Johonnett, Peter; (2001) Replication Timing Analysis of the Major Histocompatibility Complex on Human Chromosome 6. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

We employ fluorescence in situ hybridisation (FISH) to assay DNA replication in the human major histocompatibility complex (MHC). We examine whether features in the primary sequence and/or differences in transcription effect changes in replication timing. We perform these analyses on interphase chromosomes in the fixed S-phase nuclei of B-cells and fibroblasts. The MHC shows later replication in the distal end of classical class II region, termed here the "HLA-DQ/DR" locus. The rate of change in mean replication time from the class III region to the HLA-DQ/DR locus is consistent with a replication fork moving within the accepted rate of between 0.3-6Kb/min. This is contrary to the suggestion that there is a "precise switch" from early to late replication across this region. We show tissue-specific loci replicating earlier in B-cells than fibroblasts. However, the HLA-DQ/DR locus is later replicating regardless of whether or not it is expressed, providing the first evidence for an active gene cluster that is late replicating. We incubate fibroblasts with interferon-gamma in order to induce class II expression. Such cells adopt a replication timing profile similar to that in B-cells, including the late replication of the expressed HLA-DQ/DR locus. These data question the extent to which transcription influences replication time and suggest additional factors may be involved (at least in determining later replication). In the class III region, the pattern of peaks and troughs in the data are remarkably similar in each replication profile, helping us to identify a replicon in the MHC. Two-colour FISH analysis confirm the existence of this replicon and provide the first demonstration that replicons separated by >1Mb of interphase chromatin can be simultaneously replicated. We suggest that in some cells, replicons in the class II and III regions may share the same replication machinery.

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