Abstract
Macrosatellites are some of the most polymorphic regions of the human genome, yet many remain uncharacterized despite the association of some arrays with disease susceptibility. This study sought to explore the polymorphic nature of the X-linked macrosatellite DXZ4. Four aspects of DXZ4 were explored in detail, including tandem repeat copy number variation, array instability, monomer sequence polymorphism and array expression. DXZ4 arrays contained between 12 and 100 3.0 kb repeat units with an average array containing 57. Monomers were confirmed to be arranged in uninterrupted tandem arrays by restriction digest analysis and extended fiber FISH, and therefore DXZ4 encompasses 36–288 kb of Xq23. Transmission of DXZ4 through three generations in three families displayed a high degree of meiotic instability (8.3%), consistent with other macrosatellite arrays, further highlighting the unstable nature of these sequences in the human genome. Subcloning and sequencing of complete DXZ4 monomers identified numerous single nucleotide polymorphisms and alleles for the three microsatellite repeats located within each monomer. Pairwise comparisons of DXZ4 monomer sequences revealed that repeat units from an array are more similar to one another than those originating from different arrays. RNA fluorescence in situ hybridization revealed significant variation in DXZ4 expression both within and between cell lines. DXZ4 transcripts could be detected originiating from both the active and inactive X chromosome. Expression levels of DXZ4 varied significantly between males, but did not relate to the size of the array, nor did inheritance of the same array result in similar expression levels. Collectively, these studies provide considerable insight into the polymorphic nature of DXZ4, further highlighting the instability and variation potential of macrosatellites in the human genome.
Highlights
At least half of the human genome is composed of repetitive DNA [1], including transposable elements, segmental duplications and tandem repeat DNA
A comparison of a single DXZ4 monomer sequence (Accession Number HQ659112) against the assembled human genome sequence using the UCSC genome browser, reveals approximately fourteen,3 kb DXZ4 monomers covering 50 kb, arranged in tandem centered at 115 Mb on the X chromosome
In the same report [5], DNA from 17 unrelated individuals digested with EcoRI and separated by pulsed field gel electrophoresis (PFGE) revealed hybridizing DXZ4 EcoRI fragments of between 150–300 kb by Southern analysis
Summary
At least half of the human genome is composed of repetitive DNA [1], including transposable elements, segmental duplications and tandem repeat DNA. Relatively little is known about the role of macrosatellite arrays in the genome, many of which have yet to be described in detail [2]. Most macrosatellite arrays are specific to one or two chromosomal locations in the genome, and only a small number have been confirmed and characterized to some extent [2,3,4,5,6,7,8,9]. Most is known about D4Z4, a tandem array of 1–100 3.3 kb repeat units located on chromosomes 4q35 and 10q26 [10,11]. Despite links between these unusual DNA elements and disease, the role of many macrosatellites in the human genome remains unexplored
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