A series of yeast/Escherichia coliλ expression vectors designed for directional cloning of cDNAs andcre/lox‐mediated plasmid excision

摘要

AbstractA series ofSaccharomyces cerevisiae/Escherichia coliλ/plasmid expression vectors have been constructed which allow easy excision of the plasmid sequences from λ. Features of six are described, and two designated λPG15 and λAD5, are characterized in detail. Transcription of cloned sequences is controlled by the alternative promoters,ADH2, PGK, GAL10and SV40 early, and by theCYC1transcriptional terminator. UniqueEcoRI andXhoI restriction sites in the intervening polylinker make these λ vectors compatible for directional cloning of ‘ZAP’‐synthesized cDNAs. Inserted DNAs have been previously shown to have high levels of the genetic activity in bothS. cerevisiaeandE. coli, allowing these vectors to be used for genetic complementation in both species. Plasmid recovery from the λ vector is mediated by the activity of thecre‐encoded enzyme uponloxsequences flanking the plasmid and adjoining the λ arms. The plasmids contain the yeast 2 μm origin andE. colipBR322 origin, theURA3orTRP1yeast selectable markers, and ampicillin‐resistance marker inE. coli. The usefulness of the λPG15 and the λAD5 cloning vectors was demonstrated by constructing largeNeurospora crassacDNA libraries. The λPG15–N. crassalibrary was used to infectpurE,purCandtrpCmutants ofE. coli, and complemented and/or suppressed prototrophic colonies were selected. The flexibility and power of this system for cloning