged if they were included. Sequences were aligned using ClustalX (Thompson, Higgins, and Gibson 1994), and phylogenetic relationships were analyzed with PAUP* (Swofford 1999) and rates of evolution with PAML (Yang 1997).
Supplementary Material
A Logo alignment of VDEs (supplementary fig. 1), illustrating amino acid conservation at functionally important and unimportant sites, is available at Molecular Biology and Evolution online (www.mbe.oupjournals.org). Also given are plots of pairwise amino acid divergence between the two domains and between important and unimportant sites (supplementary fig. 2), full details for the maximum likelihood analysis (supplementary table 1), and the full alignment of VDE and HO sequences.
Acknowledgements
This work was supported by the Daphne Jackson Trust, Natural Environment Research Council, and the Wellcome Trust.
References
Bakhrat, A., M. S. Jurica, B. L. Stoddard, and D. Raveh. 2004. Homology modeling and mutational analysis of HO endonuclease of yeast. Genetics 166:721–728.
Berbee, M. L., and J. W. Taylor. 1993. Dating the evolutionary radiations of the true fungi. Can. J. Bot. 71:1114–1127.
Burt, A., and V. Koufopanou. 2004. Homing endonuclease genes: the rise and fall and rise again of a selfish element. Curr. Opin. Genet. Dev. 14:609–615.
Butler, G., C. Kenny, A. Fagan, C. Kurischko, C. Gaillardin, and K. H. Wolfe. 2004. Evolution of the MAT locus and its HO endonuclease in yeast species. Proc. Natl. Acad. Sci. USA 101:1632–1637.
Chatterjee, P., K. L. Brady, A. Solem, Y. Ho, and M. G. Caprara. 2003. Functionally distinct nucleic acid binding sites for a group I encoded RNA maturase/DNA homing endonuclease. J. Mol. Biol. 329:239–251.
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