Flow Cytometry and FISH to Investigate Allele-Specific Replication Timing and Homologous Association of Imprinted Chromosomes

  • Janine LaSalle
  • Marc Lalande
Part of the Methods in Molecular Biology™ book series (MIMB, volume 181)

Abstract

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, 5, 6, 7, 8, 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. This possibility was tested directly using three-dimensional (3-D) FISH in spatially intact nuclei and confocal laser scanning microscopy to

Keywords

Angelman Syndrome Rubber Cement Centrifuge Cell Homologous Association Resuspend Cell Pellet 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Humana Press Inc., Totowa, NJ 2002

Authors and Affiliations

  • Janine LaSalle
    • 1
  • Marc Lalande
    • 2
  1. 1.Medical Microbiology and Immunology, School of MedicineUniversity of CaliforniaDavis
  2. 2.Department of Genetics and Developmental Biology, School of MedicineUniversity of ConnecticutFarmington

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