Walton L. Fangman and Bonita J. Brewer Department of Genetics SK-50 University of Washington Seattle, Washington 98195 Each chromosome of a eukaryotic organism contains many replication origins (Figure 1) from which DNA is effi- ciently duplicated during S phase of the cell cycle. Recent work raises new questions about the nature of these ori- gins and their regulation. What Are Origins? Like initiation of transcription at a promoter, initiation of replication at an origin can be thought of as requiring three discrete steps: the recognition of one or more cis-acting elements by specific initiation proteins, the localized un- winding of the DNA helix, and the selection of a site for the initiation of polymerization. Continuing the analogy to a promoter, it is clear that these three steps do not necessar- ily have to occur at the same site and may differ greatly in their degree of sequence specificity. Assays to detect ori- gin function may detect one or more of these steps of initiation and thus may give different results if the events occur at different sites. Replication origins have been best defined in the yeast Saccharomyces cerevisiae. The autonomous replication sequence (ARS) assay, which detects the cis-acting se- quences required for origin function in plasmids, has al- lowed mutational analysis with increasing resolution (Mar- ahrens and Stillman, 1992). ARS elements consist of only 100-200 bp and include a conserved 11 bp core consen- sus sequence and several other less conserved elements that are required for or enhance the maintenance of a plasmid. Mapping of the site of initiation by two-dimen- sional gels (Brewer and Fangman, 1987; Huberman et al., 1987) reveals that replication initiation on plasmids occurs in the vicinity of the ARS element (within a few hundred base pairs of the ARS consensus). The exact size of the initiation zone and the locations of priming sites for DNA chain elongation are yet to be determined. Two-dimensional gel analysis of replicating chromo- somal DNA fragments has shown that many but not all ARS elements are active as origins in the chromosome, and that initiation is dependent on a functional ARS ele- ment (Dubey et al., 1991; Rivier and Rine, 1992; Desh- pande and Newlon, 1992). Quantitative assessments show that specific origins can have eff iciencies of activation that range from .90 per cell cycle (Dubey et al., 1991; Ferguson et al., 1991). The basis for differences in effi- ciency is not known. Do All Eukaryotes Have Specific Origins? A simple, reliable ARS assay has not been achieved for any eukaryote other than yeast. However, by physical means, examples of specific initiation have been observed in both Physarum and Tetrahymena (Benard and Pierron, 1992; Cech and Brehm, 1981). In these cases, the speci- ficity of initiation can be interpreted as evidence for specific