Organization and Function of Chromosomal Regions that ar

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Over the past decade, Human Artificial Chromosomes (HACs) have become important models for understanding chromosome structure and function of human centromeres and more recently were used as gene transfer vectors. A gene deficiency in human cells was successfully complemented using HAC vectors, which demonstrates their potential as therapeutic gene expression vectors. To shed light on structural requirements for formation of HACs, we constructed a library of pericentromeric and centromeric regions of the human chromosome 22. The ability of the cloned human centromeric regions to support kinetochore formation in vivo was assessed by their transfection into human cells. HACs formed efficiently with several constructs containing alphoid DNA arrays with homogeneous A-type monomers which form characteristic high order repeats. The alphoid DNA HAC constructs were mitotically stable in the absence of drug selection. In contrast to the published data, our study indicated that CENP-B binding sites may not be required for de novo kinetochore formation. We continued investigation of conditions that maximize the efficiency and selectivity of gene capture by TAR technology in order to make the procedure available to the rest of the scientific community. Specifically, we determined that up to 20% DNA divergence does not prevent efficient gene isolation. Such a tolerance to DNA divergence extended application of TAR cloning to isolation of chromosomal duplications and gene homologs. TAR cloning strategy has been also used for isolation of genomic copies of two tumor suppressor genes, PTEN and a new prostate cancer gene mapped on chromosome Xq27 that was also cloned from patient cells. During the past year, we continued to work on completion of the human chromosome 19 sequence and verification of its contig assemble. Using TAR cloning, four "unclonable" gaps were isolated and sequenced. Thus, our results helped to develop the first contiguous nucleotide sequence of human chromosome.