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 syste m. Therefore, the kanamycin/G418-resistance-encoding gene, Km(R), unde r its natural promoter cannot be expressed when integrated into the pG KL1 plasmid. However. one G418(R) 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 conta ining the Km(R) gene. In these mutant plasmids, a large part of pGKL1 has been replaced by the Km(R) gene harboring a rearranged 5'-flanking region extending over 600 bp. This new DNA sequence has been cloned a nd sequenced. The rearranged sequence allowed the Km(R) gene to be exp ressed at high level, enabling the transformant cells to grow on a med ium containing G418 at 2 mg/ml. This high level of resistance was foun d to be due to increased transcription of the Km(R) gene. Primer exten sion experiments suggested that the rearranged upstream region of Km(R ) contained transcription promoting sites recognized by the killer-pla smid-specific transcription system.