Organization /Function of Chromosomal Regions Required

尽管对染色体组织的研究和人类人工染色体（HACs）的构建具有重要意义，但着丝粒和端粒区域的特征仍然很差。其主要原因是长时间重复的着丝粒和端粒特异性DNA序列不能通过标准的YAC或BAC克隆技术克隆。在去年，我们开发了一种新的重组克隆程序，选择包括阳性和阴性遗传选择，并允许选择性地从缺乏ARS样序列的异色染色体区域分离大片段基因组DNA。应用改良TAR克隆技术成功克隆了人类5条染色体（11、13、15、22和Y）的着丝粒序列，并对其进行了物理表征。将不同的着丝粒DNA分离物用哺乳动物选择性标记进行重组，目前比较了它们在转染到人细胞时形成HACs的效率。TAR克隆策略也被用于分离两种人类疾病基因的功能基因组拷贝，即染色体8p21-p12上的前列腺癌转移抑制基因和人类端粒酶基因hTERT。这些基因的完整拷贝以前未在大插入物大小的BAC和YAC文库中发现。我们已经证明，BAC文库中这些基因的缺失是由大肠杆菌细胞的毒性序列引起的。根据我们的估计，这些毒性序列约占人类基因组的6%，并且在已发表的人类基因组草图序列中不存在。为了证明改进的TAR克隆方案在完成人类基因组序列中的实用性，我们覆盖了人类5号染色体上两个约200kb大小的空白，其中包含了新的基因。

Despite of their importance for studying the chromosome organzation and for construction for Human Artificial Chromosomes (HACs), centromeric and telomeric regions remain poorly characterized. The main reason of that is that long stretches of repeated centromere and telomere-specific DNA sequences can not be cloned by standard YAC or BAC cloning techniques. During last year we developed a new recombinational cloning procedure that selects that includes positive and negative genetic selection and allows selective isolating large fragments of genomic DNA from heterochromatic chromosomal regions lacking ARS- like sequences. A new technique (modified TAR cloning) was successfully applied for cloning and physical characterization of centromeric sequences from five human chromosomes (11, 13, 15, 22 and Y). Different centromere DNA isolates were retofitted with a mammalian selectable marker and at present they are compared on their efficiency to form HACs during transfection into human cells. TAR cloning strategy has been also used for isolation of functional genomic copies of two human disease genes, the metastasis-suppressor gene for prostate cancer from chromosome 8p21-p12, and the human telomerase gene, hTERT. Entire copies of these genes were not previously found in large insert size BAC and YAC libraries. We have shown that an absence of these genes in BAC libraries is caused by toxicity of the sequences for E. coli cells. Based on our estimate, such toxic sequences represent approximately 6% of human genome and they are not present in published human genome draft sequence. To demonstrate utility of a modified TAR cloning protocol for completion of human genome sequence, two gaps of ~ 200kb in size containing new genes from human chromosome 5 were covered.