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 is intriguing. 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 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 2002; Kim 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 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 2= 22 to 2= 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 1999; O’Neill 2004). Across the Macropodinae, the X chromosome is the most structurally divergent chromosome (Spencer 1991). The Macropodine species (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 AZD-9291 (Osimertinib) sequence classes contained within the centromeric portion of the X have been identified and characterized by sequencing, Southern hybridization, fluorescence hybridization (FISH), electrophoresis mobility shift assay (EMSA), and immunocytohistochemistry (ICHC) techniques. MATERIALS AND METHODS Microdissection and microcloning: The centromere of the X chromosome of was microdissected and microcloned AZD-9291 (Osimertinib) 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 AZD-9291 (Osimertinib) 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 g) GLCE was digested with (2004). GTG-banding was performed as per Rens (2003) with the modification of using 0.4% trypsin solution for a time of 45 sec. FISH: fibroblast cells were treated with 1 g/ml colchicine for 1 hr prior to harvesting metaphase chromosome preparations as previously described (Eldridge 1988). Slides were pretreated as previously described (Brown 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 (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, 2 SSC, 500 ng/l salmon.