A novel approach to express a heterologous gene on Kluyveromyces lactis linear killer plasmids: expression of the bacterial aph gene from a cytoplasmic promoter fragment without in-phase fusion to the plasmid open reading frame.
Using the cytoplasmically localized killer plasmids pGKL1 and pGKL2 of Kluyveromyces lactis a new linear hybrid plasmid (pWKL1) was constructed. It consists of pGKL1 into which, in addition to the previously developed cytoplasmically expressible LEU2* selection marker, a phosphotransferase encoding bacterial antibiotic resistance gene has been integrated. In the hybrid plasmid pWKL1 this gene was preceded by a putative transcriptional signal from pGKL2, i.e., the upstream conserved sequence of ORF5 (UCS5). All foreign genes, so far integrated and expressed in linear plasmids, required in addition to a cytoplasmic promoter (UCS) an in phase fusion to the corresponding open reading frame. In this study the UCS5, not containing the translational start of ORF5, and the aph coding region were intentionally not combined in phase, in order to check whether expression can occur under these conditions. Kluyveromyces lactis transformants carrying pWKL1 were highly resistant against the antibiotic geneticine (G418) and exhibited elevated levels of APH activity. Expression of the aph gene was due to the presence of the UCS5 containing fragment (110 bp). Thus, an in phase fusion was not required for expression. This is the first report that, concomitant with a selectable marker gene, foreign DNA can be integrated in a linear plasmid via homologous recombination and more importantly, that in phase fusions are not required generally for expression of heterologous genes in linear plasmids.
Meinhardt
F
F
Institut für Mikrobiologie, Westfälische Wilhelms-Universität, Münster, Federal Republic of Germany.
Wodara
C
C
Larsen
M
M
Schickel
J
J
D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D005819:Genetic Markers; D005822:Genetic Vectors; D019868:Kanamycin Kinase; D055646:Killer Factors, Yeast; D007716:Kluyveromyces / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D009183:Mycotoxins / Q000235:genetics; D016366:Open Reading Frames; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D014170:Transformation, Genetic
D004269:DNA, Bacterial; D005819:Genetic Markers; D055646:Killer Factors, Yeast; D009183:Mycotoxins; C058975:zymocin; D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase