) In multicellular eukaryotes chromosomal DNA is functionally dynamic in different cells, tissues, or stages of development. Therefore, an important aspect of investigating epigenetic mechanisms of gene expression is an understanding of the functional organization of chromosomes within their native cellular environment. Although metaphase chromosomes have been the standard subject of cytogenetic studies, interphase chromosomes are in a much more dynamic state as they undergo replication, transcription, methylation and chromatin organization during the cell cycle. The recent demonstration of a temporal and spatial association of chromosome 15q 11-13 regions that occurs in the late S phase of the cell cycle in human somatic lymphocytes was found to be deficient in cell, from patients with the parental imprinting disorders Angelman and Prader-Willi's syndromes. Homologous association was also observed at the Beckwith-Wiedemann syndrome locus at 11p15, a different imprinted locus implicated in tumorigenesis. These results demonstrate that chromosomal alteration such a uniparental disomy, deletion, or translocation can alter the normal dynamics of chromosome; during the cell cycle. However, a large scale human genome-wide search has not been performed to determine if other chromosomal domains exhibit transient interactions during the cell cycle. The existing method for examining transient chromosome interactions requires a large number of cultured primary lymphocytes, two very expensive instruments and long hours of data analysis. Cells are first fractionated on the basis of DNA content by flow cytometry, then analyzed for fluorescence in situ hybridization (FISH) signals in three- dimensionally intact nuclei by confocal laser cytometry (CLSM), then measured for 3D intersignal distance, then the data are statistically analyzed. In this proposal, a potentially more efficient and cost-effective method for the analysis of cell cycle-specific chromosome dynamics by laser scanning cytometry (LSC) is outlined so that a large-scale investigation chromosomes may be performed.
| Balmer, Damina; Goldstine, Jared; Rao, Y Manjula et al. (2003) Elevated methyl-CpG-binding protein 2 expression is acquired during postnatal human brain development and is correlated with alternative polyadenylation. J Mol Med 81:61-8 |
| LaSalle, J M; Goldstine, J; Balmer, D et al. (2001) Quantitative localization of heterogeneous methyl-CpG-binding protein 2 (MeCP2) expression phenotypes in normal and Rett syndrome brain by laser scanning cytometry. Hum Mol Genet 10:1729-40 |
| Liu, H L; Gandour-Edwards, R; Lara Jr, P N et al. (2001) Detection of low level HER-2/neu gene amplification in prostate cancer by fluorescence in situ hybridization. Cancer J 7:395-403 |
