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Nucleic Acids Research, 1986, Vol. 14, No. 17 6871-6884
© 1986

Articles

Efficient isolation of the linear DNA killer plasmid of Kluyveromyces lactis: evidence for location and expression in the cytoplasm and characterization of their terminally bound proteins

Jord C. Stam, Jan Kwakman, Michiel Meijer and Antoine R. Stuitje

Department of Electron Microscopy and Molecular Cytology, Biotechnology Centre, University of Amsterdam Plantage Muidergracht 14, 1018 TV Amsterdam, The Netherlands

Received June 13, 1986. Revised August 18, 1986. Accepted August 18, 1986.

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 kDa1 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.


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[Abstract] [Full Text]


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