THE LINEAR PLASMID PDHL1 FROM DEBARYOMYCES-HANSENII ENCODES A PROTEINHIGHLY HOMOLOGBOUS TO THE PGKL1-PLASMID DNA-POLYMERASE

Both the linear plasmids, pDHL1 (84 kb) and pDHL2 (9.2 kb), of Debaryo myces hansenii TK require the presence of a third linear plasmid pDHL3 (150 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 th e 3.5 kb NcoI-AccI fragment of pDHL2, suggesting the importance of thi s conserved region in the replication of the two smaller pDHL plasmids . The 42 kb pDHL1 fragment containing the above hybridized region was cloned and sequenced. The results showed that the cloned pDHL1 fragmen t encodes a protein of 1000 amino acid residues, having a strong simil arity to the DNA polymerase coded for by ORF1 of the killer plasmid pG KL1 from Kluyveromyces lactis. The catalytic and proof-reading exonucl ease 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 par tly similar to the alpha subunit of the K. lactis killer toxin, althou gh 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 pGKL 1-ORFs. The possible hairpin loop structure was also found just in fro nt of the ATG start codon of the pDHL1-ORFs like pGKLI-ORFs. Thus the cytoplasmic pDHL plasmids were suggested to possess a gene expression system comparable to that of K. lactis plasmids. (C) 1997 by John Wile y & Sons, Ltd.