Organization /Function of Chromosomal Regions Required

尽管它们对于研究染色体组织和构建人类人工染色体（HACs）具有重要意义，但着丝粒和端粒区域的特征仍然很差。其主要原因是长片段的重复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。在去年期间，我们开发了一种新的重组克隆程序，其选择包括阳性和阴性遗传选择，并且允许从缺乏ARS样序列的异染色质染色体区域选择性地分离基因组DNA的大片段。一种新的技术（改进的TAR克隆）成功地应用于克隆和物理特性的着丝粒序列从五个人类染色体（11，13，15，22和Y）。不同的着丝粒DNA分离物与哺乳动物选择性标记重新匹配，目前它们在转染到人细胞中期间形成HAC的效率进行比较。TAR克隆策略也已用于分离两种人类疾病基因的功能基因组拷贝，即来自染色体8 p21-p12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先前未在大插入大小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序列对大肠杆菌的毒性引起的。coli细胞。根据我们的估计，这种毒性序列约占人类基因组的6%，并且它们不存在于已公布的人类基因组草图序列中。为了证明改进的TAR克隆方案用于完成人类基因组序列的实用性，覆盖了含有来自人类5号染色体的新基因的约200 kb大小的两个缺口。

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.