Co-transformation with autonomously-replicating helper plasmids facilitates gene cloning from an Aspergillus nidulans gene library.
Autonomously-replicating, marker-less "helper" plasmids were added to transformations of Aspergillus nidulans with plasmids which normally transform by chromosomal integration. This resulted in as much as a 200-fold increase in transformation efficiency. Recovery of autonomously-replicating plasmid co-integrates indicated that co-transformation involves recombination between integrating and helper plasmids, which occurs at a high frequency. Increasing DNA sequence-homology between pairs of plasmids used in simultaneous transformations enhanced co-transformation efficiency. Using helper plasmids and an A. nidulans gene library in a normally-integrating vector, the genes adC and adD were cloned as part of such a co-integrate. In effect, the addition of helper plasmid converts an integrating into an autonomously-replicating gene library in vivo.
Gems
D H
DH
Institute of Genetics, Glasgow University, Scotland, UK.
Clutterbuck
A J
AJ
D001233:Aspergillus nidulans / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004261:DNA Replication; D004926:Escherichia coli; D015723:Gene Library; D005800:Genes, Fungal; D010957:Plasmids; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014170:Transformation, Genetic