The constitution of the centromeric portions of the sex chromosomes of the red-necked wallaby, Macropus rufogriseus (family Macropodidae, subfamily Macropodinae), was investigated to develop an overview of the sequence composition of centromeres in a marsupial genome that harbors large amounts of centric and pericentric heterochromatin. The large, C-band-positive centromeric region of the X chromosome was microdissected and the isolated DNA was microcloned. Further sequence and cytogenetic analyses of three representative clones show that all chromosomes in this species carry a 178-bp satellite sequence containing a CENP-B DNA binding domain (CENP-B box) shown herein to selectively bind marsupial CENP-B protein. Two other repeats isolated in this study localize specifically to the sex chromosomes yet differ in copy number and intrachromosomal distribution. Immunocytohistochemistry assays with anti-CENP-E, anti-CREST, anti-CENP-B, and anti-trimethyl-H3K9 antibodies defined a restricted point localization of the outer kinetochore at the functional centromere within an enlarged pericentric and heterochromatic region. The distribution of these repeated sequences within the karyotype of this species, coupled with the apparent high copy number of these sequences, indicates a capacity for retention of large amounts of centromere-associated DNA in the genome of M. rufogriseus.
D URING mitosis and meiosis in mammalian cellscentromeres are the chromosomal sites of spindle attachment, mediating the interaction between chromosomes and the cellular machinery responsible for the faithful segregation of nuclear DNA. Identification of the centromere as the site of spindle attachment is dependent on the formation of the kinetochore protein complex, a layered structure of over 30 components (reviewed in Fukagawa 2004). In eukaryotes, centromeric DNA is constitutively heterochromatic and is organized into megabases of tandem satellite DNA arrays. To date, few mammalian centromeres have been characterized in detail, leaving gaps in our understanding of the structure and evolution of this portion of the genome.Recently Schueler et al. (2001) conducted large-scale physical mapping of the human X centromere and found that it contained units of satellite sequence (171 bp in length) organized into concatomers that formed larger blocks of repeated segments several megabases in length. The long-range physical map for this region supports proposed models in which tandem arrays of satellite sequences characterize centromeric regions in higher eukaryotes (Sumner 2003). The study concluded that a correlation exists between the degeneracy of satellite sequences and the location of these sequences relative to the functional centromere: the more distal a satellite relative to the centromere, the more degenerate the sequence (Schueler et al. 2001).