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Four Evolutionary Strata on the Human X Chromosome

Author(s): Bruce T. Lahn and David C. Page

Source: Science , Oct. 29, 1999, New Series, Vol. 286, No. 5441 (Oct. 29, 1999), pp. 964-967

Published by: American Association for the Advancement of Science

Stable URL: https://www.jstor.org/stable/2899501

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964

mann, J. R. Ecker, Ce// 72, 427 (1993)] (23). The largest of 16 independent NPH3 cDNAs was se­ quenced (24) completely (GenBank accession num­ ber AF180390).

11. GenBank searches were accomplished with the gapped BLAST program [S. F. Altschul et al., Nucleic Acid Res. 25, 3389 (1997)].

12. The data are available at www.sciencemag.org/ feature/data/ 10423 58.shl

13. Single-letter abbreviations for the amino acid resi­ dues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

14. T. Patschinsky, T. Hunter, F. S. Esch, J. A. Cooper, B. M. Sefton, Proc. Natl. Acad. Sci. U.S.A. 79, 973 (1982).

15. The BTB/POZ domain was identified with SMART [J. Schultz, F. Milpetz, P. Bork, C. P. Ponting, Proc. Natl. Acad. Sci. U.S.A. 95, 5857 (1998)]. The coiled-coil structure was identified with COILS [A. Lupas, M. Van Dyke, J. Stock, Science 252, 1162 (1991)].

16. 0. Albagli, P. Dhordain, C. DeWeindt, G. LeCocq, D. LePince, Cell Growth Differ. 6, 1193 (1995); L. Ara­ vind and E. V. Koonin,J. Mo/. Biol. 285, 1353 (1999).

17. C. Cohen and D. A. D. Parry, Proteins 7, 1 (1990); A. Lupas, Trends Biochem. Sci. 21, 375 (1996).

18. Structural analyses were performed with the Protean program (DNASTAR, Madison, WI).

19. S. Fields, Methods 5, 116 (1993); S. Fields and R. Sternglanz, Trends Genet. 10, 286 (1994).

REPORTS

20. M. Nagao and K. Tanaka, J. Biol. Chem. 267, 17925 (1992).

21. M. C. Faux and J. D. Scott, Cell 85, 9 (1996); T. Pawson and J. D. Scott, Science 278, 2075 (1997); E. A. Elion, Science 281, 1625 (1998).

22. S. D. Choi, R. Creelman, J. Mullet, R. A. Wing, Weeds World 2, 17 (1995), http://genome-www.stanford. edu/ Arabidopsis/ww/home.html.

23. J. Sambrook, E. F. Fritsch, T. Maniatis, Molecular Clon­ ing: A laboratory Manual (Cold Spring Harbor Labo­ ratory Press, Plainview, NY, 1989).

24. Sequencing templates were prepared by polymerase chain reaction and sequenced with an ABl377 auto­ mated sequencer (Perkin-Elmer, Norwalk, CT).

25. Phototropism and hypocotyl growth was assayed as described previously [E. L. Stowe-Evans, R. M. Harper, A. V. Motchoulski, E. Liscum, Plant Physio/. 118, 1265 (1998)].

26. E. Liscum and R. P. Hangarter, Plant Cell 3, 685 (1991).

27. J. W. Reed, P. Nagpal, D. S. Poole, M. Furuya, J. Chory, Plant Cell 5, 147 (1993).

28. C. Bell and J. R. Ecker, Genomics 19, 137 (1994). 29. H.-G. Nam et at., Plant Cell 1, 699 (1989). 30. Information about markers AM40 and AM80 is available

at http:/ /www.biosci.missouri.edu/liscum/newmarkers. html

31. Y. Nakamura et al., DNA Res. 4, 401 (1997); http:// www.kazusa.or.jp/arabi/chr5/map/24-26Mb.html.

32. Soluble and total microsomal membrane fractions

Four Evolutionary Strata on the Human X Chromosome

Bruce T. Lahn* and David C. Paget

Human sex chromosomes evolved from autosomes. Nineteen ancestral auto­ somal genes persist as differentiated homologs on the X and Y chromosomes. The ages of individual X-Y gene pairs (measured by nucleotide divergence) and the locations of their X members on the X chromosome were found to be highly correlated. Age decreased in stepwise fashion from the distal long arm to the distal short arm in at least four "evolutionary strata." Human sex chromosome evolution was probably punctuated by at least four events, each suppressing X-Y recqmbination in one stratum, without disturbing gene order on the X chromosome. The first event, which marked the beginnings of X-Y differenti­ ation, occurred about 240 to 320 million years ago, shortly after divergence of the mammalian and avian lineages.

The human X and Y chromosomes, like those of other animals, are thought to have evolved from an ordinary pair of autosomes (J). The pseudoautosomal regions at the termini of the X and Y chromosomes still recombine during male meiosis, ensuring X-Y nucleotide se­ quence identity there. Elsewhere on the X and Y chromosomes, however, X-Y recombination has been suppressed. These nonrecombining regions of the X and Y chromosomes have become highly differentiated during evolution, and only a few X-Y sequence similarities per-

Howard Hughes Medical Institute, Whitehead Insti­ tute, and Department of Biology, Massachusetts In­ stitute of Technology, 9 Cambridge Center, Cam­ bridge, MA 02142, USA.

*Present address: Department of Human Genetics, University of Chicago, 924 East 57th Street, Chicago, IL 60637, USA. tTo whom correspondence should be addressed. E­ mail: [email protected]

sist within them. These modem X-Y gene pairs are the remaining "fossils" where extensive se­ quence identity between ancestral X and Y chromosomes once existed. The recent discov­ ery of many X-Y genes has made it possible to examine the entire group to search for patterns of human sex chromosome evolution. Thus far, the human sex chromosomes-the best charac­ terized mammalian sex chromosomes-have been found to contain 19 X-Y gene pairs (2).

We first compared the locations of all 19 pairs of genes on the human X and Y chromo­ somes (Fig. I). We determined the relative positions of the X-linked genes through radia­ tion hybrid analysis, in many cases confirming previously published localizations (3). Map po­ sitions of the Y-linked homologs were obtained principally from the literature (4-6). On the X chromosome, most of the X-Y genes map to the short arm, where they are concentrated toward the distal end. By contrast, the X-Y genes are

were separated by ultracentrifugation, followed by two-phase partitioning to enrich for plasma mem­ branes, as described previously [T. W. Short, P. Rey­ mond, W. R. Briggs, Plant Physio/. 101, 647 (1993)].

33. Antibodies against NPH1 were previously described (7). Rabbit polyclonal antisera were raised (22) against a COOH-terminal NPH3 fusion protein [CBD­ NPH3C2 (see Fig. 3A)]. CBD-NPH3 protein was ex­ pressed from pET34-Ek/LIC in Escherichia coli and purified according to manufacturer's instructions (Novagen, Madison, WI).

34. NPH1-NPH3 interaction was examined in yeast with the Matchmaker Gal4 II System (Clontech, Palo Alto, CA). Expression of fusion peptides was verified by immunoblot analysis (9, 22) with monoclonal anti­ bodies raised against the Gal4 DNA binding domain (GBD) and Gal4 activation domain (GAD) (Clontech).

35. J. H. Miller, Experiments in Molecular Genetics (Cold Spring Harbor Laboratory, Plainview, NY, 1972).

36. We thank R. Harper for data in Fig. 1; J. M. Christie and W.R. Briggs for GBD-NPH1 constructs and NPH1 antisera; D. Randall for production of NPH3 antisera; the Arabidopsis Biological Resource Center in Colum­ bus, Ohio, for BAC clones and cDNA libraries; and members of our laboratory for helpful comments on the manuscript. This work was funded by USDA National Research Initiative grant 96-35304-3709, NSF grant MCB-9723124, and University of Missouri Research Board grant RB96-055.

3 June 1999; accepted 17 September 1999

found as singletons or small clusters throughout the euchromatic portion of the Y chromosome. In general, the map order of the X-linked genes corresponds poorly to that of the Y-linked ho­ mologs. Local exceptions to this rule are pro­ vided by three small gene clusters that are present on both X and Y chromosomes (Fig. I).

We next measured, for each of the 19 X-Y gene pairs, synonymous nucleotide divergence between the X-linked and Y-linked coding re­ gions (7). Because synonymous substitutions do not alter the encoded protein, they are gen­ erally assumed to be nearly neutral with respect to selection. The statistic Ks (the estimated mean number of synonymous substitutions per synonymous site) is often used to gauge evolu­ tionary time ( 8). In the present context, Ks values provide a measure of the evolutionary time that has elapsed since the gene pairs start­ ed differentiating into distinct X and Y forms. The calculated Ks values are given in Table I, where gene pairs are listed according to map order on the X chromosome.

We noted that the 19 Ks values appeared to cluster into approximately four groups (Fig. 2): 0.94 to 1.25 (group I), 0.52 to 0.58 (group 2), 0.23 to 0.36 (group 3), and 0.05 to 0.12 (group 4). Each X-Y gene pair's Ks value differed significantly from those of all gene pairs in other groups (P :5 0.02). The most striking observation was that, on the X chromosome, the four Ks-defined groups of genes are ar­ ranged in an orderly sequence (Fig. 2). X-Y genes are stratified by age along the length of the X chromosome. By contrast, on the Y chro­ mosome, the Ks-defined groups appear to be scrambled (compare Table I and Fig. I).

What might account for the orderly stratifi­ cation of X-Y genes by age on the human X chromosome? We hypothesize that, during evo-

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lution, differentiation of the X from the Y chro­ mosome was initiated one region, or stratum, at a time. Regions were recruited in the order of their physical position, with stratum I ( contain­ ing the genes of group I) having been the first to embark on X-Y differentiation, and stratum 4 having been the most recent. Genes in the same stratum began differentiating into X and Y ho­ mologs at about the same time, accounting for their similar Ks values.

X-Y differentiation would have occurred only after X-Y recombination ceased (9). Our findings suggest that during evolution, X-Y re­ combination was suppressed regionally, begin­ ning with stratum I and subsequently expanding in discrete steps to include strata 2, 3, and 4. Chromosomal inversions, which are known to be capable of suppressing recombination across broad regions in mammals ( J 0), would appear to be the most likely mechanism. These inversions must have occurred on the evolving Y chromo­ some, where the strata have been scrambled, but not on the X chromosome, where the order of strata apparently has been preserved (Figs. I and 2). [Had the strata on the human X chromosome been extensively shuffled during evolution-as may have occurred on the mouse X chromosome after divergence of the human and murine lin­ eages (J J)-we would have observed no corre­ lation between the age ofX-Y gene pairs and the map positions of their X-chromosomal mem­ bers.] In the modern human sex chromosomes, the proximal boundary of the pseudoautosomal region is spanned by a gene that is intact on the X chromosome, but grossly interrupted on the Y chromosome (12), consistent with disruption of

REPORTS

an ancient pseudoautosomal region by a Y-chro­ mosomal inversion. We speculate that this par­ ticular event was the most recent in a series of inversions, each of which enabled X-Y differen­ tiation to begin in one stratum.

This model of staged, region-by-region ini­ tiation of X-Y differentiation also accounts for two global features of the X chromosome's gene content: (i) the concentration in strata 3 and 4 of genes with detectable Y homologs (Fig. I) and (ii) the concentration on the short arm (strata 2, 3, and 4) of genes that escape X inactivation, some with and some without Y homologs (13). Evolutionary theory predicts that once X-Y recombination ceased within a stratum, the genes on the affected portion of the Y chromosome began to decay, with most of the Y-linked genes ultimately being obliterated (J). As an adaptive response, homologous genes on the X chromosome were up-regulated, and subsequently became subject to X inactiva­ tion, processes thought to have spread during evolution on a gene-by-gene or cluster-by-clus­ ter basis (14). If decay of Y-linked genes and adaptation of X-linked homologs were gradual evolutionary processes, then one would expect the youngest X strata to exhibit the highest densities of (i) genes with detectable Y ho­ mologs and (ii) genes that escape inactivation. Both predictions are met (Fig. I) (13).

A comparison of the youngest (group 4) gene pairs with the older (groups I through 3) gene pairs illustrates certain temporal features of X-Y differentiation. We measured both synon­ ymous and nonsynonymous substitutions for each gene pair (Table I). Nonsynonymous sub-

Table 1. Sequence divergence between homologous X- and Y-linked genes.

DNA Protein Sequence Gene pair Ks KA Ks/KA divergence divergence compared

(%) (%) (nucleotides)

Group 4 GYG2/GYG2P* 0.11 0.06 1.8 7 12 525 ARSDIARSDP* 0.09 0.07 1.3 7 13 846 ARSEI ARSEP* 0.05 0.04 1.2 4 9 615 PRKXIY 0.07 0.03 2.3 5 8 1020 STSISTSP* 0.12 0.10 1.2 11 18 852 KAL1/KALP* 0.07 0.06 1.2 6 12 1302 AMELXIY 0.07 0.07 1.0 7 12 576

Group 3 TB4XIY 0.29 0.04 7.3 7 7 135 EIF7AX/Y 0.32 0.01 32 9 2 432 ZFXIY 0.23 0.04 5.8 7 7 2394 DFFRXIY 0.33 0.05 6.6 11 9 7671 DBXIY 0.36 0.04 9.0 12 9 1932 CASK/CASKP* 0.24 0.22 1.1 15 32 156 UTXIY 0.26 0.08 3.3 12 15 4068

Group 2 UBE7XIY 0.58 0.07 8.3 16 13 693 SMCXIY 0.52 0.08 6.5 17 15 4623

Group 7 RPS4XIY 0.97 0.05 19 18 18 792 RBMXIY 0.94 0.25 3.8 29 38 1188 SOX3/SRY 1.25 0.19 6.6 28 29 264

*Y copy is pseudogene. DNA 'and protein divergence refer to uncorrected nucleotide (coding region) and amino acid divergence (nonidentity).

stitutions alter the encoded protein and are con­ strained by selection. Thus, their frequency (KA, the estimated mean number of nonsynonymous substitutions per nonsynonymous site) is a func­ tion of both evolutionary time and selective constraints on the encoded proteins. The degree

X

2

1

GYG2] ARSD a ARSE PRKX STS ]b KAL1 AMELX TB4X E/F1AX ZFX

DFFRX] DBX CASK c UTX UBE1X

SMCX

RPS4X

SRY RPS4Y

ZFY

y

PRKY AMELY

[ARSEP a ARSDP

GYG2P

[DFFRY OBY

c CASKP UTY

TB4Y

b[ KALP STSP·

SMCY

EIF1AY

RBMY

Fig. 1. Map of homologous genes in nonrecombining re­ gions of human X and Y chro-

RBMX mosomes. Pseudoautosomal re- SOX3 gions of X and Y are black; het­

erochromatic region of Y is gray. Radiation hybrid analysis (3) was used to map genes on the X chromosome, which is drawn on a centiRay scale. Ks-defined stra-

ta on the X chromosome are indicated. The boundary between strata 2 and 1 is somewhere between SMCX and RPS4X; here, it is arbitrarily shown at the centromere (white oval). Genes and pseudogenes on the Y chromosome were ordered previously by analysis of naturally occurring de­ letions (4, 5). UBE1 X has a homolog on the squir­ rel monkey Y chromosome but not on the human Y chromosome (29). Brackets denote three small gene clusters {labeled a, b, c) that are present on both X and Y chromosomes.

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966

1.25

Group 1

1.00

0 .75

Ks Group2

0.50

0.25 Group3

p cen q X ch romosome map position

Fig. 2. Plot of Ks (Table 1) versus X-chromosome map position (Fig. 1) for 19 X-Y gene pairs.

of constraint can be reflected in the ratio Ksf KA; values greater than one indicate the presence of constraints on both homologs, and values in the vicinity of one are consistent with lack of con­ straint on at least one homolog (8, 15). In groups 1 through 3, 10 of 11 gene pairs exhibit KsfKA ratios of 3 or higher (Table 1), suggesting that natural selection has preserved the Y copies of these genes . Without such selection, these X-Y homologies (especially those in groups 1 and 2) would no longer be visible. By contrast, the seven gene pairs in group 4 show KsfKA ratios of 1 to 2, and in five of these pairs, the Y copy is known to be a pseudogene . Among the group 4 pairs, X-Y homology is readily apparent even in the absence of selective constraint, because there has been little time for erosion of sequence similarity. Thus, the Y -chromosomal genes of the older groups, and especially those of groups

300

~ .. ., 250 >-

15 "' C:

200 ~ g .,

150 E . ., ., g 100 ., e ., .2: so 'O :>;- X

0 .08 0 .99 Ks

/ Fig. 3. Plot of X-Y divergence time (age) versus average Ks value for X-Y gene pairs (weight­ averaged) in each stratum. The X chromosome schematic is adapted from Fig. 1. Maximum and minimum age estimates for strata 2, 3, and 4 are bracketed; these are not statistical confidence intervals. Theory predicts an approximately linear relationship between age and Ks value (8); the shaded area is calibrated with respect to stratum 2, whose age is 130 to 170 million years (21) and whose average Ks value is 0.53. By extrapolation, the age of stratum 1 is estimated between 240 and 320 million years.

REPORTS

1 and 2, are survivors of an early winnowing process that is still ongoing in group 4.

To determine the age of the Ks-defined stra­ ta, we used two methods . First, we considered published information on homologs of represen­ tative genes in diverse mammals. The maximum age of stratum 4, for example, was suggested by the prior observation that homologs of STS and KALI are pseudoautosomal or autosomal in pro­ simians (16-18). Assuming that suppression of X-Y recombination is an irreversible evolution­ ary step (14) , this implies that X-Y differentia­ tion in stratum 4 began less than 50 million years ago (Ma), when the simian and prosimian lineages diverged (J 9). Minimum ages of the strata could also be inferred. For example, STS and KALI have been shown to have X- and Y-specific homologs in both New and Old World monkeys (16, 17), suggesting that X-Y differentiation in stratum 4 began at least 30 Ma, when the New and Old World monkey lineages diverged (19, 20). Using similar logic, we in­ ferred the ages of stratum 3 (80 to 130 million years), stratum 2 (130 to 170 million years), and stratum 1 (130 to 350 million years) from prior data on gene homologs in more-distantly related species, including nonprimate mammals, mar­ supials, monotremes , and birds (21) .

These cross-species comparisons yielded reasonably precise estimates of age for strata 2, 3, and 4-the younger strata-but only crude estimates of age for stratum 1. Because this oldest stratum might contain information about the origins of mammalian sex chromosomes, its age is of great interest. Here, we used a second

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dating method, based on Ks values for X-Y gene pairs. Theory predicts that among human X-Y gene pairs, Ks values should be roughly propor­ tional to age ( 8). This expectation is met by the X-Y gene pairs of strata 2, 3, and 4 (Fig . 3). By extrapolation, we estimated that X-Y differenti­ ation began 240 to 320 Ma in stratum 1 (Fig . 3). These findings suggest that X-Y divergence be­ gan shortly after the mammalian lineage arose, having diverged from the lineage of birds (with Z-W sex chromosomes) between 300 and 350 Ma (19). [Because the sex chromosomes of birds appear to be completely unrelated to the mammalian sex chromosomes, it is thought that they arose independently, from a different auto­ somal pair (22).] Interestingly, our Ks findings indicate that SOX3 and SRY (the primary sex­ determining gene) are among the oldest known X-Y gene pairs in humans (Table 1). This find­ ing strengthens an hypothesis, by Foster and Graves , which states that an ordinary autosomal pair became sex chromosomes when mutations fashioned one allele of SOX3, originally an au­ tosomal gene, into the male-determining factor SRY (23). Indeed, formal cluster analysis of the Ks values we report suggests that the X-Y genes of group 1 might actually comprise two distinct strata, with SRY!SOX3 perhaps being older than the two other X-Y gene pairs of group 1 (RPS4XIY and RBMXIY) (24). Although the dif­ ference in Ks values between SRY!SOX3 and the two other X-Y gene pairs is not statistically significant, the evidence is suggestive .

If future studies establish that the group 1 genes are divisible into two strata, these results

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Autosome X y X y X y X y XY

! ! ! ! humans

Autosome XYin XYin XY in non-simian in birds monotremes marsupials placental mammals

Fig. 4. A proposed sequence of evolutionary events that generated four strata on the human X chromosome. Four inversions on the Y chromosome are postulated . Each inversion reduced the size of the pseudoautosomal ( X-Y recombining) region (black; for simplicity, only one pseudoautosomal region is shown for each chromosome) and enlarged the portions of the X (yellow) and Y (blue) chromosomes that did not recombine during male meiosis. Ongoing decay and loss of Y genes offset these periodic expansions of the nonrecombining region of the Y chromosome. Points of divergence from the sex chromosomes of other mammals are indicated. This model does not preclude the occurrence of (i) additional inversions or other rearrangements within the nonrecom ­ bining portion of the evolving Y chromosome or (ii) similar rearrangements on the evolving X chromosome, so long as they do not disturb the fundamental order among the four strata.

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would also help date the emergence of X inac­ tivation during mammalian sex chromosome evolution. XIST, an X-specific gene which plays a pivotal role in X inactivation (25), is located near RPS4X and therefore would be in the younger of the two strata~not in the stratum where the nascent X and Y chromosomes first differentiated. This would controvert the hypoth­ esis of Chandra, who speculated that X inactiva­ tion emerged contemporaneously with the chro­ mosomal sex-determining mechanism (26).

Consistent with our evolutionary map, Graves and colleagues have postulated that the long arm and proximal short arm of the human X chromosome are at least 170 million years old (27, 28). They have referred to this portion of the X as the "XCR" (X conserved region). Graves's XCR corresponds approximately to our strata 1 and 2. They have also postulated that the distal short arm of the human X chro­ mosome is younger. This "XAR" (X added region) was attributed to translocation of an autosome to the pseudoautosomal region of both X and Y after divergence of placental mammals from marsupials (27, 28). Our strata 3 and 4 are found within Graves's XAR.

In conclusion, we postulate that the evolution of human sex chromosomes was punctuated by at least four events, plausibly a series of inver­ sions on the Y chromosome (Fig. 4). Each event suppressed X-Y recombination in one stratum and enabled X-Y differentiation to proceed there. The first of these events, which created stratum 1, was roughly contemporaneous with the birth of the mammalian sex chromosomes and the emergence of SRY as the primary sex determinant. This occurred about 240 to 320 Ma, shortly after the mammalian and avian lineages diverged. The pseudoautosomal region was ex­ panded by translocation of autosomal material between the second and third events (which cre­ ated strata 2 and 3, respectively). The fourth event occurred relatively recently, during pri­ mate evolution, creating stratum 4, where X-Y differentiation is still in its earliest stages.

References and Notes 1. J. J. Bull, Evolution of Sex Determining Mechanisms (Ben­

jamin Cummings, Menlo Park, CA, 1983); J. A. Graves, Annu. Rev. Genet. 30, 233 (1996); B. Charlesworth, Curr. Biol. 6, 149 (1996); W.R. Rice, Bioscience 46, 331 (1996).

2. The 19 X-Y gene pairs studied include the following: GYG2/GYG2P [J. Mu, A. V. Skurat, P. j. Roach,}. Biol. Chem. 272, 27589 (1997); (6)], ARSDIARSDP, ARSE/ ARSEP [G. Meroni et al., Hum. Mo/. Genet. 5, 423 (1996)], PRKX!Y[A. Klink et al., Hum. Mal. Genet. 4,869 (1995); K. Schiebel et al., Hum. Mal. Genet. 6, 1985 (1997)], STSISTSP (16), KAL1/KALP [B. Franco et al., Nature 353, 529 (1991); R. Legouis et al., Cell 67,423 (1991); (77)], AMELXIY [Y. Nakahori, 0. Takenaka, Y. Nakagome, Genomics 9,264 (1991)], TB4XIY [H. Gonda et al.,}. /mmunol. 139, 3840 (1987); (5)], ZFXIY [D. C. Page et al., Cell 51, 1091 (1987); A. Schneider-Gadicke, P. Beer-Romero, L G. Brown, R. Nussbaum, D. C. Page, Cell 57, 1247 (1989)], EIF1AXIY [T. E. Dever et al.,}. Biol. Chem. 269, 3212 (1994); (5)], DFFRXIY [M. H. Jones et al., Hum. Mal. Genet. 5, 1695 (1996); (5)], DBXIY (5), CASK/CASKP [A. R. Cohen et al.,}. Cell Biol. 142, 129 (1998); (6)], UTX/Y(5),SMCXIY[J. Wu eta/., Hum. Mal.

REPORTS

Genet. 3, 153 (1994); A. I. Agulnik et al., Hum. Mal. Genet. 3, 879 (1994)]. RPS4XIY [E. M. Fisher et al., Cell 63, 1205 (1990)]. RBMXIY [M. Soulard et al., Nucleic Acids Res. 21, 4210 (1993); K. Ma et al., Cell 75, 1287 (1993); M. L. Delbridge, P.A. Lingenfelter, C. M. Disteche, J. A. Graves, Nature Genet. 22, 223 (1999); S. Mazeyrat, N. Saut, M. G. Mattei, M. J. Mitchell, Nature Genet. 22, 224 (1999)], SOX3/SRY [M. Stevanovic, R. Lovell-Badge, J. Collignon, P. N. Goodfellow, Hum. Mo/. Genet. 2, 2013 (1993); A. H. Sinclair et al., Nature 346, 240 (1990)]. One interspecies pair was also studied: human UBE1 X [P. M. Handley, M. Mueckler, N. R. Siegel, A. Ciechanover, A. L. Schwartz, Proc. Natl. Acad. Sci. U.S.A. 88, 258 (1991)] and squirrel monkey UBE1Y (29). In humans, UBE1Y was deleted from the Y chromosome (29). We used squirrel monkey UBE1 Y as a substitute.

3. Using polymerase chain reaction (PCR), we tested DNAs from the 93 hybrid cell lines of the GeneBridge 4 panel (Research Genetics) [G. Gyapay et al., Hum. Mo/. Genet. 5, 339 (1996)] for the presence of each of the X-linked genes. PCR conditions and primer sequences have been deposited at GenBank, where accession numbers are as follows: GYG2, G49430; ARSD, G42687; ARSE, G42688; PRKX, G42689; STS, G42690; KAL1, G42691; AMELX, G42692; TB4X, G34979; E/F1AX, G34989; ZFX, G42693; DFFRX, G34982; DBX, G34988; CASK, G49441; UTX, G34976; UBE1X, G42694; SMCX, G42695; RPS4X, AF041428; RBMX, G42696; and SOX3, G42697. Analy­ sis of the results positioned the genes with respect to the radiation hybrid map of the X chromosome con­ structed at the Whitehead/MIT Center for Genome Research [T. J. Hudson et al., Science 270, 1945 (1995); www-genome.wi.mit.edu/cgi-bin/contig/phys_map].

4. D. Vollrath et al., Science 258, 52 (1992). 5. B. T. Lahn and D. C. Page, Science 278, 675 (1997). 6. C. Sun et al., Nature Genet., in press. 7. Homologous X and Y DNA sequences were aligned by

means of MegAlign software (DNASTAR, Madison, WI). For each X-Y gene pair, estimates of the mean numbers of synonymous substitutions per synonymous site (Ks), and of nonsynonymous substitutions per nonsynony­ mous site (KA)-all corrected for multiple changes-were calculated using published algorithms (8) as implemented in GCG software (Genetics Computer Group, Madison, WI). Insertions and deletions were ignored in these cal­ culations. In the case of SOX3 and SRY, sequence similar­ ity is limited to, and our analysis was restricted to, the HMG box domain. Our analyses of other X-Y gene pairs employed all available coding sequences. Only a partial UBE1Y (squirrel monkey) coding sequence was available for comparison with its human X homolog. Sequences for all pseudogenes were extracted from genomic sequences: GYG2P, ARSDP, and ARSEP from BAC (bacterial artificial chromosome) clone 203M13 (GenBank AC002992); STSP from BAC clone NH0494J04 (GenBank AC006382); KALP from BAC clone NH0292P09 (GenBank AC006370); CASKP from BAC clone 47511 (GenBank AC004474). Se­ quences for all other genes were obtained from published cDNAs, whose GenBank accession numbers are as fol­ lows: GYG2, U94362; ARSD, X83572; ARSE, X83573; PRKX, X85545; PRKY, Y15801; STS, M16505; KAL1, M97252; AMELX, M86932; AMELY, M86933; TB4X, Ml 7733; TB4Y, AF000989; ZFX, X59739; ZFY, M30607; EIF1AX, L 18960; E/F1AY, AF000987; DFFRX, X98296; DFFRY, AF000986; DBX, AF000982; DBY, AF000984; CASK, AF032119; UTX, AF000992; UTY, AF000994; UBE1X, M58028; UBE1Y, AJ003105; SMCX, L25270; SMCY, U52191; RPS4X, M58458; RPS4Y, M58459; RBMX, Z23064; RBMY, X76059; SOX3, X71135; SRY, X53772.

8. W. H. Li, }. Mo/. Eva/. 36, 96 (1993); Molecular Evolution (Sinauer Associates, Sunderland, MA, 1997).

9. B. O. Bengtsson and P. N. Goodfellow, Ann. Hum. Genet. 51, 57 (1987).

10. L. M. Silver, Trends Genet. 9, 250 (1993); R.H. Martin et al., Hum. Genet. 93, 135 (1994); M. Jaarola, R. H. Martin, T. Ashley, Am.}. Hum. Genet. 63, 218 (1998).

11. H.J. Blair, V. Reed, S. H. Laval, Y. Boyd, Genomics 19, 215 (1994); W. J. Murphy, S. Sun, Z.-Q. Chen, J. Pecan-Slattery, S. J. O'Brien, Genome Res., in press.

12. P.A. Weller, R. Critcher, P. N. Goodfellow, J. German, N. A. Ellis, Hum. Mo/. Genet. 4, 859 (1995).

13. C. J. Brown, L. Carrel, H. F. Willard, Am.}. Hum. Genet. 60, 1333 (1997).

14. K. Jegalian and D. C. Page, Nature 394, 776 (1998). 15. KsfKA ratios can be depressed by positive selection,

which accelerates protein divergence (8). However, among the X-Y pairs shown here to have relatively low Ks/KA ratios, the abundance of Y pseudogenes ( Table 1) suggests that absence of selective con­ straint is the more significant factor.

16. P.H. Yen et al., Cell 55, 1123 (1988). 17. I. del Castillo, M. Cohen-Salmon, S. Blanchard, G.

Lutfalla, C. Petit, Nature Genet. 2, 305 (1992); B. lncerti et al., Nature Genet. 2, 311 (1992).

18. R. Toder, G. A. Rappold, K. Schiebel, W. Schempp, Hum. Genet. 95, 22 (1995).

19. S. Kumar and S. B. Hedges, Nature 392, 917 (1998); M. j. Benton, Vertebrate Paleontology (Chapman & Hall, New York, 1997).

20. D. Pilbeam, Sci. Am. 250 (no. 3), 84 (1984). 21. Stratum 3: Homologs of ZFX are autosomal in marsupials

(27), which diverged from placental mammals 130 Ma (79). For ZFX/Yand UTX/Y (and for UBE1 X/Y and SMCXIY, in stratum 2), we employed sequence-based phylogenetic analysis to determine if differentiation into X and Y forms had begun before or after mouse/human divergence. For each X-Y gene pair, we used GCG software to construct a phylogenetic tre.e relating human X, human (or mon­ key) Y, mouse. X, and mouse Y homologs. In each of the four cases, the X homologs in human and mouse formed a branch which was distinct from a second branch formed by the Y homologs in human (or monkey) and mouse. These findings suggest that X-Y differentiation of these four gene pairs began before divergence of humans and mice. This is consistent with X-Y divergence having initi­ ated before the placental mammalian radiation that oc­ curred 80 to 100 Ma. Stratum 2: Distinct X- and Y-linked forms of UBE1 have been found in both placental mam­ mals and marsupials [M. J. Mitchell, D. R. Woods, S. A. Wilcox, J. A. Graves, C. E. Bishop, Nature 359,528 (1992)], but their homologs are autosomal in monotremes (29), which diverged from placental mammals and marsupials 170 Ma (79). Stratum 1: Distinct X- and Y-linked forms of RPS4 have been found in placental mammals and mar­ supials (K. Jegalian and D. C. Page, unpublished results), but their homologs are autosomal in birds, whose lineage diverged from that of mammals 300 to 350 Ma (79). Y-specific SRY sequences have been identified in both placental mammals and marsupials [J. W. Foster et al., Nature 359, 531 t1992)].

22. A. K. Fridolfsson ·et al., Proc. Natl. Acad. Sci. U.S.A. 95, 8147 (1998).

23. J. W. Foster and J. A. Graves, Proc. Natl. Acad. Sci. U.S.A. 91, 1927 (1994).

24. Dendrograms of the 19 Ks values (Table 1) were con­ structed using five clustering algorithms (average, cen­ troid, Ward's, single linkage, and complete linkage) im­ plemented in )MP statistics software (SAS Institute, Cary, NC). The most significant branch classification (using any of the algorithms) had five clusters corre­ sponding to the four groups shown in Table 1 and Fig. 2, but with group 1 divided into subgroups 1A (SOX3/SRY) and 1 B (RPS4X/Y and RBMXIY). However, the difference in Ks value between SOX3/SRY (1.25 :+: 0.41) and RPS4X/Y (0.97 :+: 0.16) or RBMX/Y (0.94 :+: 0.15) was not statistically significant. At present, any distinction between subgroups 1A and 1 B is tentative.

25. H. F. Willard, Cell 86, 5 (1996). 26. H. S. Chandra, Proc. Natl. Acad. Sci. U.S.A. 82, 6947

(1985). . 27. J. A. Spencer, A. H. Sinclair, J. M. Watson, J. A. Graves,

Genomics 11, 339 (1991). 28. j. A. Graves, Phi/as. Trans. R. Soc. London Ser. B 350,

305 (1995); R. Toder and j. A. Graves, Mamm. Ge­ nome 9,373 (1998); J.M. Watson, j. A. Spencer, J. A. Graves, M. L. Snead, E. C. Lau, Genomics 14, 785 (1992); J. A. Spencer, j. M. Watson, J. A. Graves, Genomics 9, 598 (1991).

29. M. J. Mitchell et al., Hum. Mo/. Genet. 7,429 (1998). 30. We thank F. Lewitter for help with sequence compari­

sons, H. Skaletsky and T. Kawaguchi for help with database searches and analysis of mapping data, and P. Bain, D. Bartel, A. Bortvin, B. Charlesworth, D. Charles­ worth, A. Chess, A. Clark, C. Disteche, G. Fink, S. Gilbert, J. Graves, R. Jaenisch, K. Jegalian, E. Lander, D. Menke, W. Rice, S. Rozen, C. Tilford, and J. Wang for discussions and comments on the manuscript. Supported by NIH grant HG00257.

14 June 1999; accepted 17 September 1999

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