Genes VII

Any segment of DNA that has an origin should be able to replicate. So although plasmids are rare in eukaryotes, it may be possible to construct them by suitable manipulation in vitro. This has been accomplished in yeast, although not in higher eukaryotes.

S. cerevisiae mutants can be "transformed" to the wild phenotype by addition of DNA that carries a wild-type copy of the gene. Some yeast DNA fragments (when circularized) are able to transform defective cells very efficiently. These fragments can survive in the cell in the unintegrated (autonomous) state, that is, as self-replicating plasmids.

A high-frequency transforming fragment possesses a sequence that confers the ability to replicate efficiently in yeast. This segment is called an ARS (for autonomously replicating sequence). ARS elements are derived from authentic origins of replication; and initiation occurs at the locations of ARS elements in a chromosome.

Sequences with ARS function occur at about the same average frequency as origins of replication. Where ARS elements have been systematically mapped over extended chromosomal regions, it seems that only some of them are actually used to initiate replication. The others are silent, or possibly used only occasionally. If it is true that some origins have varying probabilities of being used, it follows that there can be no fixed termini between replicons. In this case, a given region of a chromosome could be replicated from different origins in different cell cycles.

An ARS element consists of an A PT-rich region that contains discrete sites in which mutations affect origin function. Base composition rather than sequence may be important in the rest of the region. Figure 12.10 shows a systematic mutational analysis along the length of an origin. Origin function is abolished completely by mutations in a 14 bp "core" region, called the A domain, that contains an 11 bp consensus sequence consisting of A PT base pairs. This consensus sequence is the only homology between known ARS elements (Marahrens and Stillman, 1992).

Mutations in three adjacent elements, numbered B1-B3, reduce origin function. An origin can function effectively with any 2 of the B elements, so long as a functional A element is present. (Imperfect copies of the core consensus, typically conforming at 9/11 positions, are found close to, or overlapping with, each B element, but they do not appear to be necessary for origin function.)

The ORC (origin recognition complex) is a complex of 6 proteins with a mass of ~400 kD. ORC is associated with ARS elements throughout the cell cycle, so initiation may depend on changes in its condition rather than de novo association with an origin. We discuss this in more detail in the next chapter. Counterparts to ORC are found in higher eukaryotic cells.

ARS elements satisfy the classic definition of an origin as a cis-acting sequence that causes DNA replication to initiate. Are similar elements to be found in higher eukaryotes? Difficulties in finding sequences comparable to ARS elements that can support the existence of plasmids in higher eukaryotic cells suggest the possibility that origins may be more complex (or determined by features other than discrete cis-acting sequences). There are suggestions that some animal cell replicons may have complex patterns of initiation: in some cases, many small replication bubbles are found in one region, posing the question of whether there are alternative or multiple starts to replication, and whether there is a small discrete origin. It is fair to say that the nature of the higher eukaryotic origin remains to be established (for review see DePamphlis, 1993).