The linear plasmids of Kluyveromyces lactis: Genetic and molecular analysis.

Strains of the budding-yeast Kluyveromyces lactis that produce killer-toxin contain two linear dsDNA plasmids, kl (8.9 Kb) and (13.4 Kb). The genes of kl are preceded by no known gene expression signals and transcripts are probably not polyadenylated. Classical and molecular genetic techniques have been used to attempt to investigate the mechanisms of replication, partitioning and gene expression utilised by these novel elements. Such studies have led to the preparation of a number of vectors and strains of use in this and other analyses. Data or presented which indicate the killer plasmids exist within the cytoplasm of the yeast cell, and probably do not require the nuclear RNA polymerases I, II or III for transcription of their genes. A preparation procedure has been developed for the linear plasmids, and plasmid k2, thought to encode factors necessary for the maintenance or expression of k1, has been cloned. Sequence analysis of 7,7 kb of plasmid k2 revealed the presence of several open reading frames (ORFs) organised in a similar manner to those of plasmid k1. One of these is predicted to encode a product with homology to two different subunits found within several DNA-directed RNA polymerases. Another ORF potentially encodes a polypeptide with homology to a DNA helicase encoded by, and required for accurate transcription of, the cytoplasmic poxvirus vaccinia. These two k2 products may be components of a killer plasmid-specific cytoplasmic gene expression system. Possible mechanisms of killer plasmid replication, partitioning and gene expression are discussed.