The silent mating-type loci of Saccharomyces cerevisiae, HML and HMR, are flanked by transcriptional silencers that have ARS activity (i.e., they function as replication origins when in plasmids). To test whether these ARS elements are chromosomal origins, we mapped origins near HML (close to the left telomere of chromosome III). Our results indicate that the HML-associated ARS elements either do not function as chromosomal replication origins or do so at a frequency below our detection level, suggesting that replication from a silencer-associated origin in each S phase is not essential for the maintenance of transcriptional repression at HML. Our results also imply that the ability of a DNA fragment to function as an ARS element in a plasmid does not ensure its ability to function as an efficient chromosomal replication origin. Telomere proximity is not responsible for inactivating these ARS elements, because they are not detectably functional as chromosomal origins even in genetically modified strains in which they are far from the telomere.The complex process of chromosomal DNA replication in eukaryotes is poorly understood, partly because origins of DNA replication are not well characterized. In Saccharomyces cerevisiae, autonomously replicating sequences (ARS elements) promote efficient plasmid replication and thus permit plasmids containing them to transform yeast cells at high frequency without integration into the genome. ARS elements can be classified as strong or weak, depending on the mitotic stabilities of plasmids containing them. Strong ARS elements promote high mitotic stabilities, and weak ARS elements promote lower mitotic stabilities. The properties of ARS elements suggest that they may serve as replication origins (7,47 see references 40 and 40a for the locations of these ARS elements on chromosome III)-and a weak ARS in tandemly repeated ribosomal DNA (31) are all active as chromosomal replication origins. In ribosomal DNA, however, only 5 to 30% of available ARS elements are active as origins in each cell cycle (31). Whether all the ARS elements in nonrepeated chromosomal DNA are active as replication origins was, until recently, unknown. The experiments described in this paper reveal that some ARS elements in unique DNA do not detectably function as origins in their normal chromosomal environment.In S. cerevisiae, there are two mating types, a and a. Information specifying mating types is present at three * Corresponding author. t Present address: Department of Zoology, Kutir Mahavidyalaya, Chakkey, Jaunpur-222146, U.P., India.locations on chromosome III, the HML, MAT, and HMR loci (Fig. 1). Under normal conditions, only the information at the MAT locus (either a or a) is expressed. Haploid yeast cells can switch mating type by replacing the MAT information with a copy of either the information present at HML (usually a) or the information present at HMR (usually a) (reviewed by Herskowitz [22]). The HML and HMR loci are located near the left and right telomeres of chromosome III, and infor...
The ura4 replication origin region, which is located near the ura4 gene on chromosome III of the fission yeast, Schizosaccharomyces pombe, contains multiple initiation sites. We have used 2D gel electrophoretic replicon mapping methods to study the distribution of these initiation sites, and have found that they are concentrated near three ARS elements (stretches of DNA which permit autonomous plasmid replication). To determine the roles of these ARS elements in the function of the ura4 origin region, we deleted either one or two of them from the chromosome and then assessed the consequences of the deletions by 2D gel electrophoresis. The results suggest that each of the three ARS elements is responsible for the initiation events in its vicinity and that the ARS elements interfere with each other in a hierarchical fashion. It is possible that the large initiation zones of animal cells are similarly composed of multiple mutually interfering origins.
Two dimensional gel electrophoretic techniques were used to locate all functional DNA replication origins in a 22.5 kb stretch of yeast chromosome III. Only one origin was detected, and that origin is located within several hundred bp of an ARS element.
Two-dimensional gel electrophoretic replicon mapping techniques were used to identify all functional DNA replication origins and termini in a 26.5-kbp stretch in the left arm of yeast chromosome m. Only one origin was detected; it coincided with an ARS element (ARS306), as have all previously mapped yeast origins. A replication termination region was identified in a 4.3-kbp stretch at the telomere-proximal end of the investigated region, between the origin identified in this paper and the neighboring, previously mapped, ARS305-associated origin (previously called the A6C origin). Termination does not occur at a specific site; instead, it appears to be the consequence of replication forks converging in a stretch of DNA of at least 4.3 kbp.Replication of eukaryotic chromosomes is accomplished by initiation at multiple replication origins distributed at irregular intervals along DNA molecules. Considerable indirect evidence suggests that these origins correspond to specific nucleotide sequences. However, identification of sequences which serve as origins has been a difficult task.Identification of origins in the yeast Saccharomyces cerevisiae was facilitated by the development of two-dimensional (2D) gel electrophoresis techniques which permit mapping of replication origins and termini (1, 22) and directions of replication (9, 22). These techniques have been used to show that in plasmids (1,7,14) and in chromosomes (11,12,19), replication origins correspond to ARS elements. ARS elements, or autonomously replicating sequences, were identified by Struhl et al. (28) as segments of yeast DNA capable of promoting the autonomous replication of plasmids into which they were inserted.So far, identifications of only six yeast chromosomal origins have been published. One of these, the ARS305-associated origin, is located about 40 kbp from the left end of chromosome III (Fig.
In the budding yeast, S. cerevisiae, two-dimensional (2D) gel electrophoresis techniques permit mapping of DNA replication origins to short stretches of DNA (+/- 300 bp). In contrast, in mammalian cells and Drosophila, 2D gel techniques do not permit precise origin localization; the results have been interpreted to suggest that replication initiates in broad zones (several kbp or more). However, alternative techniques (replication timing, nascent strand polarity analysis, nascent strand size analysis) suggest that mammalian origins can be mapped to short DNA stretches, just like S. cerevisiae origins. Because the fission yeast, Schizosaccharomyces pombe, resembles higher organisms in several ways to a greater extent than does S. cerevisiae, we thought that S. pombe replication origins might prove to resemble--and thus be helpful models for--animal cell origins. An attempt to test this possibility using 2D gel techniques resulted in identification of a replication origin near the ura4 gene on chromosome III of S. pombe. The 2D gel patterns produced by this S. pombe origin indeed resemble the patterns produced by animal cell origins and show that the S. pombe origin cannot be precisely located. The data suggest an initiation zone of 3-5 kbp. Some aspects of the 2D gel patterns detected at the S. pombe origin cannot be explained by the rationale of initiation in broad zones, suggesting that future biochemical and genetic studies of this complex origin are likely to provide information useful in helping to understand the apparent conflict between the 2D gel mapping techniques and other mapping techniques at animal cell origins.
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