The Linear Plasmid pDHL1 from Debaryomyces hansenii Encodes a Protein Highly Homologous to the pGKL1‐Plasmid DNA Polymerase

Both the linear plasmids, pDHL1 (8·4 kb) and pDHL2 (9·2 kb), of Debaryomyces hansenii TK require the presence of a third linear plasmid pDHL3 (15·0 kb) in the same host cell for their replication. A 3·5 kb Bam HI-PstI fragment of pDHL1 strongly hybridized by Southern analysis to the 3·5 kb NcoI-AccI fragment of pDHL2, suggesting the importance of this conserved region in the replication of the two smaller pDHL plasmids. The 4·2 kb pDHL1 fragment containing the above hybridized region was cloned and sequenced. The results showed that the cloned pDHL1 fragment encodes a protein of 1000 amino acid residues, having a strong similarity to the DNA polymerase coded for by ORF1 of the killer plasmid pGKL1 from Kluyveromyces lactis. The catalytic and proof-reading exonuclease domains as well as terminal protein motif were well conserved as in DNA polymerases of pGKL1 and other yeast linear plasmids. Analysis of the cloned fragment further showed that pDHL1 encodes a protein partly similar to the α subunit of the K. lactis killer toxin, although killer activity was not known in the DHL system. Analysis of the 5′ non-coding region of the two above pDHL1-ORFs reveal the presence of the upstream conserved sequence similar to that found upstream of pGKL1-ORFs. The possible hairpin loop structure was also found just in front of the ATG start codon of the pDHL1-ORFs like pGKL1-ORFs. Thus the cytoplasmic pDHL plasmids were suggested to possess a gene expression system comparable to that of K. lactis plasmids. © John Wiley & Sons, Ltd.