Organization and Function of Chromosomal Regions that ar

近十年来，人类人工染色体（Human Artificial chromosome, HACs）已成为了解人类着丝粒染色体结构和功能的重要模型，近年来又被用作基因转移载体。利用HAC载体成功地补充了人类细胞中的基因缺陷，证明了其作为治疗性基因表达载体的潜力。为了阐明HACs形成的结构要求，我们构建了人类22号染色体的着丝粒区和着丝粒区文库。克隆的人类着丝粒区在体内支持着丝粒形成的能力通过将其转染到人类细胞中进行评估。HACs是由几个结构体组成的，这些结构体含有均匀的a型单体，形成特征的高阶重复序列。在没有药物选择的情况下，阿尔法DNA HAC结构体在有丝分裂上是稳定的。与已发表的数据相反，我们的研究表明，CENP-B结合位点可能不需要重新形成着丝点。我们继续研究通过TAR技术最大限度地提高基因捕获效率和选择性的条件，以便使该程序可供科学界的其他人员使用。具体来说，我们确定高达20%的DNA差异不会妨碍有效的基因分离。这种对DNA分化的耐受性扩展了TAR克隆在分离染色体复制和基因同源物方面的应用。TAR克隆策略还用于从患者细胞中克隆两个肿瘤抑制基因PTEN和定位在Xq27染色体上的一个新的前列腺癌基因的基因组拷贝。在过去的一年里，我们继续完成人类第19号染色体的序列和验证其组合。利用TAR克隆，分离出4个“不可克隆”的间隙并对其进行测序。因此，我们的结果有助于开发第一个连续的人类染色体核苷酸序列。

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.