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Exceptional Chromosome Regions Workshop I

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Direct isolation of a centromeric region from the human Y chromosome by TAR cloning for structural and functional studies

Natalay Kouprina and Vladimir Larionov
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences (NIEHS), Research Triangle Park, NC 27709
kouprina@niehs.nih.gov

Isolation of specific chromosomal regions and entire genes has typically involved cloning of random fragments as BACs or YACs followed by a long and laborious process to identify the region of interest. Using the TAR cloning technique in yeast1, it is possible to directly isolate specific chromosomal regions and genes from complex genomes as large linear or circular YACs. We applied a TAR cloning technique for isolation of a centromeric region of the human mini-chromosome [delta]1 containing 5 Mb of the human chromosome Y2. This mini-chromosome was generated by two rounds of telomere-directed chromosome breakage leading to a loss of sequences from both arms of the chromosome. Despite the small size and loss of a significant part of centromeric repeats (only 140 kb of alphoid DNA left), the[delta]1 mini-chromosome segregates accurately in mitosis, suggesting that this block of alphoid DNA alone or along with the short arm flanking sequence is sufficient for a centromere function. The centromeric region containing an entire block of alphoid DNA was rescued in yeast as a circular YAC. To simplify physical analysis of the cloned material and prevent its rearrangements due to the presence of multiple repeats, the YAC was retrofitted into YAC/BAC with the NeoR gene and transferred into the E. coli cells. No detectable changes in a BAC DNA were observed during propagation in bacterial cells that allowed a complete sequencing of the block of alpha satellite repeats. Because the BAC also contains a mammalian selectable marker, it can be transferred into human cells for further functional analysis.

  1. Larionov, V., and Kouprina, N. (1999). Selective Isolation of Mammalian Genes by TAR cloning. Current Protocols in Human Genetics, volume 1, pages: 5.17.1-5.17.21
  2. Brown et al. (1996). Proc. Natl. Acad. Sci. USA 93: 7125-7130.


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