Transformation of Kluyveromyces lactis with the kanamycin (G418) resistance gene of Tn905

K. Sreekrishna, Thomas D. Webster, Robert C. Dickson

Research output: Contribution to journalArticlepeer-review

53 Scopus citations

Abstract

Direct selection of Kluyveromyces lactis resistant to the antibiotic G418 following transformation with the kanamycin resistance gene of Tn903 required the development of a procedure for producing high yields of viable spheroplasts and for the isolation of autonomous replication sequences (ARS). To obtain high yields of viable spheroplasts, cells were treated with (1) a thiol-reducing agent (l-cysteine), and (2) a high concentration of an osmotic stabilizer, 1.5 M sorbitol. Several ARS-containing plasmids were selected from a K. lactis recombinant DNA library in K. lactis and in Saccharomyces cerevisiae. Two of four ARS clones selected in K. lactis promoted transformation frequencies of 5-10 × 102 G418-resistant cells/μg of plasmid DNA. This frequency of transformation was at least twice as high as with ARS clones selected in S. cerevisiae. The stability of ARS-containing plasmids varied; after 20 generations of growth in the presence of G418, 16-38% of the cells remained resistant to the drug. In the absence of selection pressure less than 5 % of the cells retained the drug-resistance phenotype. Plasmids containing the ARS1 or 2μ replicon of 5. cerevisiae failed to transform K. lactis for G418 resistance. Inclusion of S. cerevisiae centromere, CEN4, in a K. lactis ARS recombinant plasmid did not increase the stability of the plasmid in K. lactis, and marker genes on the vector segregated predominantly 4-1:0+ through meiosis. We conclude that neither the ARS sequences or the centromere of S. cerevisiae was functioning in K. lactis.

Original languageEnglish
Pages (from-to)73-81
Number of pages9
JournalGene
Volume28
Issue number1
DOIs
StatePublished - Apr 1984

Keywords

  • Recombinant DNA
  • Saccharomyces cerevisiae
  • autonomous replication sequence
  • shuttle vectors
  • transposon
  • yeasts

ASJC Scopus subject areas

  • Genetics

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