ISOLATION AND SEQUENCE-ANALYSIS OF A GENE FROM THE LINEAR DNA PLASMIDPPACL-2 OF PICHIA-ACACIAE THAT SHOWS SIMILARITY TO A KILLER TOXIN GENE OF KLUYVEROMYCES-LACTIS

The toxin-encoding linear plasmid systems found in Pichia acaciae and Kluyveromyces lactis yeasts appear to be quite similar, both in functi on and structural organization. By Southern hybridization, a linear pl asmid of P. acaciae, pPacl-2, was found to hybridize to the second ope n reading frame (ORF2) of K. lactis plasmid pGKL1, known to encode the alpha and beta subunits of the K. lactis toxin. A 1.7 kbp segment of pPacl-2 DNA was cloned, sequenced and shown to contain four regions of strong homology to four similarly oriented regions of K. lactis ORF2. This 1.7 kbp fragment also contained an ORF of 1473 bp that could enc ode a protein of similar to 55.8 kDa. Like the a subunit gene of K. la ctis ORF2, a very hydrophobic region occurs at the N-terminus, perhaps representing a signal sequence for transport out of the cell. Unlike K. lactis ORF2, however, the encoded polypeptide is much smaller and l acks a recognizable domain common to chitinases. The structure of a to xin that includes the translation product of this P. acaciae ORF would likely be quite different from that of the K. lactis toxin. Analysis of the upstream region of the P. acaciae ORF revealed an upstream cons erved sequence identical to that found before ORFs 8 and 9 of pGKL2. A possible hairpin loop structure, as has been described for each of th e four K. lactis pGKL1 ORFs, was found just upstream of the presumed s tart codon. The similarity of the promoter-like elements found in the linear plasmid genes of these diverse yeasts reinforces the idea of th e existence of a unique, but highly conserved, expression system for t hese novel plasmids. The sequence has been deposited in the GenBank da ta library under Accession Number U02596.