Active role of a human genomic insert in replication of a yeast artificial chromosome.

Yeast artificial chromosomes (YACs) are important cloning tools for the analysis of complex genomes such as that of humans. They allow the maintenance, propagation, and analysis of regions of such genomes in an experimentally tractable system, the yeast Saccharomyces cerevisiae. Despite the fact that only one yeast origin of replication is provided by the YAC vector, YACs can maintain cloned inserts of several hundred kilobase pairs (8). However, a major drawback to the use of YACs is the high frequency of regions in the human genome that are unstable in YACs. Such unstable YACs contain inserts that cannot be stably maintained as full-length molecules and are deleted upon propagation in yeast. The instability results in gaps during physical mapping and gene hunting efforts. Examples of regions with unstable YACs or unclonable regions include the Huntington’s disease locus (3), the BRCA1 region (2), and the Bloom’s syndrome region (45). If replication initiates only from the single origin located close to the end of the YAC molecule, it is unlikely that complete replication of this molecule could occur within the length of a normal S phase (approximately 25 min), given that the replication fork rate in yeast is ∼4 kb/min at 23°C (39). YACs are often as large as native yeast chromosomes, and replication of even the smallest yeast chromosomes, which are less than 300 kb, occurs by initiation at multiple sites throughout the length of the molecule (17, 33, 52). It is therefore possible that sites within the human DNA serve as origins of replication in yeast to complete replication of large YACs and that these origins are essential for stable YAC maintenance. Early work suggested that mammalian genomes have sequences that are capable of providing autonomous replication of plasmids in yeast (51). Autonomously replicating sequences (ARSs) are identified by their ability to promote high-frequency transformation and stable extrachromosomal maintenance of plasmids (44). These A/T-rich ARSs correspond to plasmid replication origins (6). Restriction fragments from the mouse genome are capable of ARS activity in yeast, and the frequency of these sequences is estimated to be 1 per 24 kb (41). Some human sequences have been shown to have ARS activity in yeast as well (30). Given that the base pair compositions of the human genome and the yeast genome are similar (approximately 60 and 62% A/T, respectively), it may not be surprising to find that human sequences are capable of ARS activity in yeast. However, since many sequences in the yeast genome are capable of ARS activity on plasmids but are weak chromosomal replication origins (17, 52) or are not active chromosomal replication origins at all (13, 33), the ability of human sequences to provide ARS activity on a plasmid is not necessarily an indication that these same sequences act as replication origins in the context of a YAC. Using DNA fiber-fluorescent in situ hybridization on yeast cells with a YAC containing human DNA from the Duchenne muscular dystrophy gene, Rosenberg et al. (40) found structures that suggested the presence of two replication origins within the YAC insert. However, these replication origins were never confirmed by using other techniques, nor were they more precisely localized. Here we report a systematic study of how yeast replicates a large insert of human DNA in a YAC. We scanned the 240-kb YAC insert to determine whether sequences within it were initiating replication. Replication origins were found. They are discrete units that appear to have the same sequence requirements as yeast chromosomal replicators. In addition, the origin spacing, variation in efficiencies of activation, and variation in times of activation in the S phase are all similar to those of yeast chromosomes. The presence of multiple sites of replication initiation in the human insert of this stable YAC suggests that regions lacking these fortuitous sequences will have a replication defect in yeast that could jeopardize the integrity and maintenance of the molecule and could be a biological basis of YAC instability.