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.

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DURING mitosis and meiosis in mammalian cells centromeres 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).

The origins of centromere sequences and the mechanisms by which a genome retains these sequences are unknown. Although a functional centromere is defined by its ability to form the kinetochore protein complex, it is theorized that particular DNA sequences (for example, -satellites in humans) may act as a trigger for the initial protein interactions involved in the assembly of the kinetochore (WILLARD 1990). It is not known, however, whether the structure of the human centromere exemplifies that of other mammals.

By virtue of their limited opportunity for recombination, the sex chromosomes of some mammals have large amounts of constitutive heterochromatin (HAYMAN and MARTIN 1974; NOVA et al. 2002; KIM et al. 2004). The mammalian X chromosome has a high degree of genic conservation while having a highly labile arrangement, attributable in part to intrachromosomal rearrangements (GRAVES 1995; WATERS et al. 2001). This trait is exemplified by species within the Australasian marsupial subfamily of Macropodinae (wallabies and kangaroos). The Macropodinae are karyotypically diverse, with chromosome numbers ranging from 2n = 22 to 2n = 10 female/11 male. Karyotypic differences within this 58 member clade commonly involve centromere-associated rearrangements, including whole-arm reciprocal translocations, pericentric inversions, and centromere repositioning (ROFE 1978; ELDRIDGE and CLOSE 1993; GLAS et al. 1999; O'NEILL et al. 2004). Across the Macropodinae, the X chromosome is the most structurally divergent chromosome (SPENCER et al. 1991).

The Macropodine species Macropus rufogriseus (red-necked wallaby) has an exceptional amount of centric and pericentromeric heterochromatin (HAYMAN and MARTIN 1974), making isolation of macropodine centromeric DNA more amenable from this species. Therefore, we have microdissected and microcloned the C-band-positive centromeric portion of the X chromosome of this species for further analyses. Three sequence classes contained within the centromeric portion of the M. rufogriseus X have been identified and characterized by sequencing, Southern hybridization, fluorescence in situ hybridization (FISH), electrophoresis mobility shift assay (EMSA), and immunocytohistochemistry (ICHC) techniques.

Microdissection and microcloning:

The centromere of the X chromosome of M. rufogriseus banksianus was microdissected and microcloned as previously described (KAO and YU 1991). Products were size separated in a 1.2% NuSieve gel and DNA fragments between 500 and 1000 bp were subcloned into Promega pGEM-T Easy vector as per manufacturer's instructions.

Sequencing:

Candidate subclones were sequenced on an ABI 377 using Big Dye chemistry (Applied Biosystems). Sequence identity was initially determined by discontiguous MegaBLAST and TBLASTX search parameters (NCBI). Alignments and contigs were assembled using Clustal W within Vector NTI 8 suites (Informax).

Southern analyses:

Candidate subclones were analyzed by Southern hybridization. Genomic DNA (9 µg) was digested with BglII, electrophoresed on a 1% agarose gel, and subsequently transferred to Hybond N+ nylon membranes (Amersham) via salt transfer. Probes were ³²P-labeled by random priming (SAMBROOK and RUSSELL 2001). Hybridizations were performed in a BSA-free sodium phosphate buffer (CHURCH and GILBERT 1984) at 65° overnight and washed in 0.1x SSC, 0.1% SDS at 65°.

C- and G-banding:

C-banding was performed as per METCALFE et al. (2004). GTG-banding was performed as per RENS et al. (2003) with the modification of using 0.4% trypsin solution for a time of 45 sec.

FISH:

M. rufogriseus fibroblast cells were treated with 1 µg/ml colchicine for 1 hr prior to harvesting metaphase chromosome preparations as previously described (ELDRIDGE 1988). Slides were pretreated as previously described (BROWN et al. 2002).

Candidate subclones were labeled by PCR incorporation with biotin 16-dUTP (Roche). Probe preparation, hybridization, and post-hybridization washes were performed as per O'NEILL et al. (1998). Biotin was detected with FITC-avidin (yellow) (Vector Laboratories). Slides were mounted with DAPI/Vectashield (Vector Laboratories) mounting media.

Oligonucleotide digoxigenin (DIG) tailing of single-stranded CENP-B box-containing probes (see EMSA) was conducted as per manufacturer's instructions (Roche). FISH hybridizations were conducted in 30% hybridization solution (30% formamide, 2x SSC, 500 ng/µl salmon sperm DNA, 100 pmol probe) at 37° overnight. All washes were conducted at 42° and included three 30% formamide/2x SSC washes for 5 min each, followed by three 2x SSC rinses. Slides were blocked with 4x SSC/0.2%Tween-20/5%BSA before anti-DIG rhodamine (Roche) incubation at 37° for 30 min. Slides were mounted with DAPI/Vectashield (Vector Laboratories) mounting media.

Images were viewed on an Olympus AX70 fluorescence microscope equipped with a Photometrics Sensys camera and analyzed with Cytovision software (Applied Imaging) or a Leica DM6000B microscope with a DFC350FX-R2 digital camera and analyzed with Leica CW4000 Cytogenetics software (Leica Microsystems).

Gel EMSA:

A 38-bp double-stranded oligonucleotide was designed from the Mrb-sat23 clone to include the M. rufogriseus putative CENP-B box (uppercase): 5' aggtctttGTTCGTAAAATCGAGGTtttgggttagaca. The Mrb CENP-B oligo was -³²P end-labeled with polynucleotide kinase. M. rufogriseus nuclear protein was extracted from frozen fibroblast cell pellets using NucBuster protein extraction kit (Novagen). For mobility shift assay binding reactions, buffer (10 mM Tris–HCl pH 8, 10% glycerol, 1 mM EDTA, 1 mM DTT, 150 mM NaCl), 2.5 µl nuclear protein extract, and 25 M excess cold competitor oligonucleotide [cold Mrb-CENP-B oligo or AP1 oligo (Promega)] or 200 ng of human anti-CENP-B (rabbit) antibody (Santa Cruz Biotechnology) were incubated on ice for 15 min. Labeled Mrb-CENP-B oligo (0.4 pmoles) was added and incubated at room temperature for 15 more minutes prior to loading. Samples were run on a 1 mm x 15 cm, 0.25x TBE, 5% polyacrylamide gel at 200 V for 150 min at 4° following 2 hr of pre-run. The gel was dried and exposed to film overnight at –80°.

CENP-E and CREST ICHC:

Immunofluorescence in situ localization was carried out as described by SAFFERY et al. (2000) with the following modifications. The cells were fixed in 4% paraformaldehyde/KCM (120 mM KCL, 20 mM NaCl, 10 mM Tris–HCl, 0.5 mM NaEDTA, 0.1% Triton X-100). BSA at a concentration of 0.1% was used in the KB buffer (10 mM Tris–HCl, 150 mM NaCl). The anti-CENP-E antibody (supplied by T. J. Yen) was diluted 1:600 in KCM and detected using anti-rabbit AlexaFluor555 (Molecular Probes) at a final concentration of 10 µg/ml. The human CREST antisera (Antibodies, Inc.) was diluted to 7 µg/ml in KCM and detected using goat anti-human AlexaFluor555 (Molecular Probes) at a final concentration of 10 µg/ml. Chromosomes were counterstained with DAPI (0.5 µg/ml) in Vectashield mounting media (Vector Laboratories). Images were captured on Olympus AX70 fluorescence microscope equipped with a Sensys camera and analyzed with Cytovision software (Applied Imaging).

Dual ICHC/DNA–FISH:

Immunofluorescence in situ localization was carried out as described by PEREZ-BURGOS et al. (2004) with the following modifications. Rapidly growing cells were treated with media containing 2 µg/ml colchicine for 2 hr. Cells were resuspended in 0.075 M KCl and incubated at 37° for 20 min followed by a 4° incubation. The cell suspension was centrifugated onto the slides. Postcentrifugation washes were done for 20 min. Slides were fixed in 4% paraformaldehyde and then subjected to 70%/90%/100% serial ethanol dehydration. Block solution was prepared as per PANNING (2004). Anti-CENP-B and anti-trimethyl H3K9 antibodies (Upstate USA) were diluted 1:300 and 1:800 in blocking solution, respectively, and detected posthybridization using anti-rabbit AlexaFluor555 (Molecular Probes) at a final concentration of 10 µg/ml. Post-antibody washes were done with 0.2% Tween-20 in 1x PBS three times for 5 min, fixed, and dehydrated. Slides were then treated with 10 µg/ml RNase A for 5 min and denatured for 10 min at 70° in 70% formamide/2x SSC. Slides then underwent the subsequent steps of DNA–FISH as above. Mounted slides were counterstained in DAPI, imaged on a Leica DM6000B microscope with DFC350FX-R2 digital camera, and analyzed with Leica CW4000 Cytogenetics software (Leica Microsystems).

Microdissection and microcloning of the M. rufogriseus X chromosome centromere yielded 62 plasmid clones. Of these, 60 fell into three groups based on contiguous sequence alignment with an 85% minimum identity for overlapping sequence within each group, respectively. The largest representatives of each contig alignment group (Mrb-sat1, Mrb-B29, and Mrb-sat23), named for their representative clones, were chosen for Southern and FISH. The Mrb-sat1 group contained 30 clones, the Mrb-B29 group 25 clones, and the Mrb-sat23 group five clones.

The 741-bp Mrb-sat1 clone is composed of two 342-bp tandem degenerate sequence units that share 71% homology to one another (Figure 1A). This clone is 64% AT rich and has 48% identity by TBLASTX to a previously identified M. rufus Pst satellite (GenBank Accession no. MRU238844). The Mrb-B29 clone is 410 bp, characterized by 53 short tandem repeats (Figure 1B), and is 66% AT rich. The 7-mer GGAATTT, appearing 10 times, is the most common sequence within the clone. Interspersed are 43 sequence variants of this 7-mer that are either 6 or 7 bp long. Within 49 of the 53 total units, the dinucleotide "aa" core of the sequence is preserved. The Mrb-sat23 clone is 315 bp, 61% AT rich, and composed of one and a half units of a 178-bp repeat unit (Figure 1C). Within the 178-bp unit, a putative M. rufogriseus CENP-B DNA binding domain (CENP-B box) can be identified: 5' GTTCGTAAAATCGAGGT 3'. The presence of a CENP-B binding domain within a repeat of 178 bp indicates it may act as a functional centromeric repeat, akin to the human -satellite (YODA et al. 1998). However, no sequence similarity >52% was identifiable when comparing human satellite sequences to Mrb-sat23 outside of the CENP-B box. All of the other four clones in the Mrb-sat23 group possess one or two CENP-B box sequences with at most one transversion.

View larger version (28K): In this window In a new window Download PPT slide   FIGURE 1.— Schematic of DNA clones Mrb-sat1, Mrb-B29, and Mrb-sat23. Bars and open arrows indicate length and directionality of repeat units within clones; restriction enzyme sites are indicated. (A) Two full and one partial copy of the 342-bp units of Mrb-sat1. (B) Closed arrows represent 53 simple repeat units within Mrb-B29; see key. (C) One full and one partial copy of the 178-bp unit of Mrb-sat23. Solid arrow indicates 17-bp CENP-B box; shaded arrow indicates 38-bp CENP-B oligo used in EMSA and FISH reactions.

 
Southern analysis of Mrb-sat1 showed banding periodicity typical of satellites (SOUTHERN 1975) yet contained bands of varying intensity indicating that not all hybridizing restriction fragments contained relatively equivalent amounts of the sequence (Figure 2A). The Southern banding pattern of Mrb-B29 showed no ladder-like pattern; however, it hybridized intensely to many bands under stringent conditions, indicating it is present in very high copy number (Figure 2A). The Southern banding pattern of Mrb-sat23 showed ladder-like periodicity with band intensities indicative of relatively equivalent amounts of this repeat within these fragments (Figure 2A).

View larger version (45K): In this window In a new window Download PPT slide   FIGURE 2.— (A) Southern hybridization of Mrb-sat1, Mrb-B29, and Mrb-sat23 clones to male M. rufogriseus BglII digested genomic DNA. (B) M. rufogriseus metaphase chromosomes stained with DAPI (blue) and hybridized with biotin-labeled (FITC-yellow) clones. (C) Idiogram summary of FISH results for M. rufogriseus. The primary constrictions are centromeric; the secondary constriction on Xq (bottom of chromosome) is coincident with the NOR. Mrb-sat1 inset of Y chromosome under increased exposure.

 
FISH analysis of Mrb-sat1, Mrb-sat23, and Mrb-B29 to metaphase chromosomes of M. rufogriseus defined their location within the karyotype. The Mrb-sat1 sequence is sex-chromosome specific, localizing to the Xp and Xq pericentric and centric regions (Figure 2, B and C) and hybridizing faintly to the centromere of the acrocentric Y chromosome. Mrb-sat23 localizes to all centromeres of the M. rufogriseus chromosomes (Figure 2, B and C), indicating that it may be a functionally conserved component of the centromeres in this species. The Mrb-B29 sequence exists as a major constituent of the X centromere. FISH (Figure 2, B and C) showed it localized to the centric region and a portion of the pericentric region. It is present on the majority of the Y, excluding the telomeres, and localized to the pericentric region of 2p. Although the size and content of Mrb-B29 (GGAATTT and variants) are similar to telomeric sequences (T₂AG₃), no telomeric localization is seen with this probe, and no telomeric sequence is known to exist within the M. rufogriseus X centromere (METCALFE et al. 2004). The sequence and distribution of Mrb-B29 is similar to that of human satellite III, although perhaps it is present in greater relative amounts in this macropod (CHOO 1997). Together these three sequences hybridize to the entirety of the centromeric regions of both sex chromosomes. As determined in analyses of Mrb-sat1 and Mrb-B29, both the X and Y are repeat rich and possess sex-chromosome specific centromeric DNA.

Alignments of CENP-B boxes from human, Cercopithecus aethiops (African green monkey), Mus musculus, Mus caroli, and Mrb-sat23 show strong conservation of the critical 9 bp (YODA et al. 1996) between these lineages, representing 190 million years of divergence (WOODBURNE et al. 2003)(Figure 3). EMSA was conducted to determine if any protein-binding ability exists at the putative Mrb-sat23 CENP-B box. A 38-bp segment of the Mrb-sat23 oligonucleotide containing the 17-bp M. rufogriseus CENP-B box (Figure 1) bound selectively to M. rufogriseus nuclear protein and shifted upon the addition of human anti-CENP-B (rabbit) antibody (Figure 4A). This result shows that this CENP-B domain selectively binds CENP-B protein and thus implies all centromeres containing this sequence carry CENP-B binding ability. FISH localization of the 38-bp oligonucleotide containing the 17-bp M. rufogriseus CENP-B box shows identical hybridization patterns to Mrb-sat23 (Figure 4B), indicating each centromere carries the functional portion of the Mrb-sat23 sequence.

View larger version (10K): In this window In a new window Download PPT slide   FIGURE 3.— Alignment of CENP-B box identified in indicated mammalian species to the putative sequence from Mrb-sat23. Conserved bases are shown in uppercase letters; the CENP-B box bases critical to protein binding are highlighted in red. Divergent bases between marsupials and eutherians within the CENP-B box are highlighted in blue. Old World monkeys diverged from Hominidae 25 MYA (GOODMAN 1999). Mus caroli diverged from Mus musculus 5–7 MYA (KIPLING et al. 1995). Marsupials diverged from eutherians 170–190 MYA (WOODBURNE et al. 2003).

 

View larger version (29K): In this window In a new window Download PPT slide   FIGURE 4.— (A) EMSA gel. Lane 1, labeled Mrb CENP-B oligo; lane 2, labeled Mrb CENP-B oligo and M. rufogriseus nuclear protein; lane 3, as per lane 2 and including 25 M excess unlabeled Mrb CENP-B oligo; lane 4, as per lane 2 and including 25 M excess unlabeled AP1 oligo; lane 5, as per lane 2 and including anti-CENP-B antibody. Arrows in lanes 2 and 4 indicate protein-Mrb CENP-B oligo-specific complexes. Arrow in lane 5 indicates supershift of complex upon inclusion of anti-CENP-B antibody. (B) DNA–FISH of CENP-B oligo (red) sequence to metaphase chromosomes of M. rufogriseus stained with DAPI (blue).

 
ICHC on metaphase M. rufogriseus chromosomes was conducted with human anti-CENP-E antibody and human CREST antibody (EARNSHAW and ROTHFIELD 1985) to determine the localization of the functional centromere within the centromeric region. CENP-E is an outer kinetochore protein present only at active centromeres (CHOO 1997). Within M. rufogriseus the active centromere, delimited by the binding of CENP-E, is confined to discrete point locations within the expanded centromeric region (Figure 5A). The CREST antiserum, containing a mixture of antibodies to constitutive centromeric proteins such as CENP-A, CENP-B and CENP-C, localizes centromerically yet is not restricted to the point localization observed with CENP-E (Figure 5B). These data demonstrate that pericentric segments of the X chromosome support CREST binding, exceeding the boundary delineating the functional centromere.

View larger version (17K): In this window In a new window Download PPT slide   FIGURE 5.— ICHC of (A) CENP-E (green), (B) CREST (red), and (C) dual localization to M. rufogriseus metaphase chromosomes stained with DAPI (blue).

 
ICHC followed by DNA–FISH was also conducted to determine the cytological relationship between CENP-B and its presumed binding partner, Mrb-sat23. Anti-CENP-B signal is associated with Mrb-sat23 signal on all chromosomes (Figure 6A). Additionally, anti-trimethyl-histone H3-Lys9 (trimethyl-H3K9) was used to define the portion of the centromere that carries modified histones specifically targeting heterochromatin (PETERS et al. 2001). DNA–FISH with Mrb-sat23 was performed post-ICHC to delimit the relationship of this satellite with pericentric heterochromatin (Figure 6B). Both anti-trimethyl-H3K9 and Mrb-sat23 are restricted to the centromere and pericentromere on the autosomes. However, the location of this modified histone on the X is not only shared with Mrb-sat23 localization at the centromere, but also enriched at the nucleolus organizer region.

View larger version (40K): In this window In a new window Download PPT slide   FIGURE 6.— ICHC/DNA–FISH colocalizations to M. rufogriseus metaphase chromosomes. Only the X chromosome (X) and a single autosome (A) are shown. (A) ICHC of anti-CENP-B antibody (orange), DNA–FISH of Mrb-sat23 (green), and merged image. (B) ICHC of anti-trimethyl-H3K9 antibody (orange), DNA–FISH of Mrb-sat23 (green), and merged image.

 
The sequence and mapping data show that all M. rufogriseus chromosomes share a functional centromere satellite (Mrb-sat23); however, the distribution of two other centromeric repeated DNA sequences (Mrb-sat1, Mrb-B29) is restricted to the sex chromosomes. A summary of the data for the X chromosome, including C-banding, G-banding, DNA–FISH, and ICHC is shown in Figure 7. As evidenced by these data, the centromeric region of the M. rufogriseus X is a complex mixture of satellite, satellite-like, and short tandem repeated sequences and is characterized by a large pericentromere rich in these sequences.

View larger version (20K): In this window In a new window Download PPT slide   FIGURE 7.— Summary of FISH and ICHC data for the X chromosome of M. rufogriseus. (A) G-band idiogram; brackets denote the pericentric heterochromatin and a black dot marks the functional centromere. (B) C-banding. (C) Inverted DAPI stain. FISH localization of (D) Mrb-B23, (E) Mrb-sat1, and (F) Mrb-B29. ICHC of (G) anti-CENP-B and (H) anti-CENP-E.

 

The centromeric region of M. rufogriseus chromosomes, including the C-band-positive functional centromere and pericentromere (Figure 7), is among the largest within the Macropodidae (HAYMAN 1990). These centromeric expansions account for almost doubling the size of some chromosomes, including the X (HAYMAN and MARTIN 1974). While the chromatid arm sizes are equivalent between homologous chromosomes among M. rufogriseus and the other members of this clade carrying an identical 2n = 16 karyotype (HAYMAN and MARTIN 1974), the ratio of centromere length to chromatid arm length is unusually large within this species compared to that of other mammals.

Previous studies of the M. rufogriseus X chromosome indicated that the centromere contained an increased density of repeated sequences (DUNSMUIR 1976; DENNIS et al. 1980; VENOLIA and PEACOCK 1981). The work of HAYMAN and MARTIN (1974) identified M. rufogriseus as having a greater nuclear DNA content than most macropodines, characterized by a large amount of pericentromeric heterochromatin, presumably due to the addition of repetitive DNA (HAYMAN and MARTIN 1974). DUNSMUIR (1976) isolated M. rufogriseus satellite DNA by density gradient centrifugation and discovered two prominent repeated sequences representing 20% (major fraction) and 5% (minor fraction) of total DNA. Cytological examination revealed that the major fraction hybridized to pericentromeric regions of the autosomes and that the minor satellite fraction hybridized to pericentromeric regions of all the chromosomes (DUNSMUIR 1976). This work suggested the ordered arrangement of these satellite sequences into blocks rather than distributed discretely throughout the genome. These studies were important in identifying unique properties of the Macropodidae genome; however, fine mapping and sequence characterization was not possible at that time.

This current study clarifies the constitution of the C-band-positive portion of the M. rufogriseus sex chromosomes and develops a more comprehensive overview of the composition of a unique marsupial centromere. The centromeric constitution of this species differs between the sex chromosomes and the autosomes, as shown by sequencing and FISH (Figures 1 and 2). The centromeric heterochromatin of all the chromosomes carries a newly identified -like satellite, Mrb-sat23, while the sex chromosomes also harbor two other repeats, Mrb-sat1 and Mrb-B29, each varying in copy number and chromosomal distribution.

Mrb-sat1 sequence is present on the sex chromosomes, localizing to the Y as well as to the C-band-positive portion of the X, extending beyond the pericentromeric regions in Xp and Xq (Figure 7). The size, degeneracy, and distribution of Mrb-sat1 across highly heterochromatic chromosomes are consistent with the characteristics of ancient, transmuted centromeric sequences (SCHUELER et al. 2001).

Mrb-B29 hybridizes by FISH to the Y, the centromere of the X, and a pericentric portion of 2p. The clone displays an irregular Southern banding pattern, a distribution consistent with a minor interstitial element of the pericentromere (CHOO 1997). Two intense bands on the Southern, sub-2000 bp and 750 bp, demarcate two main array populations of this 7-mer simple tandem repeat. The 2p localization of Mrb-B29 demonstrates shared sequence with the sex chromosomes, indicating an association and possible translocation of material across chromosomes. Interestingly, there is an association between the X and 2p in another macropodine. Within Wallabia bicolor (swamp wallaby), the X and Y₂ chromosomes carry an autosomal translocation and fusion of chromosomes 2 and 7 (TODER et al. 1997a). While the fusion occurred between 2 and 7, with the proximal portion of 7 fused to the X, the localization of sequences to both the X and 2p in M. rufogriseus is intriguing. Future phylogenetic analyses of macropodines may allow for accurate interpretation of these data as either coincidence or the product of chromosome rearrangement in a shared ancestor.

The retention of the Mrb-sat23 satellite in the centric and pericentric regions of all the chromosomes, the 178-bp periodicity of this sequence, the inclusion of a functional CENP-B box within this sequence, and colocalization with anti-CENP-B antibody indicate this sequence is acting as the primary satellite dictating centromere function. FISH mapping of this sequence showed that this sequence resides over the entire Y chromosome, accounting for a large portion of the extensive heterochromatin found on this chromosome. The spread of Mrb-sat23 sequence throughout the extensive X chromosome pericentromere, as well as its localization to the entirety of the Y chromosome, implies that this sequence has undergone large-scale amplifications. The Southern banding pattern indicates a tandem array organization of these amplicons and implies high homogeneity of all autosomal and X chromosome sites to one another.

The colocalization of all three classes of centromeric sequences to the Y chromosome may be evidence of X–Y associations such as crossover or homogenization events at the centromeric regions between the two chromosomes (TODER et al. 1997b). Unequal crossover has been suggested to explain sex chromosome-specific amplification of constitutive heterochromatin seen in rodents (NOVA et al. 2002). The marsupial X and Y do not undergo homologous pairing (side to side) at meiosis; however, axial pairing (end to end) does occur (SOLARI and BIANCHI 1975; SHARP 1982; PAGE et al. 2003). Although no pseudoautosomal region has been identified on marsupial sex chromosomes (GRAVES et al. 1998), the X and Y of Macropus eugenii have been shown to possess homologous sequences (TODER et al. 1997b). The constitution of these regions is not known and it is speculative whether these portions are responsible for axial pairing in meiosis. Our data confirm that heterochromatic sequences in both the X and Y of M. rufogriseus are shared. Homogenization events that do not involve homologous crossover, such as replication slippage, sister chromatid exchange, and intrachromosomal recombination and transposition, can be considered probable mechanisms of satellite expansion (LAURENT et al. 1999; ALEXANDROV et al. 2001). Pericentric crossover events, for example, are thought to maintain satellite homogeneity (MASHKOVA et al. 1998).

ICHC experiments with M. rufogriseus show that CREST and CENP-B antibodies associate to a large area of pericentric and centric chromosome domains, while CENP-E localizes to the functional centromere at discrete positions central within the centromere (Figures 5 and 6A). CREST antisera contain a mixture of the constitutively associated proteins CENP-A, CENP-B, and CENP-C. CENP-A and CENP-C are outer and inner kinetochore binding proteins, respectively, present only at active centromeres, and thus the localization of CENP-A and CENP-C will be relatively equivalent to that of CENP-E. CENP-B is a constitutive protein that binds satellite DNA at a specific 17-bp CENP-B box recognition site regardless of kinetochore formation (MASUMOTO et al. 1989). CENP-B localization, found concomitant with Mrb-sat23 localization, is found within the entirety of the expanded centromeric regions of M. rufogriseus. These experiments demonstrate that larger segments of the M. rufogriseus chromosomes bind CENP-B than delimit the functional centromeres, and that these chromosomes have retained CENP-B binding sequences in the pericentric domains.

Trimethyl-H3K9, a histone-3 variant found enriched at eukaryotic pericentric heterochromatin (PETERS et al. 2001), was found at all centric and pericentric domains within the M. rufogriseus karyotype. This protein was found enriched at a second chromosomal domain on the X chromosome, specific to the NOR. Therefore, we determined that the primary functional centromere satellite identified in this study, Mrb-sat23, is not restricted to the centric core but is present across domains containing both the modified pericentric heterochromatin, as evidenced by trimethyl-H3K9 domains, and the active centric chromatin, as evidenced by CENP-B and CENP-E domains.

Hybridization of a CENP-B box sequence to all the chromosomes of this marsupial species is significant as CENP-B binding in eutherians is not considered to be required for the formation of functional centromeres in humans and mice (MASUMOTO et al. 1989; COOKE et al. 1990; KIPLING et al. 1995). The presence of a CENP-B binding-competent domain on the Y of a marsupial suggests that ancestral mammalian sex chromosomes utilized CENP-B to differentiate centromere location. This finding also illustrates that the loss of CENP-B protein binding and CENP-B box DNA on the Y are derived when found within eutherian mammals.

The EMSA and CREST data shown herein are contrary to a model presented by MALIK and HENIKOFF (2002) predicting an increase in kinetochore-binding ability with an increase in quantity of centromeric DNA. The mechanism of their model suggests an intimate association between the size of the kinetochore and the distribution of CENP-A. Our data indicate that this association may still exist in M. rufogriseus, although additional centromeric DNA present in this mammal has added no noticeable increase in the size of the kinetochore as measured by CENP-E binding ability.

Current mechanical theories of centromere maintenance include the periodic renewal of centromeric sequences by the decommissioning of corrupted centromeres and heterochromatin formation (CSINK and HENIKOFF 1998; SULLIVAN et al. 2001). Local crossover events between homologous sequences, excising looped-out sequences dense with repeated elements (REs), is one plausible method of reducing REs in their pericentric "dead" centromere hosts (LAURENT et al. 1997). Higher eukaryotes often retain remnants of ancestral centromere and telomere sequences as pericentric heterochromatin. M. rufogriseus has been able to accommodate much greater amounts of pericentric heterochromatin than is normally observed. We believe the possession of large amounts of heterochromatin in the M. rufogriseus genome is likely the result of a failure to excise extraneous pericentric heterochromatin, as only one other member of the Macropodidae, Macropus parryi, exhibits this character within the karyotype (HAYMAN and MARTIN 1974).

WICHMAN et al. (1991) observed that euchromatic rearrangements and differences in species karyotypes were accompanied by changes in composition and content of centromeric heterochromatin . That two different centric sequences of the sex chromosomes of M. rufogriseus (Mrb-sat1 and Mrb-B29) vary in distribution may be a result of their importance to the genomic architecture of the sex chromosomes. The distribution of Mrb-sat23 to centric positions on all chromosomes may be indicative of its role as a functional centromeric sequence. Comparisons of the distribution of these centromeric elements across this clade of mammals may elucidate the relationships of these sequences to the extraordinary karyotypic evolution observed in this group.

C. Giardina provided technical guidance and the staff of Macquarie University Fauna Park aided in the collection of samples used in this study. Anti-CENP-E antibody was kindly provided by T.J. Yen of the Fox Chase Center, Philadelphia. Work conducted in Australia was supported by a William J. Fulbright Scholarship to K.B. This work and R.O., K.B., G.F., and C.M. were supported by a grant from the National Science Foundation (0093250).

Sequence data from this article have been deposited with the GenBank Data Library under accession nos. AY819755, AY819756, and AY819757.

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Communicating editor: S. YOKOYAMA

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