1). Genetic linkage studies mapped a locus for hereditary prostate cancer, HPCX, to the long arm of the X chromosome at Xq27. The candidate region contains two clusters of SPANX genes, a SPANX-A/D subfamily including five members, SPANX-A1, SPANX-A2, SPANX-B, SPANX-C, and SPANX-D, and a SPANX-N subfamily including four members, SPANX-N1, SPANX-N2, SPANX-N3, and SPANX-N4, encoding cancer-testis specific antigens. Our previous analysis of the SPANX-B and SPANX-D loci identified several new DNA sequence variants resulted from gene conversion, though none of them was specific for X-linked families (Kouprina et al., 2005). During last fiscal year, we carried out mutational analysis of other SPANX genes (SPANX-A1, SPANX-A2, SPANX-C, SPANX-N1, SPANX-N2, SPANX-N3, and SPANX-N4) localized within the candidate region in the X-linked prostate cancer patients. Sequence analysis of the gene isolates did not reveal any alterations specific for the patients with hereditary prostate cancer. Thus, these and previous results indicate that either a specific combination of SPANX alleles or genomic rearrangements in the SPANX intergenic regions results in the predisposition to prostate cancer in X-linked families.2). Human artificial chromosomes (HACs) provide a unique opportunity to study kinetochore formation and to develop a new generation of vectors with potential in gene therapy. We constructed the first human artificial chromosome with a conditional centromere that can be inactivated by targeted modification of its epigenetic configuration in vivo. The HAC was built up using a DNA array based upon a synthetic alpha-satellite (alphoid) repeat containing one natural monomer, with a binding site for CENP-B (CENP-B box), and one completely synthetic monomer in which the region corresponding to the CENP-B box was replaced with a tetracycline operator (tetO). Binding of the tTA transcriptional transactivator dramatically destabilized the HAC, whilst expression of several other tetracycline-repression fusion proteins had no significant effect on HAC stability. The opportunity to selectively target different proteins into an active kinetochore and thereby regulate centromere function opens the way for an unprecedented mechanistic and structural analysis of the human kinetochore as well as for development of new HAC-based conditional gene expression systems.

Agency
National Institute of Health (NIH)
Institute
Division of Basic Sciences - NCI (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC010413-06
Application #
7337770
Study Section
(LMP)
Project Start
Project End
Budget Start
Budget End
Support Year
6
Fiscal Year
2006
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
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
Kouprina, Natalay; Noskov, Vladimir N; Koriabine, Maxim et al. (2004) Exploring transformation-associated recombination cloning for selective isolation of genomic regions. Methods Mol Biol 255:69-89
Kouprina, Natalay; Pavlicek, Adam; Mochida, Ganeshwaran H et al. (2004) Accelerated evolution of the ASPM gene controlling brain size begins prior to human brain expansion. PLoS Biol 2:E126

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