Of Work: 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 a work was focused on further optimizing of TAR cloning to make the technology more widely used in the scientific community. Set of experiments on isolation of unique chromosomal regions has demonstrated that the minimal length of a targeting sequence required for gene isolation by TAR cloning is approximately 60 bp. These observations greatly facilitate selection and generation of hooks for isolation of specific regions as well as construction of TAR vectors, because the hooks can be synthesized as oligonucleotides instead of being isolated as genomic fragments. In addition, entire copies of three human disease genes including metastasis-suppressor gene KAI1 and a paternally expressed gene Ubiq (including all regulatory regions) were successfully isolated for further functional studies. TAR cloning technique has been also applied for isolation and characterization of centromeric regions of human chromosome. As a part of this study a complete sequence of centromeric region of the human Y chromosome was determined. This centromeric fragment containing ~140 kb of alphoid DNA was used for construction of small circular Human Artificial Chromosome lacking telomeres(HAC). When transfected into human cells, it is stably maintained at 1?2 copies per cell. Development of the HAC cloning system provides new opportunities for human gene therapy and could have profound effects on our understanding of mechanisms of human chromosome segregation.
| Humble, M C; Kouprina, N; Noskov, V N et al. (2000) Radial transformation-associated recombination cloning from the mouse genome: isolation of Tg.AC transgene with flanking DNAs. Genomics 70:292-9 |
| 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 |
