Purification and molecular analysis of the Kluyveromyces lactis linear killer plasmids

The killer phenotype of the lactose-utilising yeast Kluyveromyces lactis is dependent upon the presence of two linear, dsDNA plasmids termed pGKL1 (K1) and pGKL2 (K2). Both plasmids have terminal inverted repeat sequences (TIRs) and terminal proteins (TPs) linked to their 5' ends. Structurally similar genetic elements, termed invertrons, which include human adenovirus and Bacillus subtilis bacteriophage 29, have been shown to replicate their DNA by a protein-primed mechanism. The cytoplasmic location of K1 and K2, and the finding of putative plasmid-encoded family of B DNA polymerases suggested that they encode their own protein-primed replication system.;Although many invertrons have been described in various organisms, with the exception of adenovirus and bacteriophage 29, little is known abut their biology. Difficulties arise in studying killer plasmid gene function as their structure cannot be readily modified by in vitro manipulation due to the presence of protein-linked termini. Furthermore, the killer plasmids appear to encode their own transcription mechanism, and their genes are not correctly expressed by the host transcription system when cloned into yeast on conventional nuclear located circular vectors. Although in vitro homologous recombination studies have progressed our understanding of the killer plasmids, a method for their in vitro modification and transformation has not been developed, and biochemical and molecular analysis has progressed slowly.;To advance study in these areas, a large scale procedure for the purification of the K. lactis linear killer plasmids, with intact terminal proteins (TPs), was developed using a gentle NP40-induced lysis of yeast spheroplasts, and standard chromatography methods.