A series of yeast/Escherichia coli lambda expression vectors designed for directional cloning of cDNAs and cre/lox-mediated plasmid excision.
A series of Saccharomyces cerevisiae/Escherichia coli lambda/plasmid expression vectors have been constructed which allow easy excision of the plasmid sequences from lambda. Features of six are described, and two designated lambda PG15 and lambda AD5, are characterized in detail. Transcription of cloned sequences is controlled by the alternative promoters, ADH2, PGK, GAL10 and SV40 early, and by the CYC1 transcriptional terminator. Unique EcoRI and XhoI restriction sites in the intervening polylinker make these lambda vectors compatible for directional cloning of 'ZAP'-synthesized cDNAs. Inserted DNAs have been previously shown to have high levels of the genetic activity in both S. cerevisiae and E. coli, allowing these vectors to be used for genetic complementation in both species. Plasmid recovery from the lambda vector is mediated by the activity of the cre-encoded enzyme upon lox sequences flanking the plasmid and adjoining the lambda arms. The plasmids contain the yeast 2 microns origin and E. coli pBR322 origin, the URA3 or TRP1 yeast selectable markers, and ampicillin-resistance marker in E. coli. The usefulness of the lambda PG15 and the lambda AD5 cloning vectors was demonstrated by constructing large Neurospora crassa cDNA libraries. The lambda PG15-N. crassa library was used to infect purE, purC and trpC mutants of E. coli, and complemented and/or suppressed prototrophic colonies were selected. The flexibility and power of this system for cloning of cDNAs is discussed.
Brunelli
J P
JP
Department of Genetics and Cell Biology, Washington State University, Pullman 99164-4234.
Pall
M L
ML
D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D005819:Genetic Markers; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D018076:DNA, Complementary; D005819:Genetic Markers