Nucleic Acids Research, 2003, Vol. 31, No. 3 922-934
© 2003 Oxford University Press
Cloning of human centromeres by transformation-associated recombination in yeast and generation of functional human artificial chromosomes
1 Laboratory of Biosystems and Cancer, Center for Cancer Research, National Cancer Institute (NCI, NIH), Building 37, Room 5032, Bethesda, MD 20892, USA, 2 Laboratory of Genetic Disease Research, Human Genome Research Institute (NHGRI, NIH), Bethesda, MD, USA, 3 National Center for Biotechnology Information, National Institutes of Health, Bethesda, MD 20894, USA, 4 Division of Molecular and Cell Genetics, Department of Molecular and Cellular Biology, Faculty of Medicine, Tottori University, Yonago, Japan, 5 Discovery Research Laboratories I, Pharmaceutical Research Division, Takeda Chemical Industries, Ltd, 10 Wadai Tsukuba, Ibaraki 300-4293, Japan, 6 Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences (NIEHS, NIH), Research Triangle Park, NC, USA and 7 Institute of Genetics, Nottingham University, Queen’s Medical Centre, Nottingham NG7 2UH, UK
*To whom correspondence should be addressed. Tel: +1 301 496 7941; Fax: +1 301 480 2772; Email: kouprinn{at}mail.nih.gov
Human centromeres remain poorly characterized regions of the human genome despite their importance for the maintenance of chromosomes. In part this is due to the difficulty of cloning of highly repetitive DNA fragments and distinguishing chromosome-specific clones in a genomic library. In this work we report the highly selective isolation of human centromeric DNA using transformation-associated recombination (TAR) cloning. A TAR vector with alphoid DNA monomers as targeting sequences was used to isolate large centromeric regions of human chromosomes 2, 5, 8, 11, 15, 19, 21 and 22 from human cells as well as monochromosomal hybrid cells. The alphoid DNA array was also isolated from the 12 Mb human mini-chromosome 
Yq74 that contained the minimum amount of alphoid DNA required for proper chromosome segregation. Preliminary results of the structural analyses of different centromeres are reported in this paper. The ability of the cloned human centromeric regions to support human artificial chromosome (HAC) formation was assessed by transfection into human HT1080 cells. Centromeric clones from Yq74 did not support the formation of HACs, indicating that the requirements for the existence of a functional centromere on an endogenous chromosome and those for forming a de novo centromere may be distinct. A construct with an alphoid DNA array from chromosome 22 with no detectable CENP-B motifs formed mitotically stable HACs in the absence of drug selection without detectable acquisition of host DNAs. In summary, our results demonstrated that TAR cloning is a useful tool for investigating human centromere organization and the structural requirements for formation of HAC vectors that might have a potential for therapeutic applications.
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