Yeast (Saccharomyces cerevisiae) transposons (Ty elements) are excised from up to 20% of supercoiled plasmids during transformation of yeast cells. The excision occurs by homologous recombination across the direct terminal repeats (deltas) of the Ty element, leaving behind a single delta in the transforming plasmid. Only the initial transforming plasmid is susceptible to excision, and no high frequency excision is observed in plasmids that have become established in transformed cells or in plasmids that are resident in cells undergoing transformation. High frequency excision from plasmids during yeast transformation is not specific for Ty elements and can be observed with other segments of plasmid DNA bounded by direct repeats. The frequency of Ty excision from supercoiled plasmids is greatly reduced when the host yeast cells contain the rad52 mutation, a defect in double-strand DNA repair. When linear or ligated-linear plasmid DNAs containing a Ty element are used for transformation, few or no excision plasmids are found among the transformant colonies. These results suggest that when a yeast cell is transformed with a supercoiled plasmid, the plasmid DNA is highly susceptible to homologous recombination for a short period of time.
Two yeast (Saccharomyces cerevisiae) mutants that tolerate centromere (CEN) plasmids at high copy number have been isolated. The mutations relieve the restraint normally imposed on plasmid copy number by a cloned CEN sequence. Our CEN plasmids specify resistance to G418 and are high copy plasmids only when the mutant host cells are grown on medium containing this antibiotic. The high copy number of the plasmids is independent of the specific cloned CEN sequence and recovered plasmids show no alteration in structure or function of the CEN DNA. The efficiency with which CEN plasmids go to high copy number is increased if the mutant cell is cotransformed by another CEN plasmid. The genomic mutation responsible for the high copy number (COP) is dominant and stable, and it segregates in a Mendelian manner. Homozygous COP/COP a/a diploids do not tolerate CEN plasmids at high copy number, suggesting that the mutation is regulated by mating type. The genomic DNA from both mutant cells contains an altered transposon (Ty) restriction fragment that cosegregates with the COP phenotype in crosses of mutant and wild-type strains. The mutations may be transposon-mediated events that identify a gene involved in centromere or mitotic spindle function.Plasmids containing a cloned centromere (CEN) from yeast (Saccharomyces cerevisiae) function as minichromosomes in yeast cells (1). These cloned CEN sequences retain both the structural and functional characteristics of chromosomal centromeres. Structurally, plasmid-borne CEN sequences have the same distinctive chromatin structure that occurs in the centromere region of yeast chromosomes (2). Functionally, CEN plasmids exhibit three characteristics of chromosomes in yeast cells: they are mitotically stable in the absence of selective pressure; they segregate during meiosis in a Mendelian manner; and they are found at low copy number in the host cell (1). The low copy number of CEN plasmids is not altered by the presence in cis of the yeast 2-,um plasmid amplification system that normally drives plasmids to high copy number (3). When wild-type yeast cells are forced to maintain multiple CEN plasmids bearing independently selectable markers, the cells grow slowly, and cell viability is decreased, indicating a toxic effect from the excess CEN plasmids (4). We have been using the copy control function of cloned CEN sequences in an attempt to identify gene products, such as components of the spindle or kinetochore, that interact with yeast centromeres. We have constructed (5) a plasmid system that permits rapid detection of high copy number plasmids by selecting host cells on antibiotic medium. These plasmids contain the phosphotransferase gene from the bacterial transposon TnS (6) under control of a weak promoter. The product of the phosphotransferase gene inactivates antibiotic G418 by phosphorylation (7), thereby conferring antibiotic resistance upon host cells in proportion to plasmid copy number (5). Using this plasmid system we have obtained two mutant yeast strains that to...
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