Identification of imprinted genes using a novel screening method based on asynchronous DNA replication

Genomic imprinting refers to the process of epigenetic change that occurs during germ cell development that results in either maternal- or paternal-specific gene expression. Identification of imprinted genes is of primary importance to the understanding of imprinting mechanisms and the role of specific imprinted genes in human disease. Recently, it has been established that chromosomal regions known to contain imprinted genes replicate asynchronously. We propose a novel screening method to identify imprinted genes based on replication asynchrony as a marker for imprinted domains. Dividing human cells were pulse-labeled with BrdU and separated into different fractions of S-phase by flow cytometry. A library of late-replicating inter-Alu sequences should be enriched in gene-associated sequences that replicate early on one chromosome and late on the other homologue. Clones were analyzed for replication timing by hybridization to inter-Alu replication profiles. Candidates for replication asynchrony exhibited broad or biphasic replication timing, and these were analyzed for chromosomal location by hybridizations to inter-Alu products from a hybrid mapping panel. Initial screening of 123 clones resulted in 3 asynchronously-replicating clones that localized to single chromosomes. Chromosome 17 and chromosome 19 candidates might be located in regions thought to be imprinted by synteny with mouse chromosomes. A chromosomemore » 15 clone was further characterized because of its possible localization to the Prader-Willi/Angelman locus. This sequence was localized outside the region deleted in Prader-Willi patients, and was found to be expressed in human cell lines. Replication asynchrony for this sequence appears to be polymorphic because cells derived from some individuals indicated synchronous replication. This appears to be the first example of a polymorphism in replication asynchrony.« less