Box 13.1Replication of the yeast genome

Microarray analysis is being used to examine the dynamics of origin firing and fork migration in replicating yeast chromosomes.

Research into genome replication is entering a new phase. Attention is still focused on the detailed biochemical events occurring at individual replication forks, but these studies are now being supplemented by new experimental strategies that have been made possible by the availability of complete genome sequences. These new strategies, which are being pioneered with the yeast Saccharomyces cerevisiae, are providing a global view of the pattern and dynamics of replication across entire genomes. Research during the 1990s had shown that, as in all eukaryotes, replication of individual yeast chromosomes follows a consistent pattern from one cell cycle to the next, the region around the centromere usually being one of the first parts of a chromosome to be replicated, and the telomeres being replicated towards the end of S phase. But these features of genome replication had been extrapolated from studies of just a few origins, and it was generally agreed that further progress would require a new experimental strategy that enabled the activities of many origins to be studied at the same time. The new strategy that has been devised combines an established approach to genome replication - labeling with heavy isotopes - with the latest advances in microarray technology.

Compared with other eukaryotes, yeast has a relatively small genome (see Table 2.1). This is a major advantage in global studies of genome activity because it means that microarrays representing all of the genome can be prepared quite easily, using a set of cDNAs or oligonucleotides specific for every one of the 6000 yeast genes. In Section 3.2.3 we saw how cDNA probing of microarrays has been used to follow the global pattern of gene expression during the yeast sporulation pathway. Can a similar approach be used to follow the pattern of origin activation during S phase?

Microarray analysis is based on hybridization probing, so to use a microarray to follow the pattern of genome replication it will be necessary to devise a way of separating unreplicated DNA from replicated DNA, so that one or the other fraction can be used as a hybridization probe. If, for example, a sample of replicated DNA could be obtained from cells that had just entered S phase, then this DNA could be used to probe the microarray in order to identify those genes that have already been replicated at this early stage. A second probe, prepared from replicated DNA from slightly later in S phase, would identify the next set of genes to be replicated, and so on. When data have been collected for all of S phase, the gene identities could be correlated with the genome map to chart the pattern of replication for each chromosome. But how can samples of replicated DNA be prepared? The problem has already been solved, way back in 1958 by Meselson and Stahl, who used heavy-isotope labeling to distinguish between old and new DNA during replication of the Escherichia coli genome (Section 13.1.1). By growing bacteria in medium containing ¹⁵NH₄Cl they prepared cells containing ¹⁵N-labeled double-stranded DNA. Transfer to normal medium, followed by one round of genome replication, gave rise to daughter cells whose DNA molecules were hybrids, comprised of one strand of ¹⁵N-DNA and one strand of ¹⁴N-DNA (see Figure 13.3A). A similar approach can be used with yeast: grow the cells in medium with heavy nitrogen, transfer to normal medium, and allow the cells to enter S phase. Extract the DNA, treat it with a restriction endonuclease, and fractionate by density gradient centrifugation. Two bands are seen. One is made up of fragments of ¹⁵N-¹⁵N-DNA, derived from the unreplicated component of the genome. The other band is ¹⁴N-¹⁵N-DNA and hence is derived from the regions that have undergone replication. Purify the ¹⁴N-¹⁵N-DNA, label it with a radioactive or fluorescent marker, and apply to the microarray (see figure on facing page).