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Science 13 April 2007:
Vol. 316. no. 5822, pp. 243 - 246
DOI: 10.1126/science.1140615
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Reports

Evolutionary Formation of New Centromeres in Macaque

Mario Ventura1*, Francesca Antonacci1*, Maria Francesca Cardone1, Roscoe Stanyon2, Pietro D'Addabbo1, Angelo Cellamare1, L. James Sprague3, Evan E. Eichler3, Nicoletta Archidiacono1 and Mariano Rocchi1{dagger}

1 Department of Genetics and Microbiology, University of Bari, 70126 Bari, Italy.
2 Department of Animal Biology and Genetics, University of Florence, Florence 50125, Italy.
3 Howard Hughes Medical Institute, Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA 98195, USA.


Figure 1 Fig. 1. All macaque (left) or human (right) chromosomes harboring ENCs. Quinacrine mustard banded macaque (left) and human (right) chromosomes are also displayed. Details are reported at www.biologia.uniba.it/macaque. Some macaque chromosomes are shown upside down to facilitate comparison in accordance with the orientation in the whole-genome assemblage. The Ns in red circles flank ENCs; the As in black circles flank ancestral centromeres. Synteny block conservation is indicated by the color. The arrows on the left of some macaque synteny blocks indicate that these blocks have opposite sequence polarity, with respect to the corresponding block in humans. These annotations, obviously, are affected by the upside down position of some chromosomes. The Human Genome Project has assumed as "+" sequence polarity the 5'->3' strand that starts from the tip of the short arm of each chromosome. The assumption that centromeres were conserved leads to a violation of this rule for macaque chromosomes 1 (1), 2 (3), 4 (6), 10 (20/22), 13 (2p), and 18 (18) (corresponding human chromosomes in parentheses). [View Larger Version of this Image (39K GIF file)]
 

Figure 2 Fig. 2. A reconstruction of chromosome 6 evolution in primates [modified from (9)]. The letters indicate the specific BAC clones used in the study (9) and reported in table S9. PA, primate ancestral; CA, Catarrhini ancestral; and A.C., ancestral centromere. The letter N in a red circle represents a novel centromere. A square bracket encompasses the Old World monkey–specific (macaque included) inversion of K-L-M markers, with respect to the human form. MMU4 is upside down, with respect to the correct position as reported in Fig. 3, to allow for an easy synteny comparison. L2, human BAC RP11-474A9. [View Larger Version of this Image (40K GIF file)]
 

Figure 3 Fig. 3. (A to C) Partial metaphases showing examples of FISH experiments on macaque and human chromosomes, with the use of the nonduplicated BAC clones (A) CH250-209i5 and (B) CH250-215i15 and the duplicated clone (C) CH250-284C24. (Mapping details are given in Fig. 4, A and B.) (D) Partial metaphases showing FISH on stretched chromosomes with the use of the duplicated BAC clones CH250-188G18 (red) and CH250-67B18 (green), containing alphoid sequences. The arrows indicate MMU4. [View Larger Version of this Image (40K GIF file)]
 

Figure 4 Fig. 4. (A) BAC clone contig map of the MMU4 region corresponding to the HSA6 DNA segment from bp 144,712,821 (right) to 146,765,547 (left) (UCSC March 2006 release). Human and macaque STSs (table S4) were used to screen high-density filters of the macaque BAC library CH250. Positively hybridizing BACs were then used in FISH experiments on macaque and human metaphases, and a contig tiling path of the BACs (blue segments) was defined. The analysis discovered single and duplicated regions around the MMU4 pericentromeric region. The marker positions of the STSs in the human sequence are reported in the top line. STSs in red are localized in the MMU4 single-copy region; blue STSs markers are within the region that is duplicated around the MMU4 centromere. This duplicated region is illustrated in detail in (B). Vertical red lines represent macaque STSs derived from BAC-end sequences. The yellow vertical region represents a gap that is probably composed of sequences refractory to cloning. (B) Details of the BAC contig assembly of the duplicated pericentromeric region of MMU4. FISH analysis of the macaque BAC clones indicated that duplicated blocks were present on both sides of the centromere. To further characterize these duplicated pericentromeric regions, we sequenced and aligned appropriate STS-amplified products from different clones with the MegAlign software (31). The sequence analysis allowed for the classification of the duplicated STSs and the corresponding BACs into seven separate blocks: three on the q arm (Dq1-3) and four on the p arm (Dp1-4) (table S7). The top blue line shows the mapping of the STS markers in human. The first nonduplicated STS, on both sides, is shown in red. The MMU line depicts all of the STS that were used in the study, arranged according to their inferred position in macaque. Macaque STSs derived from BAC-end sequences are shown in brown. Dq1, Dq2, and Dq3 and Dp1, Dp2, Dp3, and Dp4 indicate the duplicated subsets on the q-arm side (Dq-block) and on the p-arm side (Dp-block) of the centromere, respectively. Some STSs are apparently missing in some blocks. Their positions are indicated by brown arrowheads above the macaque STS line. The number of missing STSs is also indicated. Sequenced BAC ends are represented by small rectangles at the borders of the BACs. White and red diagonally striped rectangles stand for monomeric alpha satellite sequences; unfilled red rectangles stand for repetitive nonsatellite elements; and blue rectangles stand for single-copy sequences. The absence of a rectangle indicates that the end sequencing failed twice. Sequenced PCR products of specific STS primers are represented by colored circles. These sequences were used for the classification of duplicated blocks. The colored circles indicate STSs whose PCR products have been sequenced. Circles related to the same STS are in the same color only if their sequences are perfectly matched. A full description of the BAC contig assembly is reported in the SOM text. [View Larger Version of this Image (50K GIF file)]
 

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