Department of Biology, Galton Laboratory, University College London, London, England
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Abstract |
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Introduction |
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DAZ is located on the Y chromosome, but it is closely related to the autosomal gene DAZL1. While DAZL1 is present in all vertebrates, DAZ is found only in Old World Monkeys. Thus, DAZ [Yq11.23 ] is believed to have evolved via translocation of DAZL1 [3p24] to the Y chromosome (Saxena et al. 1996
; Gromoll et al. 1999
) some time after the divergence of Old and New World monkeys; Kumar and Hedges (1998)
dated that divergence to about 40 MYA. After the translocation event, DAZ underwent a series of rearrangements and a modified copy was amplified, yielding a Y gene cluster.
DAZ and DAZL1 have a functional role in fertility. Both DAZ and DAZL1 are expressed exclusively in germ cells (Cooke et al. 1996
; Ruggiu et al. 1997
; Gromoll et al. 1999
), and in humans DAZ expression is highest in spermatogonia (Menke, Mutter, and Page 1997
). Experimental elimination of DAZL1 in mice results in termination of germ cell development beyond the spermatoginial stage (Ruggiu et al. 1997
). Moreover, Y-encoded human DAZ can compliment the sterile phenotype of DAZL1 null mice, yielding a partial recovery of spermatogenesis, which suggests the same or similar target mRNA for DAZ and DAZL1 during spermatogenesis (Slee et al. 1999
). Although the specific functions of DAZ and DAZL1 are unknown, the presence of RNA recognition motifs suggests that these genes could be involved in controlling the cell cycle switch from mitotic to meiotic cell division (Gromoll et al. 1999
); this cell cycle switch is controlled by RNA-binding proteins in yeast (Watanabe et al. 1997
).
Surprisingly, a recent evolutionary analysis of the DAZ family (DAZ and DAZL1 genes) indicated a lack of functional constraints on DAZ. Agulnik et al. (1998)
found a high rate of nonsynonymous substitution, similar rates between exons and introns, and similar rates among the three codon positions. They concluded that there were no functional constraints on evolution of DAZ and that patterns of sequence divergence were due to neutral drift. They hypothesized that Y-linked DAZ played little role in human spermatogenesis.
The nonsynonymous-to-synonymous rate ratio (dN/ dS) provides a sensitive measure of selective pressure on the protein. However, when selection pressure varies among amino acid sites, the average dN/dS ratio might not be very informative about the evolutionary processes affecting the gene. The objective of this study was to investigate the role of both purifying and positive selection on the DAZ gene family by using maximum-likelihood methods that accommodate differences in selective pressures among sites (Nielsen and Yang 1998
; Yang et al. 2000
). Our findings indicated that DAZ was not free of functional constraints and that other explanations for its rapid rate of nonsynonymous evolution must be considered. There has been considerable debate as to whether rapid evolution in gene families is caused by positive Darwinian selection after gene duplication (Ohta 1993
) or by relaxation, but not complete loss, of functional constraints in redundant genes (Kimura 1983
; Li 1985
). In the latter case, a new function might evolve when formerly neutral substitutions convey a selective advantage in a novel environment or genetic background (Dykhuizen and Hartl 1980
). We also examined variable selective pressures among lineages (Yang 1998
; Yang and Nielsen 1998
), and our findings suggest that both models could have played a role in the evolution of the DAZ gene family.
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Materials and Methods |
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We implemented four nested models of variable selective pressures among branches (Yang 1998
; Yang and Nielsen 1998
). Model A was the simplest and assumed the same
ratio for all branches. Models B and C were based on the prediction that a gene family evolves under different selective pressures following gene duplication. Model B assumed two
ratios: one for the branch predating the translocation to the Y chromosome (fig. 1
; branch a), and a second for branches postdating the translocation (branches bg). Model C assumed three
ratios: one for branch a, one for DAZL1 branches postdating the translocation (branches bd), and one for all DAZ branches (branches eg). Model D (free ratios) assumed an independent
ratio for each branch of a topology and was employed to evaluate the potential for positive selection in any one branch of the tree.
ML models (Yang and Nielsen 2000
; Yang et al. 2000
) also permit testing and identification of selective pressures at individual codon sites. We implemented three such models: M3 (discrete), M7 (beta), and M8 (beta&
). M3 assumed two site classes with the proportions f0 and f1 and ratios
0 and
1 estimated from the data. M7 assumed that
ratios were distributed among sites according to a beta distribution. Depending on parameters p and q, the beta distribution can take a variety of shapes within the interval (0, 1). M8, an extension of M7, added an extra class of sites having an
parameter freely estimated from the data. Positive selection was indicated when an
parameter of M3 or M8 was >1. The likelihood ratio test was used to compare a one-ratio model (M0) with M3 and to compare M7 with M8. If there were sites with
> 1, Bayesian methods were used to calculate the posterior probability that a site fell into each site class; sites with high probabilities for
> 1 were likely to be under positive Darwinian selection (Yang et al. 2000
).
All ML analyses of codon models were performed using the codeml program of the PAML package (Yang 1999
). The models employed correction for transition/ transversion rate bias and codon usage bias, features of DNA sequence evolution that have a significant effect on the estimation of substitution rates (Yang and Nielsen 1998, 2000
).
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Results |
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Because positive selection at any one point in the phylogeny could have affected our results, we applied the free-ratios model (model D) to the same data. The likelihood score under model D was significantly better than that obtained for model B (2 = 14.2, df = 5, P = 0.014). Branches b, d, and g exhibited
values >1 (table 1
). Use of the simpler but less realistic F3x4 model, which calculates codon frequencies by using base composition at the three codon positions, produced similar results. Note that
for branch g was slightly less than 1 under the F3x4 model (
6 = 0.99), whereas it was greater than 1 under the F61 model (
6 = 1.144).
Variable Selection Pressure Among Sites
Phylogenetic analysis of data set 2 under the nucleotide model GTR+d recovered a topology in which divergent copies of DAZ from the same species were not monophyletic, indicating that divergent copies of DAZ originated in an early amplification event and persisted in multiple lineages (fig. 2A
). This result is similar to that obtained in a previous analysis of the DAZ gene family (Agulnik et al. 1998
). We also inferred a tree topology from synonymous divergences (fig. 2B
). This tree, although different from the tree obtained from the nucleotide analysis, also indicated that some copies of DAZ originated from an early amplification event and persisted to the present day. Both trees also indicate a clear bifurcation between all DAZL1 and DAZ sequences, supporting the hypothesis that a single translocation event gave rise to the Y-encoded DAZ. To investigate the impact of tree topology, models of variable
values among sites were analyzed using both topologies in figure 2
(table 2 ). The small size of data set 2 (291 bp; 97 codons) prevented use of the parameter-rich model of empirical codon frequencies (the F61 model), and the F3x4 model was used instead.
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Discussion |
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Based on an evolutionary analysis of the DAZ gene family, Agulnik et al. (1998)
concluded that there were no functional constraints on the evolution of DAZ in primates and questioned the role of DAZ in human spermatogenesis. Our findings, however, indicate that the majority of sites in DAZ are subject to purifying selection. This notion is supported by the observation of intact reading frames for all the sampled exons of DAZ; there are no frameshift mutations or premature stop codons. Moreover, complementation of sterile-phenotype DAZL1 mice by human DAZ strongly suggests a functional role for human DAZ in spermatogenesis (Slee et al. 1999
). These observations, taken together with expression patterns of DAZ, lead us to conclude that DAZ is not free from functional constraints in primates and that the DAZ gene is likely to have functional importance in human spermatogenesis.
The pairwise approach used by Agulnick et al. (1998)
is the most common method of computing synonymous and nonsynonymous rates. This approach, however, averages rates over all sites and also over the entire time interval that separates a pair of sequences. Agulnick et al. (1998)
did not observe dN/dS ratios in excess of 1 in most comparisons because evolution by positive selection occurred at a subset of sites and only in certain lineages of DAZ. This example is not unique, as the pairwise approach also failed to detect positive selection in HIV (Leigh Brown 1997
; Crandall et al. 1999
; Zanotto et al. 1999
). Moreover, the same effect led to the incorrect conclusion that the
-casein gene was free from functional constraint (Ward, Honeycutt, and Derr 1997
). These studies indicate that for some proteins the traditional approach to estimating the dN/dS ratio might not provide a sensible measure of selective pressure.
Gene duplication is considered an important mechanism for functional divergence (Ohno 1970
; Ohta 1993
). However, the process by which duplicated genes acquire new functions is less clear. There is often an acceleration of the rate of evolution following gene duplication (Li 1985
; Ohta 1993, 1994
). Accelerated rates could initially be driven by positive Darwinian selection for functional divergence (Ohta 1993, 1994
) or by relaxation of selective constraints. In the latter case, it is thought that random fixation of neutral changes eventually leads to a novel function in one or both copies. This model was referred to as the "Dykhuizen-Hartl effect" by Zhang, Rosenberg, and Nei (1998)
. Our findings are consistent with both models. The elevated rate of nonsynonymous substitution in autosomal DAZL1 following its duplication appears to result from the action of positive Darwinian selection. However, elevated rates of nonsynonymous substitution in DAZ immediately following its origin via the translocation event appear to result from decreased levels of purifying selection, suggesting a possible role for the Dykhuizen-Hartl effect in the early stages of DAZ evolution.
Our findings are consistent with several studies of gene families in which positive Darwinian selection was shown to be at least partially responsible for a rate increase following gene duplication (Ohta 1993, 1994
; Zhang, Rosenberg, and Nei 1
998; Duda and Palumbi 1999
; Rooney and Zhang 1999
; Schmidt, Goodman, and Grossman 1999
). However, in the only other case in which the relative contribution of both models was investigated, the Dykhuizen-Hartl effect was ruled out (Zhang, Rosenberg, and Nei 1998
). Although in this respect our findings appear to differ, it is important to point out that the method we employed to accommodate variation in selective pressures among lineages calculated
as an average across all sites. Because an episode of positive selection at a subset of sites could elevate
at a specific branch without causing it to exceed 1, it is not possible to completely rule out the positive- selection model. More complex models which can simultaneously accommodate rate variation among sites and lineages are under development and might be useful in distinguishing between positive selection and the Dykhuizen-Hartl effect.
Darwinian selection appears to be a relatively common feature of mammalian reproductive proteins (Karn and Nachman 1999
; Rooney and Zhang 1999
; Wyckoff, Wang, and Wu 2000
). Our findings indicate the DAZ gene family represents another example of this pattern. However, not all lineages of the DAZ gene family are presently evolving by positive Darwinian selection; both DAZ and DAZL1 of M. fascicularis are evolving by purifying selection. With regard to the difference between humans and M. fascicularis, it is interesting to note that the mature DAZ protein of humans has only 1 processed copy of exon 8, whereas the mature DAZ protein of M. fascicularis has 10 processed copies of exon 8. The DNA sequence for DAZ in both species contains multiple copies of both exons 7 and 8. However, at some point in the human lineage, a mutation disabled the splice sites of exons 8A and 8D. Because all but one copy of exon 8 in present-day human DAZ are descended from the disabled copies of exons 8A and 8D, the mature DAZ protein of humans includes only one processed copy of exon 8 (Gromoll et al. 1999
). Because the open reading frame was preserved in M. fascicularis despite several duplication and rearrangement events, multiple copies of exons 7 and 8 must have evolved functional importance during divergence of this gene family (Gromoll et al. 1999
). It is tempting to speculate that in the human lineage a loss of processing of all but one copy of exon 8 initiated adaptive evolution at other sites in both DAZ and DAZL1 to maintain proper spermatogenesis. Additional sequences of DAZ and DAZL1 from a variety of primate species are needed to understand the role of positive selection in functional divergence of the DAZ gene family.
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Acknowledgements |
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Footnotes |
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1 Keywords: DAZ
DAZL1
gene family
maximum likelihood
codon model
positive selection
2 Address for correspondence and reprints: J. P. Bielawski, Department of Biology, University College London, 4 Stephenson Way, London NW1 2HE, United Kingdom. j.bielawski{at}ucl.ac.uk
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literature cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Agulnik, A. I., A. Zharkikh, H. Boettger-Tong, T. Bourgeron, K. McElreavey, and C. E. Bishop. 1998. Evolution of the DAZ gene family suggests that Y-linked DAZ plays little, or a limited, role in spermatogenesis but underlines a recent African origin for human populations. Hum. Mol. Genet. 7:13711377
Cooke, H. J., M. Lee, S. Kerr, and M. Ruggiu. 1996. A murine homologue of the human DAZ gene is autosomal and expressed only in male and female gonads. Hum. Mol. Genet. 5:513516
Crandall, K. A., C. R. Kelsey, H. Imanichi, H. C. Lane, and N. P. Salzman. 1999. Parallel evolution of drug resistance in HIV: failure of nonsynonymous/synonymous substitution rate ratio to detect selection. Mol. Biol. Evol. 16: 372382
Duda, T. F., and S. R. Palumbi. 1999. Molecular genetics of ecological diversification: duplication and rapid evolution of toxin genes of the venomous gastropod Conus. Proc. Natl. Acad. Sci. USA 96:68206823
Dykhuizen, D., and D. L. Hartl. 1980. Selective neutrality of 6PGD allozymes in E. coli and the effects of genetic background. Genetics 96:801817
Elliott, D. J., and H. J. Cooke. 1997. The molecular genetics of male infertility. Bioessays 19:801809
Ferlin, A., E. Moro, A. Garolla, and C. Foresta. 1999. Human male infertility and Y chromosome deletions: role of the AZF- candidate genes DAZ, RBM and DFFRY. Hum. Reprod. 14:17101716
Goldman, N., and Z. Yang. 1994. A codon based model of nucleotide substitution for protein-coding DNA sequences. Mol. Biol. Evol. 11:725736
Gromoll, J., G. F. Weinbauer, H. Skaletsky, S. Schlatt, M. Rocchietti-March, D. C. Page, and E. Nieschlag. 1999. The Old World monkey DAZ (Deleted in AZoospermia) gene yields insights into the evolution of the DAZ gene cluster on the human Y chromosome. Hum. Mol. Genet. 8: 20172024
Karn, R. C., and M. W. Nachman. 1999. Reduced nucleotide variability at an androgen-binding protein locus (Abpa) in house mice: evidence for positive natural selection. Mol. Biol. Evol. 16:11921197[Abstract]
Kimura, M. 1983. The neutral theory of molecular evolution. Cambridge University Press, Cambridge, England
Kumar, S., and B. Hedges. 1998. A molecular timescale for vertebrate evolution. Nature 392:917920
Leigh Brown, A. J. 1997. Analysis of HIV-1 env gene reveals evidence for a low effective number in the viral population. Proc. Natl. Acad. Sci. USA 94:18621865
Li, W.-H. 1985. Accelerated evolution following gene duplication and its implications for the neutralist-selectionist controversy. Pp. 333352 in T. Ohta and K. Aoki, eds. Population genetics and molecular evolution. Japan Scientific Press, Tokyo
Menke, D. B., G. L. Mutter, and D. C. Page. 1997. Expression of DAZ, an azoospermia factor candidate, in human spermatogonia. Am. J. Hum. Genet. 60:237241[ISI][Medline]
Nielsen, R., and Z. Yang. 1998. Likelihood models for detecting positively selected amino acid sites and applications to the HIV-1 envelope gene. Genetics 148:929936
Ohno, S. 1970. Evolution by gene duplication. Springer-Verlag, Berlin
Ohta, T. 1993. Pattern of nucleotide substitution in growth hormone-prolactin gene family: a paradigm for evolution by gene duplication. Genetics 134:12711276
. 1994. Further examples of evolution by gene duplication revealed through DNA sequence comparisons. Genetics 138:13311337
Rooney, A. P., and J. Zhang. 1999. Rapid evolution of primate sperm protein: relaxation of functional constraint or positive Darwinian selection? Mol. Biol. Evol. 16:706710[Abstract]
Ruggiu, M., R. Speed, M. Taggart, S. J. McKay, F. Kilanowski, P. Saunders, J. Dorin, and H. J. Cooke. 1997. The mouse DAZL1a gene encodes a cytoplasmic protein essential for gametogenesis. Nature 389:7377
Saxena, R., L. G. Brown, T. Hawkins et al. (11 co-authors). 1996. The DAZ gene cluster on the human Y chromosome arose from an autosomal gene that was transposed, repeatedly amplified and pruned. Nat. Genet. 14:292299[ISI][Medline]
Saxena, R., J. W. A. de Vries, S. Repping, R. K. Alagappan, H. Skaletsky, L. G. Brown, P. Ma, E. Chen, J. M. N. Hoovers, and D. C. Page. 2000. Four DAZ genes in two clusters found in the AZFc region of the human Y chromosome. Genomics 67:256267
Schmidt, T. R., M. Goodman, and L. I. Grossman. 1999. Molecular evolution of the COX7A gene family in primates. Mol. Biol. Evol. 16:619626[Abstract]
Shinka, T., and Y. Nakahori. 1996. The azoospermic factor on the Y chromosome. Acta Paediatr. Jpn. 38:399404[Medline]
Slee, R., B. Grimes, R. M. Speed, M. Taggart, S. M. Maguire, A. Ross, N. I. McGill, P. T. Saunders, and H. J. Cooke. 1999. A human DAZ transgene confers partial rescue of the mouse Dazl null phenotype. Proc. Natl. Acad. Sci. USA 96:80408045
Swofford, D. L. 2000. PAUP*. Phylogenetic analysis using parsimony (*and other methods). Version 4. Sinauer, Sunderland, Mass
Ward, T. J., R. L. Honeycutt, and J. N. Derr. 1997. Nucleotide sequence evolution at the kappa-casein locus: evidence for positive selection within the family Bovidae. Genetics 147:18631872
Watanabe, Y., S. Shinozaki-Yabana, Y. Chikashige, Y. Hiraoka, and M. Yamamoto. 1997. Phosphorylation of RNA-binding protein controls cell cycle switch from mitotic to meiotic in fission yeast. Nature 386:187190
Wycoff, G. J., W. Wang, and C.-I. Wu. 2000. Rapid evolution of male reproductive genes in the descent of man. Nature 403:304309
Yang, Z. 1994a. Estimating the pattern of nucleotide substitution. J. Mol. Evol. 39:105111
. 1994b. Maximum likelihood phylogenetic estimation from DNA sequences with variable rates over sites: approximate methods. J. Mol. Evol. 39:306314
. 1998. Likelihood ratio tests for detecting positive selection and application to primate lysozyme evolution. Mol. Biol. Evol. 15:568573[Abstract]
. 1999. Phylogenetic analysis by maximum likelihood (PAML). Version 2. University College London, England
Yang, Z., and R. Nielsen. 1998. Synonymous and nonsynonymous rate variation in nuclear genes of mammals. J. Mol. Evol. 46:409418[ISI][Medline]
. 2000. Estimating synonymous and nonsynonymous substitution rates under realistic evolutionary models. Mol. Biol. Evol. 17:3243
Yang, Z., R. Nielsen, N. Goldman, and A.-M. K. Pederson. 2000. Codon-substitution models for heterogeneous selection pressure at amino acid sites. Genetics 155:431449
Zanotto, P. M. de A., E. G. Kallas, R. F. de Souza, and E. C. Holmes. 1999. Genealogical evidence for positive selection in the nef gene of HIV-1. Genetics 153:1077 1089
Zhang, J., H. F. Rosenberg, and M. Nei. 1998. Positive Darwinian selection after gene duplication in primate ribonuclease genes. Proc. Natl. Acad. Sci. USA 95:37083713