Molecular and genetic studies on the Kluyveromyces lactis killer plasmids

Killer strains of K. lactis contain two linear, cytoplasmic linear dsDNA plasmids, known as k1 and k2. These plasmids have been cloned and sequenced and sequence analysis has demonstrated that these plasmids encode 14 large open reading frames. Evidence has suggested that these plasmids replicate through a protein-primed replication mechanism, similar to Adenovirus. This study has investigated the identity of some of the constituents of this replication system, specifically the relationship between the two putative DNA polymerases encoded by the k1 and k2 plasmids and the k1 and k2 terminal proteins. Two gene hybrids, a Glutathione S-Transferase k20RF2 and a Maltose Binding Protein- k20RF2 were constructed and an antibody raised against the GST-k20RF2 protein was shown to cross react with the MBP-k20RF2 protein. Thus demonstrating the presence of anti-k20RF2 antibodies. ORF1 of k1 has been disrupted using a LEU2 marker and demonstrated to be an essential gene for the maintenance of the k1 plasmid. ORF1 shares significant homology to the class B (viral) DNA polymerases and this ORF is believed to encode a k1 specific DNA polymerase. Finally, a hybrid k20RF2-k1ORFl DNA polymerase was constructed and used in a set of experiments which suggested that the terminal protein and DNA polymerase domains of the putative k1 and k2 DNA polymerases are not totally independent entities, as a reconstituted DNA polymerase with a k20RF2 N-terminal domain and a k1ORFl C-terminal domain could not replace the functionality of k20RF2 in vivo.