Open AccessEfficient isolation of the linear DNA killer plasmid ofKluyveromyces lactis: evidence for location and expression in the cytoplasm and characterization of their terminally bound proteins
Author(s) -
Jord C. Stam,
J. H. J. M. Kwakman,
Michiel Meijer,
Antoine R. Stuitje
Publication year - 1986
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/14.17.6871
Subject(s) - biology , kluyveromyces lactis , plasmid , dna , cytoplasm , kluyveromyces , extrachromosomal dna , microbiology and biotechnology , shuttle vector , gene , origin of replication , recombinant dna , saccharomyces cerevisiae , genetics , vector (molecular biology)
Differential centrifugation of an osmotic lysate of K. lactis protoplasts showed that the linear DNA killer plasmids of K. lactis, pGKL1 and pGKL2, are almost exclusively present in the cytoplasmic fraction. This fractionation procedure allows the rapid isolation of large amounts of plasmid DNA without contamination by chromosomal and mitochondrial DNA. With these DNA preparations the size of the terminally bound proteins was estimated to be 28 and 36 kDal for pGKL1 and pGKL2, respectively. The entire pGKL1 sequence (except for 21 base pairs at the right terminus) was cloned in a shuttle vector that permits autonomous replication in the nucleus of K. lactis. However, killer gene expression could not be established in transformants. In connection with the observed cytoplasmic localization, this result suggests that gene expression of the killer DNA plasmids is entirely cytoplasmic.