DIRECT ISOLATION OF CHROMOSOMAL REGIONS FOR THE STUDY OF HUMAN GENES

工作总结：分离整个基因通常涉及克隆细菌(如BAC或PAC)或酵母(如YAC)中的随机片段，然后是一个漫长而费力的过程来识别感兴趣的区域。利用最近在酿酒酵母中发展起来的TAR(转换相关重组)克隆技术，可以利用完整或部分序列信息直接从复杂基因组中分离出特定的染色体区域和基因作为大的线性或环状YAC。去年，这项新技术成功地应用于从人类和小鼠基因组中分离出七个不同的基因和特定区域。对于四个被重复序列丰富的基因(包括乳腺癌基因BRCA1和BRCA2)，已经证明了功能拷贝的分离，这表明基因分离是高保真的。焦油克隆技术也被应用于从Y人类染色体Delta1的缺失形式中分离出一个着丝粒区域，该区域仅包含~100kb的Alphid DNA。尽管着丝粒重复序列的大小很小，并且丢失了相当大的一部分，但在有丝分裂中，增量1染色体Y准确地分离，这表明仅有或与短臂侧翼序列一起的α-DNA片段足以实现着丝粒功能。利用焦油克隆的改良版本，着丝粒区域在酵母中被挽救为一组环状YAC。YAC分离株含有完整的Alphid DNA片段，用于构建第一个小环状人类人工染色体(HAC)。它含有YAC和BAC盒，NeoR可选择的哺乳动物标记，以及来自增量1染色体Y的整个Alphid DNA块。当将其导入人类细胞时，它稳定地保持在每个细胞1？2个拷贝。HAC克隆系统的成功开发为人类基因治疗提供了新的机遇，并可能对我们理解人类着丝粒区域的组织和动点功能产生深远的影响。-非整倍体，染色体缺失，DNA，DNA修复，基因组载体，酿酒酵母

Summary Of Work: Isolation of entire genes has typically involved cloning of random fragments in bacteria (as BACs or PACs) or in yeast (as YACs) followed by a long and laborious process to identify the region of interest. Using the recently developed TAR (Transformation- Associated Recombination) cloning technique in the yeast Saccharomyces cerevisiae, it is possible to directly isolate specific chromosomal regions and genes from complex genomes as large linear or circular YACs using a complete or a partial sequence information. During last year the new technique was successfully applied for isolation of seven different genes and specific regions from human and mouse genomes. For four genes enriched by repeats (including breast cancer genes BRCA1 and BRCA2) isolation of functional copies has been demonstrated, suggesting a high fidelity of gene isolation. TAR cloning technique has been also applied for isolation of a centromeric region from deleted form of the Y human chromosome, delta 1, containing only ~100 kb of alphoid DNA. Despite the small size and loss of a significant part of centromeric repeats, the delta 1 chromosome Y segregates accurately in mitosis, suggesting that the block of alphoid DNA alone or along with the short arm flanking sequences is sufficient for a centromere function. Using a modified version of TAR cloning the centromeric region was rescued in yeast as a set of circular YACs. YAC isolates containing an entire block of alphoid DNA were used for construction of the first small circular Human Artificial Chromosome (HAC). It contains YAC and BAC cassettes, the NeoR selectable mammalian marker, and entire block of alphoid DNA from the delta 1 chromosome Y. When transfected into human cells, it is stably maintained at 1?2 copies per cell. Successful development of the HAC cloning system provides new opportunities for human gene therapy and could have profound effects on our understanding of organization of a human centromeric region and kinetochores function. - Aneuploidy, Chrosome Deletion, DNA, DNA Repair, Genome Vectors, Saccharomyces Cerevisiae