Yeast centromere sequences do not confer mitotic stability on circular plasmids containing ARS elements of Tetrahymena thermophila rDNA.
We have previously demonstrated that a 657 bp TaqI-XbaI and a 427 bp XbaI-XbaI fragment from the 5' non-transcribed spacer of the extrachromosomal ribosomal DNA of Tetrahymena thermophila function as autonomously replicating sequences (ARS) in Saccharomyces cerevisiae. These fragments are adjacent to each other in a region that encompasses the in vivo origin of bidirectional replication of rDNA. The presence of a yeast centromere (CEN) fragment does not confer mitotic stability on these plasmids. A sensitive yeast colony colour assay (Hieter et al. 1985a) has been used to evaluate the cis-acting effect of each ARS segment on the pattern of inheritance of a plasmid containing CEN5:URA3:SUP4. Colonies of transformed cells obtained both in the presence and absence of selection were red with no detectable white or pink sectors. The lack of sectoring indicates that both plasmids are lost at an extremely high rate, likely due to 1:0 segregation events. We conclude that while these ARS elements confer a high frequency transformation phenotype, they lack a function which is required in cis for the maintenance of mitotic stability in the presence of a centromere. This missing cis-acting function may result in the inability of the plasmids to be brought under the control of cell-cycle regulated replication.
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Department of Biology, York University, Toronto, Ontario, Canada.
Pearlman
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D000818:Animals; D002503:Centromere / Q000502:physiology*; D002875:Chromosomes / Q000502:physiology*; D004261:DNA Replication; D004262:DNA Restriction Enzymes; D004275:DNA, Ribosomal / Q000235:genetics*; D008938:Mitosis; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013768:Tetrahymena / Q000166:cytology / Q000235:genetics*
D004275:DNA, Ribosomal; D004262:DNA Restriction Enzymes