Analysis of replication timing properties of human X-chromosomal loci by fluorescence in situ hybridization.

We have used fluorescence in situ hybridization on interphase nuclei of normal female cells to compare the replication timing patterns of genes on the human X chromosome that are known to escape X inactivation with those that are inactivated. By this procedure it was possible not only to determine the relative time of replication of the earlier-replicating allele for different loci but also to estimate the degree of asynchrony of replication of the two alleles for each individual locus. Loci such as HPRT and FRAXA, which are normally inactivated, displayed a high degree of replication asynchrony, whereas loci that are not inactivated (ZFX and RPS4X) were found to replicate very synchronously. Interestingly, examination of XIST, which is expressed only from the inactive X chromosome, by this procedure revealed that it also replicated asynchronously, with the expressed copy apparently replicating first. Therefore, by examining different loci from the X chromosome it was determined that there is a strict correlation between the expression and relative time of replication of individual genes.