Flow cytometry and FISH to investigate allele-specific replication timing and homologous association of imprinted chromosomes.
Chromosome replication banding studies show that homologous regions on a pair of autosomes generally replicate at the same time in S phase (1). Izumikawa et al. first observed that this was not the case for the imprinted chromosomal region 15q11-q13 (2). This observation has been confirmed in other replication banding studies (3) as well by the fluorescence in situ hybridization (FISH) replication assay (4-9). The latter technique has also been used to observe DNA replication asynchrony in association with allelic inactivation of genes such as those encoding olfactory receptors and the cytokine, interleukin 2 (10,11). The latter genes are not imprinted but display random silencing of an allele in individual cells. In imprinted regions, DNA replication was generally observed to occur earlier on the paternal homologue (5,6,9,12,13). The patterns of allele-specific replication in the cells of Prader- Willi (PWS) and Angelman syndrome (AS) patients, however, have generally been synchronous (5,6,14). Furthermore, an investigation of the kinetics of allele-specific replication timing in the GABRB3/A5 cluster on 15q11-13 revealed that cells from PWS and AS have lost the strict replication timing observed on the parental chromosomes of normal cells (12). These results suggested the requirement of a biparental contribution for the regulation of replication asynchrony and lead to the hypothesis that allelic cross-talk, perhaps via pairing of homologous chromosomes, might play a role in the imprinting process.
LaSalle
J
J
Medical Microbiology and Immunology, School of Medicine, University of California, Davis, CA, USA.
Lalande
M
M
D000483:Alleles; D000818:Animals; D002478:Cells, Cultured; D004261:DNA Replication / Q000235:genetics*; D005434:Flow Cytometry / Q000379:methods*; D018392:Genomic Imprinting / Q000235:genetics*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*