A new system for amplifying 2 μm plasmid copy number in Saccharomyces cerevisiae

Abstract

The yeast 2 microns plasmid is found in the nucleus of almost all Saccharomyces cerevisiae strains. Its replication is very similar to that of chromosomal DNA. Although the plasmid does not encode essential genes it is stably maintained in the yeast population and exhibits only a small, though detectable, loss rate. This stability is achieved by a plasmid-encoded copy-number control system which ensures constant plasmid levels. For the investigation of 2 microns replication, a yeast strain that is absolutely dependent on this plasmid was constructed. This was achieved by disruption of the chromosomal CDC9 gene, coding for DNA ligase and providing this essential gene on a 2 microns-derived plasmid. This plasmid is absolutely stable under all growth conditions tested. Using the temperature-sensitive mutant allele cdc9-1 we have developed an artificial control system which allows one to change the copy number of 2 microns-derived plasmids solely by changing the incubation temperature.

A Saccharomyces cerevisiae autoselection system for optimised recombinant protein expression

“…A second approach uses hosts such as srb1 or pgk1 mutants that cannot grow on normal rich medium without complementation from plasmid SRB1 or PGK1 genes (Ayub et al, 1992;Rech et al, 2002). In a third strategy where plasmid shuffling was used to replace CDC9 and FBA1 maintenance plasmids (Unternahrer et al, 1991;Compagno et al, 1993) shuffling was 'passive' and required screening for loss of the maintenance plasmids. Here, a more active, 5FOA-driven plasmid shuffling step provides a simpler and more reproducible means for establishment of stable expression clones.…”

“…Autoselection was first described in 1983 for an E. coli system where plasmids carrying a λ phage repressor prevented killing by λ prophage that could not make its own repressor (Rosteck and Hershberger, 1983). Since then there have been several reports of yeast autoselective systems (Loison et al, 1986(Loison et al, , 1989Unternahrer et al, 1991;Ayub et al, 1992;Napp and Da Silva, 1992;Rech et al, 2002;Compagno et al, 1993;Compagno et al, 1996;Hsieh and Da Silva, 1998;van Rensburg et al, 1998). However, only one system, relying on the rescue of lethality of fur1 ura3 mutants by URA3 plasmids (Loison et al, 1986), has any history of multiple, yet limited, usage after the initial publication (for example (Loison et al, 1989;van Rensburg et al, 1996;van Rensburg et al, 1997;van Rensburg et al, 1998;Dussossoy et al, 1999;Setati et al, 2001;La Grange et al, 2001;Gorgens et al, 2004)).…”

2‐μm vectors containing the Saccharomyces cerevisiae metallothionein gene as a selectable marker: Excellent stability in complex media, and high‐level expression of a recombinant protein from a CUP1‐promoter‐controlled expression cassette in cis

“…To circumvent this need, autoselection systems have been devised (e.g. Unternahrer et al, 1991;Rech et al, 1992;Napp and Da Silva, 1993, and references therein). Albeit elegant, such systems are not ideally suited for routine work since they usually require both the use of house-keeping plasmids (see, however, Rech et al, 1992) and the introduction ofsometimes extensive (Napp and Da Silva, 1993)genetic alterations into the host.…”

Selection of yeast cells with a higher plasmid copy number in a Saccharomyces cerevisiae autoselection system

“…In such systems, selection of the transformed population occurs independently of the growth medium composition, allowing the use of relatively inexpensive complex media which are preferred in industry for commercial-scale fermentations. Different autoselection systems have been developed for Saccharomyces cerevisiae host cells (Loison et al, 1986;Piper and Curran, 1990;Unternahrer et al, 1991;Rech et al, 1992;Ayub et al, 1992;Ludwig et al, 1993). The efficiency of these systems is related to the opportunity to use rich media which yield increased recombinant protein productions (Loison et al, 1989;Napp and Da Silva, 1993).…”