Construction of a yeast expression system with positive selection for gene insertion in the absence of a specific phenotype.
A positive-selection system of cloned inserts in Escherichia coli has been devised using a streptomycin-resistant (Smr) gene and streptomycin-dependent (Smd) E. coli. A vector, pHA394, based on the yeast expression vector pAM82 has the Smr gene, which inactivates streptomycin (Sm) and invalidates the streptomycin dependence, resulting in a very low transformation efficiency. Replacement of the Smr gene by the recombinant vector allows high-frequency transformation. This system was applied to the lysozyme gene. After the yeast secretion signal was fused to the lysozyme gene using an intermediate vector, pHA474, the Smr gene of pHA394 was replaced by the fusion gene, followed by transformation of Smd E. coli. Analysis of the transformants showed that the plasmid gene contained 100% of the lysozyme gene.
Hashimoto
Y
Y
Graduate School of Pharmaceutical Sciences, Kyushu University 62, Fukuoka, Japan.
Miki
T
T
Mukae
M
M
Ueda
T
T
Imoto
T
T
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D003001:Cloning, Molecular / Q000379:methods*; D015169:Colony Count, Microbial; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D005822:Genetic Vectors; D009113:Muramidase / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics*; D013307:Streptomycin / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D009113:Muramidase; D013307:Streptomycin