High frequency recombination and the expression of genes cloned on chimeric yeast plasmids: Identification of a fragment of 2-μm circle essential for transformation.

We have carried out experiments aimed at explaining the observed variations in transformation frequencies when Saccharomyces cerevisiae or Saccharomyces carlbergensis are transformed with chimeric plasmids that contain one of 4 possible EcoRI fragments of the yeast 2-μm circle. These plasmids fall into 2 classes when used to transform 2 different yeast his3 auxotrophs, one (strain LL20) harbours indigenous 2-μm circle, and the other (strain YF233) is devoid of this plasmid. Hybrid plasmids containing either the 2.4 mega-dalton (mD) R-form EcoRI fragment (pYF88) or the l.4 mD L-form EcoRI fragment (pYF177) of 2-μm circle transform either of the two hosts at a high frequency (50,000 colonies per Mg in LL20 and 10,000 colonies per μg in YF233). Hybrid plasmids containing the 1.5 mD R-form EcoRI fragment (pYF87) or the 2.5 mD L-form EcoRI fragment (pYF178) of the 2-μm circle transform LL20 at a reduced frequency (6,000-16,000 colonies per μg) and YF233 at extremely low frequencies (1-5 colonies per μg). All plasmids retrieved from strain YF233 that had been transformed with pYF88 or pYF177 were identical to the original transforming plasmid. Of the plasmids retrieved from strain LL20 that had been transformed with pYF87 and pYF178, approximately half had acquired an extra copy of the 2-μm circle. Of the plasmids retrieved from strain LL20 that had been transformed with pYF88 and pYF177, an average of only approximately 13% had acquired an extra copy of 2-μm circle. Taken together, these observations indicate that the transformation of yeast by a plasmid lacking the ability to replicate (pYF87 and pYF1780) occurs by the recombinational acquisition of 1 copy of the host 2-μm circle, which serves to supply the incoming plasmid with missing essential sequences. A comparison of 2-μm circle DNA fragments carried by pYF88 and pYF177 indicates that the region of 2-μm circle required for high frequency transformation is a 1.2 mD segment that is common to the 2.4 mD R-form and 1.4 ml) L-form EcoRI fragments. This region extends from the EcoRI cut site adjacent to the PstI site, through to the end of the inverted repeat. However, the inverted repeat sequence alone is not sufficient to bestow high frequency transformation of yeast.