Search

Fluorescence in situ hybridization (FISH) has been shown to discriminate between unreplicated and replicated regions of the genome in interphase nuclei, based on the number of specific fluorescent signals that can be detected. By examining the replication status of hybridizing sequences in large numbers of individual cells from an asynchronously growing population, it is possible to deduce a relative order of replication of different sequences. The availability of well-mapped genomic probes and the ability to compare results from different cell lines make this a convenient approach with which to map domains of replication timing control at any chromosomal position and to relate this to various patterns of gene expression. Since there appear to be important but poorly understood correlations among replication timing, chromatin structure, and transcriptional competence in mammalian cells, this provides a valuable approach to understanding these interrelationships at the molecular level. The procedures for using FISH to examine replication timing in mammalian nuclei are described here in detail, and the advantages and limitations of the approach are discussed. Some other strategies for using high-resolution FISH on chromatin fibers to examine replication properties of specific sequences in situ are also described.

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.