Creation of a functional promoter by rearrangement in a Kluyveromyces lactis linear plasmid

The expression of genes from cytoplasmic killer plasmids in the yeast Kluyveromyces lactis depends on their own specific transcription system. Therefore, the kanamycin/G418-resistance-encoding gene, KmR, under its natural promoter cannot be expressed when integrated into the pGKL1 plasmid. However, one G418R transformant clone was isolated. The resistance was due to the presence of two modified plasmids, k1-kan2a (10.4 kb) and k1-kan2b (5.2 kb) which were derivatives of pGKL1 containing the KmR gene. In these mutant plasmids, a large part of pGKL1 has been replaced by the KmR gene harboring a rearranged 5'-flanking region extending over 600 bp. This new DNA sequence has been cloned and sequenced. The rearranged sequence allowed the KmR gene to be expressed at high level, enabling the transformant cells to grow on a medium containing G418 at 2 mg/ml. This high level of resistance was found to be due to increased transcription of the KmR gene. Primer extension experiments suggested that the rearranged upstream region of KmR contained transcription promoting sites recognized by the killer-plasmid-specific transcription system.