Replication of a linear mini-chromosome with terminal inverted repeats from the Kluyveromyces lactis linear DNA plasmid k2 in the cytoplasm of Saccharomyces cerevisiae.

The k1 and k2 linear DNA plasmids of Kluveromyces lactis replicate in the cytoplasm under the control of plasmid-encoded genes. These plasmids can also replicate autonomously in the cytoplasm of mitochondrial DNA-deficient strains of Saccharomyces cerevisiae. Essential for replication are plasmid-specific terminal inverted repeats (TIRs) to which a terminal protein (TP) is attached at the 5' ends. A plasmid was constructed with k2 TIRs in opposite orientations and with a selectable marker (URA3) under the control of k1UCS2 (upstream conserved sequence 2, the promoter of k1 open reading frame 2) in between the TIRs. Transformation of k1- and k2-containing S. cerevisiae with a fragment generated by releasing the TIR-flanked fragment from the plasmid by restriction digestion was very efficient, despite the absence of a TP. Transformation was also achieved with a fragment generated by PCR. Southern blotting demonstrated that transformants contained multiple copies of DNA fragments with the same size as the transforming DNA, supporting the hypothesis that these were replicating linear mini-chromosomes. The high frequency of transformation strongly suggests that these mini-chromosomes readily replicate supported by k2. Derivatives with a heterologous gene, firefly luciferase (LUC), expressed luciferase at high levels provided the gene was adjacent to a cytoplasmic plasmid promoter (k2UCS5).