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 kinetochore?s function. - Aneuploidy, Chrosome Deletion, DNA, DNA Repair, Genome Vectors, Saccharomyces Cerevisiae
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| Kouprina, N; Nikolaishvili, N; Graves, J et al. (1999) Integrity of human YACs during propagation in recombination-deficient yeast strains. Genomics 56:262-73 |
| Larionov, V (1999) Direct isolation of specific chromosomal regions and entire genes by TAR cloning. Genet Eng (N Y) 21:37-55 |
