On the level of plasmid-bearing cells in transformed cultures of Saccharomyces cerevisiae.
LC1, a YIP5-derived plasmid containing a human DNA fragment with ARS activity in yeast, has been used to study the replication of ARS plasmids in Saccharomyces cerevisiae. ARS plasmids carried in yeast hosts are normally mitotically unstable. In transformed cultures the fraction of cells that contain plasmid, measured by plating on selective media, is lower than would be expected from measured rates of plasmid loss. In the case of S. cerevisiae carrying either the plasmid LC1 or YRP17, the assay yields values of the order of 10-20% or 30-50% respectively. We have found that by doing a double nutritional upshift that involves conditioned medium and casamino acids, a population of cells can be defined that carry plasmid but are unable to grow on media that select for the plasmid marker. Thus the total fraction of cells that can be shown to contain plasmid increases to greater than 70%. To distinguish between the inability of plasmid to replicate in these cells and lack of expression of the selectable gene, cultures grown from single cells were analysed for the presence of plasmid DNA. In a substantial fraction of the population, plasmid DNA could be detected only by polymerase chain reaction and not by standard blotting and hybridization. These results suggest that plasmid is unable to replicate in these cells. Growth kinetics experiments with transformed cultures are consistent with the notion that only a small fraction of the cells contains plasmid capable of replication upon dilution into selective medium. Possible explanations for the phenomena observed are discussed.
Guerrini
A M
AM
Dipartimento di Biologia Cellulare e dello Sviluppo, Universitá La Sapienza, Rome, Italy.
Boglione
C
C
Ascenzioni
F
F
Donini
P
P
D003470:Culture Media; D004261:DNA Replication; D004271:DNA, Fungal / Q000032:analysis* / Q000096:biosynthesis; D015966:Gene Expression Regulation, Fungal; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D014170:Transformation, Genetic
D003470:Culture Media; D004271:DNA, Fungal