Expression of a foreign KmR gene in linear killer DNA plasmids in yeast.
The killer plasmids of the yeast Kluyveromyces lactis, pGKL1 and 2 (k1 and k2 for short), are linear double-stranded DNAs. The expression of genes of these plasmids is thought to depend on their own transcription system. Cloning the plasmid genes in conventional circular vectors is therefore not suitable for transcriptional studies, because such vectors use the host nuclear transcription system. In vitro modification of the linear plasmid genomes in order to introduce transcription reporter genes has been difficult because the structure of the plasmids, with covalently bound terminal proteins, does not allow their manipulation in vitro and amplification in Escherichia coli. We introduced the kanamycin/G418 resistance gene, KmR, into the k1 plasmid in vivo, by transforming the yeast with the linearized KmR gene bordered with short k1 sequences (part of the region encoding the toxin) to allow homologous recombination with the resident k1. In the linear recombinants obtained, however, the KmR was not expressed, while it was expressed if carried on circularized plasmids. By replacing the native promoter of KmR by the ORF1 promoter from k1, the KmR gene could be expressed in linear recombinants and conferred on the host a high level of resistance to the drug. All the linear recombinant plasmids were extremely stable under nonselective conditions. As a rare event, the integration of KmR produced a palindromic rearrangement of the k1 plasmid.
Institut Curie, Centre Universitaire, Orsay, France.
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C
Tanguy-Rougeau
X J
XJ
Chen
M
M
Wésolowski-Louvel
H
H
Fukuhara
D004271:DNA, Fungal / Q000235:genetics*; D005800:Genes, Fungal; D007716:Kluyveromyces / Q000235:genetics*; D010641:Phenotype; D010957:Plasmids; D011995:Recombination, Genetic; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D004718:Saccharomycetales / Q000235:genetics*; D014170:Transformation, Genetic
D004271:DNA, Fungal