and ScLCD1 ). Indeed, 20.7% (35/169) of the genes that did not confer any DDR phenotypes gave Emb or Ste phenotypes (data not shown). 20. S. Xanthoudakis, T. Curran, EMBO J. 11, 653 (1992). 21.  L. Michaux et al., Genes Chromosomes Cancer 15, 38 (1996). 22.  R. L. Cai, Y. Yan-Neale, M. A. Cueto, H. Xu, D. Cohen, J. Biol. Chem. 275 , 27909 (2000). 23.  F. al-Khodairy, T. Enoch, I. M. Hagan, A. M. Carr, J. Cell Sci. 108, 475 (1995). 24.  S. Muller, C. Hoege, G. Pyrowolakis, S. Jentsch, Nature Rev. Mol. Cell Biol. 2, 202 (2001). 25.  J. C. Venter et al., Science 291, 1304 (2001). 26. E. S. Lander et al., Nature 409, 860 (2001). 27. S. M. Elbashir et al., Nature 411, 494 (2001). 28. M. Vidal, P. Legrain, Nucleic Acids Res. 27, 919. (1999). 29. A. Davy et al., EMBO Rep. 2, 821 (2001). 30.  A. J. M. Walhout, M. Vidal, Nature Rev. Mol. Cell. Biol. 2, 55 (2001). 31. A. F. Demburg, J. Zalevsky, M. P. Colaiacovo, A. M. Villeneuve, Genes Dev.  14, 1578 (2000). 32.  We thank J. Reid, A. J. M. Walhout, J. Lamb, M. Tewari, R. Abraham, and S. van den Heuvel for comments on the
Construction and Analysis of a
Human-Chimpanzee
Comparative Clone Map
Asao Fujiyama,¹.²*† Hidemi Watanabe,¹*† Atsushi Toyoda,¹* Todd D. Taylor,¹* Takehiko Itoh,³* Shih-Feng Tsai,⁴.5* Hong-Seog Park,6* Marie-Laure Yaspo,7* Hans Lehrach,7 Zhu Chen,8* Gang Fu,8* Naruya Saitou,2* Kazutoyo Osoegawa,9 Pieter J. de Jong, 9 Yumiko Suto,¹⁰ Masahira Hattori,¹*† Yoshiyuki Sakaki 1.11*÷ The recently released human genome sequences provide us with reference data to conduct comparative genomic research on primates, which will be important to understand what genetic information makes us human. Here we present a first-generation human-chimpanzee comparative genome map and its initial analysis. The map was constructed through paired alignment of 77,461 chim- panzee bacterial artificial chromosome end sequences with publicly available human genome sequences. We detected candidate positions, including two clusters on human chromosome 21 that suggest large, nonrandom regions of difference between the two genomes. With the draft human genome sequences ( 1 , 2 ), comparative genomics has become a pow- erful approach to extract genetic information from large stretches of nucleotide sequences ¹RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi-ku,  Yokohama,  Kanagawa  230-0045,  Japan. ²National Institute of Genetics, Yata 1111, Mishima, Shizuoka 411-8450, Japan. ³Mitsubishi Research Insti- tute, 3-6, Otemachi 2-chome Chiyoda-ku, Tokyo 100- 8141, Japan. 4National Yang-Ming University, 128, Yen- chiu, Yuan Road, Sec. 2, Taipei 115, Taiwan. "National Health Research Institutes, 128, Yen-chiu, Yuan Road, Sec. 2, Taipei 115, Taiwan. 6Genome Research Center, KRIBB, 52 Oun-dong, Yusong-gu, Daejeon 305-333, Ko- rea.  7Max Planck Institute for Molecular Genetics, Ih- nestrasse 73, D-14195 Berlin-Dahlem, Germany. "Chi- nese National Human Genome Center at Shanghai, 351, Guo Shou Jing Road, Shanghai 201203, China. 2Chil- dren's Hospital Oakland Research Institute, BACPAC Re- sources, 747 52nd Street, Oakland, CA 94609–1809, USA.  ³⁰Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Hongou, Bunkyou-ku, Tokyo 113-0033, Japan. 11Human Genome Center, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. *These authors form the International Consortium for the Sequencing of Chimpanzee Chromosome 22. †To whom correspondence should be addressed. E- mail: afujiyam@gsc.riken.go.jp, watanabe@gsc.riken. go.jp, hattori@gsc.riken.go.jp, sakaki@gsc.riken.go.jp through identification of conserved regions that are most likely functionally important. Genomic information is also the most valu- able resource for understanding the genetic differences between species, a basis for deci- phering how genome information is pro- cessed into phenotypes. Because chimpan- zees are our closest relatives, the differences between us are less than with any other spe- cies, yet these differences are more likely to manuscript; D. Haber for valuable information; members of the Vidal lab for support; and the Caenorhabditis Genetics Center (CGC) for providing worm strains. S.J.B. was supported by Human Frontiers Science Project and Tosteson Postdoctoral Fellowships. A.G. was supported by the Max Planck Society (E. Nigg) and by Deutsche Forschungsgemeinschaft grant 703/1-1. Supported by grants 5R01HG01715-02 (National Human Genome Re- search Institute), PO1CA80111-02 and 7 R33 CA81658- 02 (National Cancer Institute), and 232 (Merck Genome Research Institute) awarded to M.V. 5 September 2001; accepted 30 October 2001 be important. It has been estimated that the sequence identity between human and chim- panzee is within the range of 98 to 99% (3–12). Thus, comparisons between humans and chimpanzees are the most efficient and effective approach to understand what makes us human. In this report we present the construction and analysis of a first-generation human- chimpanzee comparative genomic map based on the alignments of 77,461 chimpanzee bac- terial artificial chromosome (BAC) end se- quences (BESs) to human genomic sequences obtained from the public databases. To pre- pare the BESs, we used two independently prepared BAC libraries, PTB1 and RPCI-43 ( Table 1 ) ( 13 ). Briefly, we sequenced 64,116 BAC clones (roughly 3.3 times coverage of the currently available human contiguous genomic sequence) that produced 114,421 valid BESs ( 13 ). The BESs were then aligned with the RefSeq human genome contigs [Na- tional Center for Biotechnology Information (NCBI)] through NCBI-BLAST. The number of BESs having an alignment longer than 50 base pairs (bp) with ≥90% identity was 77,461 ( 13 ). Out of this number, 49,160 BESs from 24,580 clones formed paired ends where each pair was derived from the same clone. Only one end could be successfully aligned from the remaining 28,301 clones. The remaining 36,960 BESs that were not mapped to the human genome were catego- rized into three different classes: (i) those corresponding to repeat sequences (1168
sequenced (53,240 BESs from PTB1 and 24,221 BESs from RPCI-43 are mapped). ↑Number of total BESs, §Number of mapped BESs derived from the same BAC clone. Long BES pairs (>300 kb) ↑Number of mapped BESs. were not used for the mapping. ||Number of the singleton (lone) BESs mapped on the human genome sequence. ¶Contains hits to sequences not included in the NCBI contigs or hits to mRNAs only. BESs) or showing hits to human sequences not included in the NT contigs (20,376 BESs), (ii) those matched only with sequenc- es from several species other than human (515 BESs), and (iii) the 14,901 BESs that did not match with human sequences, which either correspond to unsequenced human re- gions or are from chimpanzee regions that have diverged substantially from humans or did not match for other unknown reasons. The BESs mapped with high confidence ( 13 ) were used to calculate the difference between the chimpanzee and human genomes at the nucleotide level. The number of sites in valid alignments (nucleotide sites that have PHRED quality values q ≥ 30) was 19,813,086. Out of this number, 19,568,394 sites were identical to their human counter- parts for a mean percent identity of 98.77. This value is consistent with previous obser- vations ( 3 , 9 , 10 ); however, our calculation comes from a much larger random compari- son of slightly less than 1% of the total genome. The distribution of the percent iden- tity of BESs (q ≥ 30) that have ≥ 100 sites per bin is depicted in Fig. 1. The distribution was skewed to 100% identity instead of the normal distribution, and hence the mode of the difference is at around 0.8%. Although most of the BESs have higher identity, we also found the existence of many low-identity BESs in the genome. To construct a human-chimpanzee compar- ative map, we aligned 49,160 paired BESs (practically equivalent to BAC clones) from 24,580 chimpanzee BACs to the RefSeq human genome contigs (build 24, July 2001) construct- ed by NCBI ( 13 ). In addition, 28,301 singleton BAC ends were mapped onto the human ge- nome, even though sequences of the opposite ends were either repeats, low-similarity hits, or not found in the current database ( Table 1 ). The exact positions of these clones will become clearer through the progress of the human ge- nome sequencing and/or the sequencing of the corresponding chimpanzee clones in the near future. The entire map showing the relative positions of the chimpanzee BAC clones (with links to the corresponding human BAC se- quences) along the human genome contigs (NT contigs, NCBI) and LocusLink (NCBI) infor- mation and a comprehensive cross reference table that includes corresponding positions in the human genome and links to the human BAC sequences can be freely accessed through the Web (14). We found that 48.6% of the whole human genome was covered by the chimpanzee BACs (Table 2). One of the reasons for this apparently low coverage is that we used rath- er stringent conditions for the calculation; that is, BAC clones were incorporated into the calculation only when they had two se- quenced ends in the same NT contig with the correct orientation. Probably because the ori- entation of draft sequences within the NT contig is sometimes incorrect, 70% of the total paired ends fit the condition. The cov- erage for chromosomes 14, 20, 21, and 22 was substantially higher. This difference cor- relates closely with the quality of the human genome sequences used as reference where finished chromosomes and those with longer contigs display higher BAC coverage. We also tested 1 Mb of the human draft sequenc- es corresponding to positions 8012178 to 8426236 and 18502342 to 19012063 of chro- mosome 21, which we were able to retrieve from the public portion of the Celera data- base; however, we observed no substantial differences between the mapping results ob- tained through the public database and that of Celera. Theoretically speaking, the probabil- ity of coverage assuming totally random nu- cleotide distribution was calculated at around 0.7 ( 15 ); thus, we concluded that the actual coverage of about 70% for these essentially
↑Number of BESs (paired and single) allocated to the particular chromosome. There are 191 BESs orientation. remaining that are not assigned at this time point. finished chromosomes is reasonable. Rela- tively lower coverage of chromosome X (about half of the other unfinished chromo- somes) can be explained by the haploid status of the chromosome in the chimpanzee BAC libraries. In contrast, we can only speculate as to why the Y chromosome coverage is so much lower (4.8%) as compared with the other chromosomes (Table 2). One possibil- ity is that the pseudoautosomal region of the Y chromosome confounded our matching al- gorithm. Another hypothesis may be that the human Y sequences in the DNA databank are so different that the BESs in this study could not find many valid matches. We will have to wait until sequencing of the human Y chro- mosome is finished to answer some of these questions. Alternatively, we will also need an independent approach, such as the construc- tion and analysis of chimpanzee Y chromo- some–specific libraries prepared from sorted chromosomes, generating a complementary somes. (A, D, and G) Clone names and positions (in parentheses) on human chromosomes determined by the BES analysis (13) [FISH data found in the clone server (www.ncbi.nlm.nih.gov/ genome/clone/) were also used]. (B, E, and H) FISH mapping onto human chromosomes. (C, F, and I) FISH mapping onto chimpanzee chromosomes. Chromosome numbers in the parentheses (expressed in Roman letters) are expressed as phylogenetic chromosome number ( X = 10) (18). Arrowheads designate the positions of hybridization signals. Magnification of each panel is variable. tool to help fully resolve the comparative mapping for the Y chromosome. Because the genomic sequence of human chromosome 21 is finished to a high degree ( 16 ), we analyzed the chimpanzee-human re- lationship of this chromosome by combining the BES mapping information with a se- quence-tagged site (STS)–based approach ( 17 ). We identified 18 STSs that amplified products from human DNA but not from that of chimpanzee (shown as circles in Fig. 2). Because we used genomic DNA isolated from three chimpanzee individuals, two males and one female, the effects of relatively larger polymorphisms among the chimpanzee genomes should be minimized. These 18 primer sets, together with the flanking STSs, were further tested with other primates in- cluding gorilla. Out of these, amplification products appeared exclusively in humans from seven primer pairs (filled green circles in Fig. 2 whose positions in human chr21 are about 7.2, 8.5, 10.0, 11.6, 11.8, 18.1, and 29.3 Mb from the centromeric end, respectively) ( 16 , 17 ), suggesting that these loci might correspond to insertions that are specific to the human lineage. Nonhuman primate spe- cific deletions cannot be ruled out but seem less likely because this deletion would have had to occur in all primates but humans. The remaining 11 primer pairs fail to amplify any products from chimpanzee DNAs but showed positive signals in some of the other primates, suggesting the existence of deletions or mu- tation sites at those positions in the chimpan- zee genome. Although one cannot exclude the possibility that these findings are mere reflection of the statistical variation, the re- gion around these sites remains to be the primary target of further investigations in the future. Simple extrapolation of these results suggests that there might be more than sev- eral hundred such sites in the entire chimpan- zee or human genomes, again including sta- tistical variations. To identify the boundaries of possible genomic rearrangements, we then searched for candidate clones containing chromosomal breakpoints by inspecting the mapping re- sults. If a clone contains one of the break- points of a large genomic rearrangement, the paired BESs of the clone should map to sites separated by an interval longer than expected or the experimentally determined insert size ( 13 ). We identified one such example, clone PTB-053J22. One of the BESs and 62% of the finished sequence of this clone matched (>99%) to the 12q15 human BAC clone (AC005294; clone GSHB-410F4 contained in NT_029419.1), and the other BES and 37% of the sequence matched (>98%) to the 12p12 BAC (AC011604; clone RP11-80N2 contained in NT_009700.5). Sequences of these three clones are in finished status and the location of the human clones has been confirmed by both electron–polymerase chain reaction (e-PCR) and fluorescent in situ hybridization (FISH) analyses (http://ncbi. nlm.nih.gov/genome/clone). In  addition, PTB-053J22 is fully included in another in- dependently sequenced chimpanzee BAC clone (AC007214, RP43-135M19). Thus, it is highly likely that PTB-053J22, which we detected through this study, contains one of the breakpoints corresponding to the human (or vice versa in chimpanzee) chromosomal inversion between 12p12 and 12q15. In ad- dition, this region in human chromosome 12 or in chimpanzee chromosome 10 is known to be inverted in gorilla and chimpanzee as opposed to human and orangutan ( 8 , 18 ). We found several genes around the PTB-053J22 BESs in the following order, SCL21A14 (sol- ute carrier, organic anion transporter, family 21, member 14), <36 kb>, PTB-053J22-F, <5 kb>, SLC21A8 (solute carrier, organic anion transporter, family 21, member 8, <cen>, DYRK2 (dual-specificity tyrosine- Y-phosphorylation regulated kinase 2), <330 kb>, PTB-053J22-R, <168 kb>, and IFNG (interferon-γ), based on the annotations on the corresponding NT contigs. The effect of the inversion on these genes should be the target of future studies. To independently test our mapping proce- dure, we selected 15 chimpanzee BAC clones mapped to human chromosomes 1 to 8 by the BES alignment procedure ( 13 ) and subjected them to FISH analysis with both human and chimpanzee M-phase cell spreads (Fig. 3). As shown, 13 clones showed single locus signals at the corresponding positions on both human and chimpanzee chromosomes, and two clones, RP43-50L24 and RP43-60K09, showed similar signals at two loci on the human and chimpanzee chromosomes, sug- gesting that the mapping procedure we used in this study is reliable. We believe that the whole genome chimpanzee/human compara- tive map built here by the BES alignment procedure is reasonably accurate and useful for future studies. Recent development of the human-mouse comparative map ( 19 , 20 ) also supports our approach. Users of this map should still be careful in applying the information because the possi- bility remains that assignment of particular clones in the NT contig is incorrect or that inter- or intrachromosomally duplicated re- gions may be included within an insert. How- ever, the quality of the map, and thus its usefulness, should increasingly improve as the finishing of the human genome sequence proceeds. References and Notes 1. International Human Genome Sequencing consor- tium, Nature 409, 860 (2001). 2. J. C. Venter et al., Science 291, 1304 (2001). 3. M.-C. King, A. C. Wilson, Science 188, 107 (1975). 4. A. Jauch et al., Proc. Natl. Acad. Sci. U.S.A. 89, 8611 (1992). 5. B. Dutrillaux, Hum. Genet. 48, 251 (1979). 6. W. Burrows, O. A. Ryder, Nature 385, 125 (1997). 7. M. Goodman et al., Mol. Phylogenet. Evol. 9, 585 (1998). 8. E. Nickerson, D. L. Nelson, Genomics 50, 368 (1998). 9.  J. G. Hacia et al., Nature Genet. 18, 155 (1998). 10.  H. Kaessmann, V. Wiebe, S. Paabo, Science 286, 1159 (1999). 11. P. Gagneux, A. Varki, Mol. Phylogenet. Genet. 18, 2 (2001). 12. S. Paabo, Science 291, 1219 (2001). 13. Methods to construct the BAC end sequences (BESs) library and the comparative map can be seen at http://hgp.gsc.riken.go.jp or Science Online (14). All sequence reads were submitted to the DNA Data- bank of Japan (DDBJ) under accession numbers AG029037 to AG186569 and AG186783 to AG187837. 14. The supplemental data are available at hgp.gsc.riken. go.jp or www.sciencemag.org/cgi/content/full/295/ 5552/131/DC1. 15. The probability equation, N — ln(1 — P)/ln(1 — 1/n), where N is the number of clones, in this case 24,580, and n is the total size (2.7 Gb in this case) divided by the size of fractions. The estimated insert size of the BAC libraries is about 130 to 150 kb. The calculated P value is 0.694 (130-kb insert) or 0.745 (150-kb insert). 16. M. Hattori et al., Nature 405, 311 (2000). 17. In total, 752 primer pairs were designed from the hu- man chromosome 21 sequence at distances of about 50 kb (sequences of the primer pairs can be seen at Science Online) (14). For the reactions successful only for hu- man DNA, we expanded the test to include two addi- tional genomic DNA preparations from different indi- viduals of chimpanzee (male and female) and five other primates, including gorilla ( Gorilla gorilla ), orangutan (Pongo pygmaeus), Old World monkey (Macaca fas- cicularis), New World monkey ( Ateles geoffroys ), and prosimian ( Lemur catta ). 18. International System for Human Cytogenetic No- menclature (1985) (ISCN1985) (Karger, Basel, Swit- zerland, 1985), pp. 95–109. 19.  D. Butler, Nature 413, 444 (2001). 20. See http://mouse.ensembl.org. 21. We thank all the members and associates (individual names can be seen at our Web site) of the Human Genome Research Group, RIKEN Genomic Sciences Center, for their enthusiastic support and stimulating discussions. Special thanks to RIKEN GSC and the National Institute of Genetics for supporting the international workshop on chimpanzee genomics, held in Tokyo, March 2001. This work was supported in part by special grants from the Ministry of Educa- tion, Culture, Sports, Science and Technology for Genome Research. 8 August 2001; accepted 13 November 2001
Nucleotide Variation Along the
Drosophila melanogaster Fourth
Chromosome
Wen Wang,¹ Kevin Thornton,² Andrew Berry,³ Manyuan Long¹.²* The Drosophila melanogaster fourth chromosome, believed to be nonrecom- bining and invariable, is a classic example of the effect of natural selection in eliminating genetic variation in linked loci. However, in a chromosome-wide assay of nucleotide variation in natural populations, we have observed a high level of polymorphism in a ~200-kilobase region and marked levels of poly- morphism in several other fragments interspersed with regions of little vari- ation, suggesting different evolutionary histories in different chromosomal domains. Statistical tests of neutral evolution showed that a few haplotypes predominate in the 200-kilobase polymorphic region. Finally, contrary to the expectation of no recombination, we identified six recombination events within the chromosome. Thus, positive Darwinian selection and recombination have affected the evolution of this chromosome. Detecting evolutionary forces that shape the structure of genetic variation at the genomic level often relies on understanding the effects of natural selection on nearby linked loci ( 1–4 ), for which the fourth chromosome of Drosoph- ila melanogaster is a classical model system (5–7). It has been thought to undergo no mei- otic recombination except under certain exper- imental conditions (e.g., the interchromosomal effect introduced for the purpose of mapping) ¹Department of Ecology and Evolution, ²Committee on Genetics, University of Chicago, 1101 East 57 Street, Chicago, IL 60637, USA. ²Museum of Compar- ative Zoology, Harvard University, 26 Oxford Street, Cambridge, MA 02138, USA. *To whom correspondence should be addressed. E- mail: mlong@midway.uchicago.edu ( 8 , 9 ). Two genetic models—"selective sweep" by the hitchhiking effect ( 1 , 10 ), in which an advantageous allele is selected for and fixed in species rapidly, and the background selection model, in which deleterious mutations are se- lected against ( 2 , 10 )—predicted a lack of vari- ation throughout the chromosome, which has been supported by limited data ( 6 , 11 ). We have reexamined the level of variation and recombi- nation in the fourth chromosome using a chro- mosome-wide assay of nucleotide variation. We first investigated within-species nucle- otide variation in two adjacent regions from the 102F cytological position of the fourth chromosome of D. melanogaster: (i) 4257 base pairs (bp) of the CG11091 locus, and (ii) 847 bp of an intron of the toy gene. CG11091 and toy are separated by ~10 kb. We directly