Evolutionary origin of higher-order repeat structure in alpha-satellite DNA of primate centromeres

Abstract

Alpha-satellite DNA (AS) is a main DNA component of primate centromeres, consisting of tandemly repeated units of ~170 bp. The AS of humans contains sequences organized into higher-order repeat (HOR) structures, in which a block of multiple repeat units forms a larger repeat unit and the larger units are repeated tandemly. The presence of HOR in AS is widely thought to be unique to hominids (family Hominidae; humans and great apes). Recently, we have identified an HOR-containing AS in the siamang, which is a small ape species belonging to the genus Symphalangus in the family Hylobatidae. This result supports the view that HOR in AS is an attribute of hominoids (superfamily Hominoidea) rather than hominids. A single example is, however, not sufficient for discussion of the evolutionary origin of HOR-containing AS. In the present study, we developed an efficient method for detecting signs of large-scale HOR and demonstrated HOR of AS in all the three other genera. Thus, AS organized into HOR occurs widely in hominoids. Our results indicate that (i) HOR-containing AS was present in the last common ancestor of hominoids or (ii) HOR-containing AS emerged independently in most or all basal branches of hominoids. We have also confirmed HOR occurrence in centromeric AS in the Hylobatidae family, which remained unclear in our previous study because of the existence of AS in subtelomeric regions, in addition to centromeres, of siamang chromosomes.

Keywords: alpha-satellite DNA; centromere; evolutionary origin; higher-order repeat; hominoids.

Figure 1.

Strategy for the detection of HOR signs. Transposition of the Tn5 transposon to DNA molecules of an AS-containing fosmid clone was induced, and two clones carrying Tn5 at different positions were selected. Each clone was sequenced using two primers that annealed to Tn5 and oriented outward. The sequence reads (∼900 bp, including the tail region of a low reliability) were combined, and a longer sequence (∼1800 bp) was obtained. These sequences were compared between the two clones by the dot-matrix analysis. When a line spanning more than two basic units was found, it was regarded as a sign of HOR.

Figure 2.

Dot-matrix analysis of clone partial sequences for signs of HOR. The criterion in the dot-matrix analysis was that a 19-nucleotide match should exist over a window of 20 nucleotides. Examples of the results are shown. HooFos09 and NomFos1B3 exhibited a line spanning more than two basic repeat units, and HooFos03 and NomFos3F5 did not.

Figure 3.

Confirmation of deletion clone insert size. Deletion clones of various sizes were prepared from the HooCam09 and NomCam1B3 clones. Out of these pools, clones of sizes adequate for sequencing analysis were selected. This photograph is an example of gel electrophoresis in which deletion clones of insert sizes of 5.3–0.5 kb, originating from HooCam09, are contained. The 2.1-kb vector plasmid carried two cutting sites for restriction endonuclease XhoI just outside of the cloning site, and the insert fragments in the original clones did not have a site for this enzyme. Each deletion clone was digested with XhoI and divided into two fragments: the 2.1-kb plasmid vector and its insert fragment.

Figure 4.

Pairwise comparisons of basic repeat unit sequences. The comparisons were made using the MEGA5 program under default settings. The horizontal and vertical axes of each matrix represent consecutive basic repeat units contained in the AS contig sequence. Cells showing nucleotide identities of 90–95 and >95% are indicated by yellow and red, respectively. The same matrices containing the identity values in the cells are shown in Supplementary Figs S1–S3.

Figure 5.

FISH analysis of chromosomes for locations of AS. The left panels show FISH images obtained using a fluorescence scanner, and the right panels are images of the same chromosome spreads stained with 4′,6-diamidino-2-phenylindole (DAPI). In the left panels, red signals indicate hybridization of the probe DNA to chromosome DNA. The combination of chromosome spread and probe is shown on each left panel. The bar in the panels represents 10 µm. All experimental procedures were the same as those described in our previous work.

Figure 6.

Neighbour-joining phylogenetic tree of within-species consensus sequences. The consensus sequences of AS of Hoolock hoolock, Hylobates agilis and Nomascus siki were obtained by sending the contig sequences, which we determined in the present study, to the Consensus Maker program (version 2.0.0; http://www.hiv.lanl.gov/content/sequence/CONSENSUS/consensus.html). The consensus sequences obtained are included in the respective DDBJ files. The consensus sequences for Nomascus leucogenys, Symphalangus syndactylus, human, and orangutan AS were obtained from the respective references. The neighbour-joining phylogenetic tree of these within-species consensus sequences was made using the ClustalW program contained in MEGA.^.