Stable multicopy integration of vector sequences in Hansenula polymorpha.
Plasmids without an origin of replication, but bearing the URA3 gene of Saccharomyces cerevisiae as a selective marker for transformation, are shown to replicate autonomously in Hansenula polymorpha, indicating that parts of the S. cerevisiae URA3 gene can fulfil an autonomous replication and stabilization function in H. polymorpha. Such plasmids, replicated in low copy number in monomeric conformation, could be rescued in E. coli, and showed a low mitotic stability under selective and non-selective conditions. Selective propagation of such transformants, however, led to the integration of plasmid sequences into the H. polymorpha genome. The integration event usually occurred in high copy number (approx. 30-50) at a single non-homologous site of the genome. The plasmid sequences were found to be present in tandem array and stable under non-selective conditions. In contrast, the use of homologous URA3 gene under similar conditions led to low-copy-number transformants.
Gatzke
R
R
Institut für Mikrobiologie, Heinrich-Heine-Universität Düsseldorf, Germany.
Weydemann
U
U
Janowicz
Z A
ZA
Hollenberg
C P
CP
D004261:DNA Replication / Q000235:genetics; D004271:DNA, Fungal / Q000032:analysis; D005784:Gene Amplification; D005800:Genes, Fungal / Q000235:genetics; D005819:Genetic Markers; D005822:Genetic Vectors / Q000235:genetics*; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000235:genetics*; D010843:Pichia / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014170:Transformation, Genetic / Q000235:genetics*
D004271:DNA, Fungal; D005819:Genetic Markers; D009964:Orotidine-5'-Phosphate Decarboxylase