* Departamento de Genética
Instituto Cavanilles de Biodiversidad and Biología Evolutiva, Universidad de Valencia, Valencia, Spain
Correspondence: E-mail: ignacio.marin{at}uv.es.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: gene duplication homeotic genes homeobox mutual information
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Multiple forces may be involved in the maintenance or elimination of duplicated genes. The classical model to explain duplicates maintenance postulates that, after a period of redundancy, positive selection generates functional divergence and the emergence of a new function for one of the duplicates (Ohno 1970). However, evidence for positive selection acting on Hox genes has not generally been found (e.g., Hughes and Hughes 1993). Moreover, knockout mice phenotypic analyses has provided proof of extensive redundancy among Hox genes in a vertebrate (reviewed in St-Jacques and McMahon [1996]). Finally, it has been shown that, keeping intact the cis-acting regulatory sequences, the coding regions of some Hox genes can substitute for each other efficiently, both in vertebrates and in Drosophila (Greer et al. 2000; Hirth et al. 2001). These results have led to the formulation of alternative hypotheses to explain Hox genes maintenance (reviewed in Force et al. [1999]; Massingham, Davies, and Liò [2001]). Particularly, acquisition of differential expression patterns by subfunctionalization (Force et al. 1999) and quantitative effects, dependent on the total number of genes within a paralog group (Greer et al. 2000) have been suggested to be the main forces that explain evolutionary conservation of paralogous Hox genes.
Even in favorable cases, the determination of the selective regimes acting on coding regions, and most especially the detection of directional, positive selection, is a complex task that requires using both very sensitive methods of detection and the right genes as models. This work is focused on the determination of the impact of natural selection on the coding regions of Hox7 genes. Hox paralog group VII is particularly suitable for this type of study. First, it is in many species the paralog group with the minimum number of genes. Thus, exceptionally, a single Hox7 gene seems to exist in condrichthyan or actinopterygian fishes (and whether any Hox7 genes exist in Fugu rubripes is unclear [Aparicio et al. 1997; Amores et al. 1998; Snell, Scemama and Stellwag 1999; Kim et al. 2000]). Also, mammals have only two Hox7 genes (called Hoxa-7 and Hoxb-7). This is a rare feature, shared only with paralog group II. Finally, the tetraploid species Xenopus laevis has three group VII genes, one Hoxa-7 and two Hoxb-7 genes (Bisbee et al. 1977). This is again an exceptional case where we can check whether positive selection occurred after a recent duplication. Selective forces were analyzed using several advanced methods. First, we explored, using maximum-likelihood methods based on a phylogenetic approach, whether variation on the selective forces acting on the whole set of Hox7 sequences occurred. Second, we analyzed, using a similar approach, whether selection varied in different vertebrate lineages. Third, we confirmed the results obtained in the second type of analysis by pairwise comparisons among all the Hox7 genes. Finally, we search for regions under functional differentiation after the Hoxa-7/Hoxb-7 gene duplication. Notably, our results are compatible with the classical view of maintenance of duplicated genes by functional diversification of their protein products (Ohno 1970).
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The nucleotide sequences of these genes were conceptually translated and a multiple-sequence alignment of their encoded proteins was generated using the default parameters of ClustalX version 1.83 (Thompson et al. 1997). The alignments were inspected, and minor changes made when necessary to improve alignment, using GeneDoc version 2.6 (Nicholas and Nicholas 1997). From these final protein alignments, we generated the corresponding nucleotide-sequence alignment to be used for the rest of analyses. Given the high heterogeneity in the conservation of amino acid sequences between the N-terminal and homeobox regions, an analysis of the distribution of amino acid substitutions was performed to obtain an accurate phylogenetic tree. The gamma shape parameter () was estimated using the program GAMMA (Gu and Zhang 1997). Thereafter, a protein phylogenetic tree based on gamma-corrected distances among sequences was inferred by the neighbor-joining (NJ) routine (Saitou and Nei 1987) available in MEGA program version 2.1 (Kumar et al. 2001). A total of 1,000 bootstrap replicates were performed under the gamma distribution model using NJ in MEGA to determine the reliability of each node of the phylogenetic tree. The highly supported tree topology obtained was the expected according to our knowledge of the evolution of Hox genes and phylogenetic relationships among the analyzed species. For subsequent analyses, those positions containing gaps in any of the sequences were eliminated.
Analyses of Selective Constraints Acting on Hox7 genes
We used three different methods to determine selective constraints acting on Hox7 genes: (1) general tests for heterogenous selective constraints among sites, (2) general tests for variable ratios, and (3) pairwise comparisons.
Tests for Heterogeneous Selective Constraints Among Amino Acidic Sites in Hox7 Sequences
We followed the strategies described in recent articles (e. g., Yang et al. 2000b), based on previous works by Nielsen and Yang (1998). To determine the selective forces acting on Hox7 genes, six codon-based models (Yang et al. 2000a), implemented in version 3.0 of the PAML package (Yang 2000) were analyzed. These models explore whether, given a certain phylogenetic tree, variable selective constraints among amino acid sites are present. For each model, a maximum-likelihood (ML) approach was used to estimate , the ratio of nonsynonymous (dN) to synonymous (dS) nucleotide substitution rates per codon (
= dN/dS) (Nielsen and Yang 1998; Yang et al. 2000b). Then, the basic model of a single
for the whole set of codons and sequences (usually called M0 [Goldman and Yang 1994]) was compared with more complex models (called M1 to M3, M7, and M8). These models are divided into two groups. M1, M2, and M3 assume discrete distributions. The "neutral" model 1 (M1) considers only two
values (
= 0 and
= 1), the "selection" model 2 (M2) includes three classes of codons, namely those with
= 0 and
= 1 plus a third class of codons with an
value estimated from the data, and, finally, in the "discrete" model 3 (M3), the number of different
ratios that can be estimated from the data is unconstrained. For these models, p0, p1, and p2 (see table 1) refer to the proportions of the different codon classes. The other models, M7 (ß model) and M8 (ß +
model), assume continuous distributions for
values. M7 does not allow for positively selected sites, whereas M8 takes into account putative positive selection at specific codon sites (Yang et al. 2000a). In these models, p and q refer to the parameters of the beta distribution, and p0 refers to the proportions of sites following the beta distribution. In summary, M2, M3, and M8 may detect positively selected sites, if present.
|
Tests for Variable Ratios Among Vertebrate Lineages
ML methods were also used to establish whether selection varied among branches of the phylogenetic tree. We compared the M0 model, which assumes a single parameter value for the entire tree, with the "free-ratio" model, which accepts independent
parameter values for each branch (Yang 1998) and with a model with four different
values, which we have called the "four-ratio" model. For this last model, we assigned particular
values for three branches that are related to duplication events. A fourth rate was assigned for all the other branches. Posterior Bayesian probabilities for codons were estimated for the free-ratio model, using the CODEML program from the PAML package.
Pairwise Comparisons of Hox7 Sequences
Maximum-likelihood estimates of dN and dS for pairs of sequences were obtained using the discrete models M0 and M3. A maximum of four different types of codons, according to their values, were allowed to be estimated by the program, but they were pooled when the program detected only two or three different classes. Number of degrees of freedom was established according to the number of estimated
parameters. The difference of the log-likelihood values between nested models were tested by the LRT (Huelsenbeck and Crandall 1997). As above, posterior probabilities for codons were estimated in those comparisons were positive selection was detected.
Detection of Functional Divergence after the Hoxa-7/Hoxb-7 Gene Duplication
Wang and Gu (2001) defined two different types of amino acidic divergence after gene duplication. Type I functional divergence refers to changes in functional constraints in one of the genes, resulting in high conservation in the sequences of different species for one paralog, while the other paralog evolves more freely. This type of divergence can be measured following the method developed by Gu (1999): a maximum-likelihood procedure is used to calculate a coefficient of functional divergence (), and it is determined whether such coefficient is large enough as to reject the null hypothesis of no functional differentiation. If the null hypothesis is rejected, a posterior probability for functional divergence for each position in the alignment is calculated. To implement this procedure, we used the program DIVERGE version 1.04 (Gu and Vander Velden 2002; available at http://xgu1.zool.iastate.edu/cgi-bin/download.cgi). We established a cutoff value for significance (P = 0.6) according to the effect that it had on the
parameter the elimination of the sets of amino acids with values above a certain posterior probability (see Wang and Gu 2001). We found that the elimination of amino acids with P > 0.6, led to a value of
that is essentially zero.
Type II divergence (Wang and Gu 2001) refers to amino acids that show gene-specific conservation; that is, although conservation is substantial within each paralog in different species, different amino acids are conserved in each of the two paralogs. To detect this type of divergence, we computed the mutual information content of our protein alignment using MatrixPlot (see Gorodkin et al. [1999]). We then established the positions in our alignment that had the maximum mutual information content when compared with those that show gene-specific amino acids (e.g., position 4 in figure 1). To establish whether the amino acidic changes detected by this procedure may have occurred immediately after the Hoxa-7/Hoxb-7 duplication and before the divergence among the analyzed species, we compared the mutual information results with the posterior Bayesian probabilities for codons established according to the "free-ratio" model (see above) for the branch connecting Hoxa-7 genes with Hoxb-7 genes.
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Interpretation of our results depends on how Hoxa-7 and Hoxb-7 genes originated. This question is related to determining the origin of the multiple Hox gene clusters found in vertebrates. So far, several hypotheses have been proposed to explain the evolution of those clusters. First, several authors proposed that mammalian Hox gene clusters originated in two successive rounds of duplication, perhaps associated with two full-genome duplications, in such a way that the current four clusters derive from two ancestral clusters, a proto AB cluster and a proto CD cluster. This model is often referred to as [(AB)(CD)] (Schughart, Kappen and Ruddle 1989; Kappen and Ruddle 1993; Zhang and Nei 1996; reviewed in Málaga-Trillo and Meyer [2001]; see also Gu, Wang, and Gu [2002] for a comprehensive analysis on whether genomic duplications actually occurred). However, alternative models, involving three rounds of duplication have been also proposed. Zhang and Nei (1996) indicated two of those scenarios: [(B(A(CD))] or [A(B(CD))]. Bailey et al. (1997) suggested a different one: [D(A(BC))]. Recently, Force, Amores, and Postlethwait (2002), although favoring the two-round hypothesis, proposed two additional alternatives with three steps: [D(C(AB))] and [C(D(AB))]. Largely, the discussion depends on the data used. In this case, sequence similarity of Hox genes (Zhang and Nei 1996), similarity of collagen genes, closely linked to each of the Hox clusters (Bailey et al. 1997), or parsimony analyses of the patterns of presence/absence of Hox genes in different clusters (e. g., Force, Amores, and Posthethwait 2002) yield contradictory results. It was expected that studying organisms that diverged from tetrapods before any of the cluster duplications occurred would sort out which of those alternatives is correct. However, despite considerable advances, the situation is still unclear. Thus, a chondrichthyan, the horn shark Heterodontus francisci, has at least two clusters, one of them quite similar to the mammalian A cluster and the other related to the mammalian D cluster (Kim et al. 2000). However, the possibility of this species having up to four clusters has not been excluded (cited in Irvine et al. [2002]). An even more distant relative, the agnathan Petromyzon marinus, a sea lamprey, has been recently studied by two groups who found that it contains three or, more likely, four Hox clusters (Force, Amores, and Postlethwait 2002; Irvine et al. 2002). Although it seems that part of this complexity arose by cluster duplications specific for agnathan fishes, it still does not fully contribute to establishing the most likely history for the four tetrapod Hox clusters.
We think however that, if indeed any of the six hypotheses detailed above are correct, these complications do not greatly affect the interpretation of our results. Fortunately, all them are quite similar from the point of view of the origin of A and B cluster genes. In three of those alternatives, [(AB)(CD)], [D(C(AB))], and [C(D(AB))], Hoxa-7 and Hoxb-7 genes would have emerged from an ancestral "protoa-7/b-7" gene. In those cases, we can suggest, according to our results, that positive natural selection acted on one or both of those genes after the duplication event, precisely as Ohno's classical model postulates. In the other three cases, [(B(A(CD))], [A(B(CD))], and [D(A(BC))], it is significant that Hoxa-7 and Hoxb-7 genes are still separated by a single step (i. e., one duplication event). Thus, no matter when the putative Hoxc-7 and Hoxd-7 genes arose and became lost, we still can postulate that strong positive selection may have occurred just after the genes that gave rise to the modern Hoxa-7 and Hoxb-7 genes originated by duplication.
In summary, we suggest that, no matter what the precise evolutionary history of Hox7 genes turns to be, the most likely explanation for our results would be that the duplication that originated the gene lineages that gave rise to the Hoxa-7 and Hoxb-7 genes was followed by a period in which those two lineages diversified under positive selection. We also suggest that tetraploidization in the Xenopus lineage may have been followed by a similar episode in which positive selection acted on one of the Hoxb-7 duplicates (most likely Hoxb-7b [fig. 2]) or even on both of them (see the positive pairwise comparisons in table 3). After those periods of positive selectiondriven processes, strong purifying selection predominated, being in fact the main force in the long term. This may explain why some values, as the one for the Xenopus Hoxb-7b branch or the one that groups all tetrapod Hoxa-7 genes, have values in the free-ratio model that are lower than 1 but still several times higher than almost all the other branches (see figure 2). It cannot be excluded that positive selection has contributed also to the substantial divergence of the Hoxa-7 gene of the fish Morone (Snell, Scemama, and Stellwag 1999) that we excluded from our analyses. Additional studies whenever other actynopterygian Hoxa-7 sequences are available may shed light on this intriguing possibility.
No matter how they originated, that Hoxa-7 and Hoxb-7 genes existed before the split of Actinopterygia and Sarcopterygia can be considered an established fact. On one hand, two closely related actinopterygian species (belonging to the Morone and Oreochromis genera) have been found to contain a Hoxa-7 gene (Snell, Scemama, and Stellwag 1999; work cited in Málaga-Trillo and Meyer [2001]), while a more distant relative, Danio rerio, has a Hoxb-7 gene (Amores et al. 1998). On the other hand, both tetrapods and the coelacanth Latimeria (Koh et al. 2003) have both Hoxa-7 and Hoxb-7 genes. A significant problem is then to explain the loss of Hox7 genes in actinopterygian fish lineages at the same time that we suggest that the two paralogs diversified immediately after the gene duplication that originated them, and thus they may have been already functionally different in the ancestor of all actynopterygian. A possibility is that Hox7 genes may have become dispensable as a consequence of the additional round of duplication that occurred in actinopterygian fishes. This additional duplication may have happened soon after the Actinopterygia/Sarcopterygia split, if it is confirmed that species that are basal in the actinopterygian tree have more than four Hox gene clusters, as suggested by data obtained for the bichir, Polypterus (Ledje, Kim, and Ruddle 2002). This hypothesis moreover implies that genes of paralog groups other than group VII must be able in some cases to compensate for lack of Hox7 gene function. In this context, considering the phenotypes of mouse that are null mutants for Hox7 genes is significant. Mouse Hoxa-7 knockout mutants are normal, whereas only a small percentage of Hoxb-7 null mutant mice have skeletal abnormalities, and even double mutants are often normal (Chen, Greer, and Capecchi 1998). All these results contrast with the complex patterns of expression detected for both Hoxa-7 and Hoxb-7, which led to predictions of multiple roles for these genes (Mahon, Westphal, and Gruss 1988; Vogels, de Graaff, and Deschamps 1990). Lack of phenotypes in single mutants does not demonstrate identical functions for both paralogs (Nowak et al. 1997). In addition, our results suggest that mammalian Hox7 genes cannot be redundant, because they both show strong selective constraints. Thus, the subtle phenotypic effect in double mutants suggests that apparent redundancy caused by homeostatic effects due to the action of Hox genes that belong to paralog groups other than group VII may be occurring (see Rijli and Chambon [1997] for a discussion).
It is interesting to compare our results to those of Hughes and Hughes (1993). These authors analyzed whether Xenopus, mouse, or human Hox7 genes evolved under directional selection, finding negative results. However, their analyses were based on procedures (Nei and Gojobori 1986) that allow detection of positive selection only when it involves the whole sequence of the gene. With more refined methods, we have been able to detect positive selection acting on a limited number of amino acids and in a phylogenetic context. Interestingly, a recent work presented evidence for positive selection for three duplicated Hox genes in zebrafish using a different methodology (Van de Peer et al. 2001). These results suggest that analyzing whether episodes of positive selection on coding regions contribute to explaining the evolution of most or even all Hox genes is an attractive research program.
Finally, recent data suggest that changes in coding regions of the homeotic gene Ultrabithorax may have been critical in the modifications of body plans found in arthropods and onychophorans (Galant and Carroll 2002; Ronshaugen, McGinnis, and McGinnis 2002). Significant changes map to the C-terminal region of the protein, outside of the homeobox. These results, together with our data, suggest that new functions (or significant modifications of preexisting ones) may be acquired by relatively simple changes affecting particular regulatory regions present in HOX proteins. In particular, it has been shown that the N-terminal region where we have detected most of the significant changes acts as a modulator of the repressive action of the HOXA-7 homeodomain (Schnabel and Abate-Shen 1996), and deletions of any of two parts of it (corresponding to positions 40 to 86 and 88 to132 in figure 1) altered HOXB-7 function on granulocyte differentiation (Yaron et al. 2001). In this context, the data showing that HOXB-7 protein interacts through its N-terminus with the cactus-related protein IB-
(Chariot et al. 1999) are most interesting. They suggests that the modulatory action of this region may be caused by regulating HOX proteins interactions with certain partners. These partners, as classically shown for cofactors such as Exd/Pbx (reviewed in Mann and Affolter [1998]), determine the ability to bind specific sequences, and thus activate or repress Hox target genes. However, an interesting difference is that Exd/Pbx factors bind to highly conserved regions in HOX proteins, as the YPWM motif that is located just before the homeodomain (positions 139 to 142 in figure 1). Thus, in this case, they could potentially interact with both Hoxa-7 and Hoxb-7 gene products. Our results suggest that interactions through regions in the less conserved N-terminal half of HOX proteins may contribute to differentiating the functions of very close relatives, such as the HOXA-7/HOXB-7 pair.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
William Jeffery, Associate Editor
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Akaike, H. 1974. New look at statistical-model identification tree. IEEE T. Automat. Contr. AC19:716-723.[ISI]
Amores, A., A. Force, and Y. L. Yan, et al. (13 co-authors). 1998. Zebrafish hox clusters and vertebrate genome evolution. Science 282:1711-1714.
Aparicio, S., S. Hawker, A. Cottage, Y. Mikawa, L. Zuo, B. Venkatesh, E. Chen, R. Krumlauf, and S. Brenner. 1997. Organization of the Fugu rubripes Hox clusters: evidence for continuing evolution of vertebrate Hox complexes. Nat. Genet. 16:79-83.[ISI][Medline]
Bailey, W. J., J. Kim, G. P. Wagner, and F. H. Ruddle. 1997. Phylogenetic reconstruction of vertebrate Hox cluster duplications. Mol. Biol. Evol. 14:843-853.[Abstract]
Bisbee, C.A., M. A. Baker, A. C. Wilson, H. A. Irandokht, and M. Fischberg. 1977. Albumin phylogeny for clawed frogs (Xenopus). Science 195:785-787.[ISI][Medline]
Chariot, A., F. Princen, J. Gielen, M. P. Merville, G. Franzoso, K. Brown, U. Siebenlist, and V. Bours. 1999. IB-
enhances transactivation by the HOXB7 homeodomain-containing protein. J. Biol. Chem. 274:5318-5325.
Chen, F., J. Greer, and M. Capecchi. 1998. Analysis of Hoxa7/Hoxb7 mutants suggests periodicity in the generation of the different sets of vertebrae. Mech. Dev. 77:49-57.[CrossRef][ISI][Medline]
Finnerty, J. R, and M. Q. Martindale. 1998. The evolution of the Hox cluster: insights from outgroups. Curr. Opin. Genet. Dev. 8:681-687.[CrossRef][ISI][Medline]
Force, A., A. Amores, and J. H. Postlethwait. 2002. Hox cluster organization in the jawless vertebrate Petromyzon marinus. J. Exp. Zool. 294:30-46.[CrossRef][ISI][Medline]
Force, A., M. Lynch, F. B. Pickett, A. Amores, Y. Yan, and J. Postlethwait. 1999. Preservation of duplicate genes by complementary, degenerative mutations. Genetics 151:1531-1545.
Galant, R., and S. B. Carroll. 2002. Evolution of a transcriptional repression domain in an insect Hox protein. Nature 415:910-913.[CrossRef][ISI][Medline]
Goldman, N., and Z. Yang. 1994. A codon-based model of nucleotide substitution for protein-coding DNA sequences. Mol. Biol. Evol. 11:725-736.
Gorodkin, J., H. H. Staerfeldt, O. Lund, and S. Brunak. 1999. MatrixPlot: visualizing sequence constraints. Bioinformatics 15:769-770.
Greer, J. M., J. Puetz, K. R. Thomas, and M. R. Capecchi. 2000. Maintenance of functional equivalence during paralogous Hox gene evolution. Nature 403:661-665.[CrossRef][ISI][Medline]
Gu, X. 1999. Statistical methods for testing functional divergence after gene duplication. Mol. Biol. Evol. 16:1664-1674.
Gu, X., Y. Wang, and J. Gu. 2002. Age distribution of human gene families shows significant roles of both large- and small-scale duplications in vertebrate evolution. Nat. Genet. 31:205-209.[CrossRef][ISI][Medline]
Gu, X., and K. Vander Velden. 2002. DIVERGE: phylogeny-based analysis for functional-structural divergence of a protein family. Bioinformatics 18:500-501.
Gu, X., and J. Zhang. 1997. A simple method for estimating the parameter of substitution rate variation among sites. Mol. Biol. Evol. 14:1106-1113.[Abstract]
Hirth, F., T. Loop, B. Egger, D. F. B. Miller, T. C. Kaufman, and H. Reichert. 2001. Functional equivalence of Hox gene products in the specification of the tritocerebrum during embryonic brain development of Drosophila. Development 128:4781-4788.
Huelsenbeck, J. P., and K. A. Crandall. 1997. Phylogeny estimation and hypothesis testing using maximum likelihood. Annu. Rev. Ecol. Syst. 28:437-466.[CrossRef][ISI]
Hughes, M. K., and A. L. Hughes. 1993. Evolution of duplicate genes in a tetraploid animal, Xenopus laevis. Mol. Biol. Evol. 10:1360-1369.[Abstract]
Irvine, S. Q., J. L. Carr, W. J. Bailey, K. Kawasaki, N. Shimizu, C. T. Amemiya, and F. H. Ruddle. 2002. Genomic analysis of Hox clusters in the sea lamprey Petromyzon marinus. J. Exp. Zool. 294:47-62.[CrossRef][ISI][Medline]
Kappen, C., and F. H. Ruddle. 1993. Evolution of a regulatory gene family: HOM/HOX genes. Curr Opin. Genet. Dev. 3:931-938.[Medline]
Kim, C.-B., C. Amemiya, W. Bailey, K. Kawasaki, J. Mezey, W. Miller, S. Minoshima, N. Shimizu, G. Wagner, and F. Ruddle. 2000. Hox cluster genomics in the horn shark, Heterodontus francisci. Proc. Natl. Acad. Sci. USA 97:1655-1660.
Koh, E. G., K. Lam, A. Christoffels, M. V. Erdmann, S. Brenner, and B. Venkatesh. 2003. Hox gene clusters in the Indonesian coelacanth, Latimeria menadoensis. Proc. Natl. Acad. Sci. USA 100:1084-1088.
Krumlauf, R. 1994. Hox genes in vertebrate development. Cell 78:191-201.[ISI][Medline]
Kumar, S., K. Tamura, I. B. Jacobsen, and M. Nei. 2001. MEGA: molecular evolutionary genetics analysis. Version 2.1. Distributed by the authors (www.megasoftware.net).
Ledje, C., C. B. Kim, and F. H. Ruddle. 2002. Characterization of Hox genes in the bichir, Polypterus palmas. J. Exp. Zool. 294:107-111.[CrossRef][ISI][Medline]
Mahon, K. A., H. Westphal, and P. Gruss. 1988. Expression of homeobox gene Hox 1.1 during mouse embryogenesis. Development 104:(Suppl.): 187-95.[ISI][Medline]
Málaga-Trillo, E., and A. Meyer. 2001. Genome duplications and accelerated evolution of Hox genes and cluster architecture in teleost fishes. Am. Zool. 41:676-686.[ISI]
Mann, R. S., and M. Affolter. 1998. Hox proteins meet more partners. Curr. Opin. Genet. Dev. 8:423-429.[CrossRef][ISI][Medline]
Massingham, T., L. J. Davies, and P. Liò. 2001. Analysing gene function after duplication. Bioessays 23:873-876.[CrossRef][ISI][Medline]
McGinnis, W., and R. Krumlauf. 1992. Homeobox genes and axial patterning. Cell 68:283-302.[ISI][Medline]
Nei, M., and T. Gojobori. 1986. Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Mol. Biol. Evol. 3:418-426.[Abstract]
Nicholas, K. B., and H. B. Nicholas, Jr. 1997. Distributed by the authors. (www.cris.com/ketchup/genedoc.shtml).
Nielsen, R., and Z. Yang. 1998. Likelihood models for detecting positively selected amino acid sites and applications to the HIV-1 envelope gene. Genetics 148:929-938.
Nowak, M. A., M. C. Boerlijst, J. Cooke, and J. Maynard Smith. 1997. Evolution of genetic redundancy. Nature 388:167-171.[CrossRef][ISI][Medline]
Ohno, S. 1970. Evolution by gene duplication. Springer-Verlag, Berlin.
Pollard, S. L., and P. W. H. Holland. 2000. Evidence for 14 homeobox gene clusters in human genome ancestry. Curr. Biol. 10:1059-1062.[CrossRef][ISI][Medline]
Prince, V. E. 2002. The Hox paradox: more complex(es) than imagined. Dev. Biol. 249:1-15.[CrossRef][ISI][Medline]
Rijli, F. M., and P. Chambon. 1997. Genetic interactions of Hox genes in limb development: learning from compound mutants. Curr. Opin. Genet. Dev. 7:481-487.[CrossRef][ISI][Medline]
Ronshaugen, M., M. McGinnis, and W. McGinnis. 2002. Hox protein mutation and macroevolution of the insect body plan. Nature 415:914-917.[CrossRef][ISI][Medline]
Ruddle, F. H., J. L. Bartels, K. L. Bentley, C. Kappen, M. T. Murtha, and J. W. Pendleton. 1994. Evolution of Hox genes. Annu. Rev. Genet. 28:423-432.[CrossRef][ISI][Medline]
Saitou, N, and M. Nei. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406-425.[Abstract]
Schnabel, C. A., and C. Abate-Shen. 1996. Repression by HoxA7 is mediated by the homeodomain and the modulatory action of its N-terminal-arm residues. Mol. Cell. Biol. 16:2678-2688.[Abstract]
Schughart, K., C. Kappen, and F. H. Ruddle. 1989. Duplication of large genomic regions during the evolution of vertebrate homeobox genes. Proc. Natl. Acad. Sci. USA 86:7067-7071.[Abstract]
Snell, E. A., J. L. Scemama, and E. J. Stellwag. 1999. Genomic organization of the Hoxa4-Hoxa10 region from Morone saxatilis: implications for Hox gene evolution among vertebrates. J. Exp. Zool. (Mol. Dev. Evol.) 285:41-49.
St-Jacques, B., and A. P. McMahon. 1996. Early mouse development: lessons from gene targeting. Curr. Opin. Genet. Dev. 6:439-444.[CrossRef][ISI][Medline]
Thompson, J.D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins. 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 24:4876-4882.[CrossRef]
Van de Peer, Y., J. S. Taylor, I. Braasch, and A. Meyer. 2001. The ghost of selection past: rates of evolution and functional divergence in anciently duplicated genes. J. Mol. Evol. 53:436-446.[CrossRef][ISI][Medline]
Vogels, R., W. de Graaff, and J. Deschamps. 1990. Expression of the murine homeobox-containing gene Hox-2.3 suggests multiple time-dependent and tissue-specific roles during development. Development 110:1159-1168.[Abstract]
Wang, Y., and X. Gu. 2001. Functional divergence in the caspase gene family and altered functional constraints: statistical analysis and prediction. Genetics 158:1311-1320.
Yang, Z. 1998. Likelihood ratio tests for detecting positive selection and application to primate lysozyme evolution. Mol. Biol. Evol. 15:568-573.[Abstract]
Yang, Z. 2000. Phylogenetic analysis of maximum likelihood (PAML). Version 3. University College London, England.
Yang, Z., R. Nielsen, N. Goldman, and A. M. K. Pedersen. 2000a. Codon substitution models for heterogeneous selection pressures at amino acid sites. Genetics 153:1077-1089.[ISI]
Yang, Z., R. Nielsen, N. Goldman, and A. M. K. Pedersen. 2000b. Codon-substitution models for heterogeneous selection pressure at amino acid sites. Genetics 155:431-449.
Yaron, Y., J. K. McAdara, M. Lynch, E. Hughes, and J. C. Gasson. 2001. Identification of novel functional regions important for the activity of HOXB7 in mammalian cells. J. Immunol. 166:5058-5067.
Zhang, J., and M. Nei. 1996. Evolution of Antennapedia-class homeobox genes. Genetics. 142:295-303.