Abstract

Using the recently developed TAR (Transformation-Associated Recombination) cloning technique, 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. In addition, entire copies of four human genes including metastasis-suppressor gene KAI1, a paternally expressed gene Ubiq and the telomerase reverse transcriptase hTERT gene 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.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
Division of Basic Sciences - NCI (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC010413-01
Application #
6423821
Study Section
(LGD)
Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
2000
Total Cost
Indirect Cost

  Institution

Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code

  Publications

Kouprina, Natalay; Earnshaw, William C; Masumoto, Hiroshi et al. (2013) A new generation of human artificial chromosomes for functional genomics and gene therapy. Cell Mol Life Sci 70:1135-48
Kouprina, Natalay; Lee, Nicholas C O; Pavlicek, Adam et al. (2012) Exclusion of the 750-kb genetically unstable region at Xq27 as a candidate locus for prostate malignancy in HPCX1-linked families. Genes Chromosomes Cancer 51:933-48
Kouprina, Natalay; Larionov, Vladimir (2008) Selective isolation of genomic loci from complex genomes by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae. Nat Protoc 3:371-7
Nakano, Megumi; Cardinale, Stefano; Noskov, Vladimir N et al. (2008) Inactivation of a human kinetochore by specific targeting of chromatin modifiers. Dev Cell 14:507-22
Leem, S-H; Yoon, Y-H; Kim, S I et al. (2008) Purification of circular YACs from yeast cells for DNA sequencing. Genome 51:155-8
Kouprina, Natalay; Noskov, Vladimir N; Pavlicek, Adam et al. (2007) Evolutionary diversification of SPANX-N sperm protein gene structure and expression. PLoS ONE 2:e359
Okamoto, Yasuhide; Nakano, Megumi; Ohzeki, Jun-ichirou et al. (2007) A minimal CENP-A core is required for nucleation and maintenance of a functional human centromere. EMBO J 26:1279-91
Kouprina, Natalay; Noskov, Vladimir N; Solomon, Greg et al. (2007) Mutational analysis of SPANX genes in families with X-linked prostate cancer. Prostate 67:820-8
Leem, Sun-Hee; Kouprina, Natalay; Grimwood, Jane et al. (2004) Closing the gaps on human chromosome 19 revealed genes with a high density of repetitive tandemly arrayed elements. Genome Res 14:239-46
Grimwood, Jane; Gordon, Laurie A; Olsen, Anne et al. (2004) The DNA sequence and biology of human chromosome 19. Nature 428:529-35

Showing the most recent 10 out of 13 publications