Saccharomyces cerevisiae mutants that tolerate centromere plasmids at high copy number.

Abstract

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...

A system for the analysis of yeast ribosomal DNA mutations.

“…The elevated copy number is also not due to poor transcription of the selectable marker as was reported for CEN-ARS plasmids carrying the his3-A4 allele (3) since highand low-copy-number transformants have the same mitotic division rate under selective growth conditions (data not shown). We rather think that S. cerevisiae MG34 used for transformation is less stringent in one of the factors involved in the accommodation of CEN-bearing plasmids upon transformation, for instance, the product of the recently identified cop gene (21). Preliminary transformation experiments with S. cerevisiae HR2 as a host did not reveal high-copy-number transformants with pORCS plasmids.…”

Latent Effects of Hsp90 Mutants Revealed at Reduced Expression Levels

“…In addition, the copy number of the CEN plasmids utilized in this study is regulated, as cells maintaining multiple CEN plasmids grow slowly [60]. In addition, the low copy number of CEN plasmids is dominant to the addition of high copy genetic elements [61] and genetic alterations that increase CEN abundance are rare [62]. Nonetheless, CEN plasmids are not as stable as chromosomally encoded DNA, which may lead to a small amount of noise in our measurements.…”

Deletion of Budding Yeast MAD2 Suppresses Clone-to-Clone Differences in Artificial Linear Chromosome Copy Numbers and Gives Rise to Higher Retention Rates

“…We employed the classic genetic fluctuation test and developed a qPCR-based method to determine the artificial linear chromosome copy numbers per cell in a population ( Figure 1 ). We initially applied the assay to measure the average number of artificial circular plasmids per cell as a control, where previous reports have established circular chromosomes are carried at 1–2 copies per cell on average [ 3 , 4 , 5 , 6 , 7 ]. Our initial results were in good agreement with this, where average chromosome copy numbers ranged between (0.96 ± 0.23) up to (2.19 ± 0.96) in a variety of wild type MAD2 and mutant mad2∆ haploid and diploid clones ( Table S1 ).…”

“…There is broad interest in using budding yeast artificial short-linear chromosomes as a tool to study the mechanisms of chromosome segregation in mitosis and as a technology to carry large fragments of exogenous DNA for recombinant protein expression, for bioengineering of entire metabolic pathways, where all of the pathway enzymes are carried on a single large artificial chromosome, or for amplification of exogenous DNA clones, such as fragments of the human or mouse genomes or other organisms [ 1 , 2 ]. Artificial linear chromosomes also have the advantage of containing telomeres, a centromere, and an origin of replication all of which can contribute to a high rate of chromosome segregation fidelity relative to other smaller circular plasmids that lack all of these elements [ 3 , 4 , 5 , 6 , 7 ]. These chromosome elements also allow artificial linear chromosomes to maintain a presence in a population of yeast even in the absence of selection, at least for short durations of time.…”