Migration of the yeast linear DNA plasmid from the cytoplasm into the nucleus in Saccharomyces cerevisiae.
The Kluyveromyces linear plasmids, pGKL1 and pGKL2, carrying terminal protein (TP), are located in the cytoplasm and have a unique gene expression system with the plasmid-specific promoter element termed UCS, which functions only in the cytoplasm. In this study we have developed an in vivo assay system in Saccharomyces cerevisiae which enables the detection of a rare migration of the yeast cytoplasmic plasmid to the nucleus, using a pGKL1-derived cytoplasmic linear plasmid pCLU1. pCLU1 had both the UCS-fused LEU2 gene (a cytoplasmic marker) and the native URA3 gene (a nuclear marker) and therefore its cytoplasmic-nucleo localized could be determined by the phenotypic analysis of the marker. The nuclearly migrated plasmids were often detected as linear plasmids having the telomere sequence of the host yeast at both ends, although circular plasmids were also found. The circular form was produced by the the terminal fusion of pCLU1. Insertion of a Ty element into a nuclearly migrated plasmid was observed, allowing the ROAM-regulated expression of the adjacent nuclearly silent UCS-fused LEU2 gene. The nuclearly located plasmids, whether linear or circular, were less sensitive to UV-mediated curing than pGKL and pCLU1.
Gunge
N
N
Kumamoto Institute of Technology, Japan.
Fukuda
K
K
Takahashi
S
S
Meinhardt
F
F
D001483:Base Sequence; D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism* / Q000528:radiation effects; D003593:Cytoplasm / Q000378:metabolism*; D004270:DNA, Circular / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D004271:DNA, Fungal / Q000378:metabolism* / Q000528:radiation effects; D004274:DNA, Recombinant / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D007716:Kluyveromyces / Q000235:genetics; D008969:Molecular Sequence Data; D010641:Phenotype; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism / Q000528:radiation effects; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000378:metabolism* / Q000528:radiation effects; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D016615:Telomere; D014466:Ultraviolet Rays
D004270:DNA, Circular; D004271:DNA, Fungal; D004274:DNA, Recombinant