A series of yeast/Escherichia coli λ expression vectors designed for directional cloning of cDNAs and cre/lox‐mediated plasmid excision

Abstract

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.

β-lapachone Activates a Mre11p-Tel1p G1/S Checkpoint in Budding Yeast

“…To generate pYPGE15-XRS2 HA, a KpnI-SalI fragment of the YCpT-XRS2-HA construct containing the C terminal HA tagged XRS2 gene (kindly provided by Dr. Katsunori Sugimoto) was cloned into KpnI SalI linearized pYPGE15. 25 This construct expressed appropriate sized proteins and complemented a xrs2 null mutant concerning sensitivity to DNA damage. Standard methods for yeast culture and manipulations were used.…”

The Putative Plasma Membrane Na+/H+ Antiporter SOS1 Controls Long-Distance Na+ Transport in Plants

“…cerevisiae strains ANT3 (⌬ena1::HIS3::ena4, ⌬nha1::LEU2), GX1 (⌬ena1::HIS3::ena4, ⌬nhx1::TRP1), AXT3 (⌬ena1::HIS3::ena4, ⌬nha1:: LEU2, ⌬nhx1::TRP1), and W⌬3 (⌬trk1::LEU2, ⌬trk2::HIS3) are derivatives of W303 and have been described elsewhere (Madrid et al, 1998;Quintero et al, 2000). The entire SOS1 open reading frame was cloned into the yeast expression vector pYPGE15 under the control of the constitutive gene promoter PGK1 (Brunelli and Pall, 1993). K ϩ acquisition was determined in arginine-phosphate medium (AP medium; 10 mM L-arginine, 8 mM PO 4 H 3 , 2 mM MgSO 4 , 0.2 mM CaCl 2 , 2% glucose ϩ vitamins and trace elements, pH 6.5), which is essentially free of alkali cations, supplemented with various concentrations of KCl as noted (Ramos et al, 1994).…”

The Anaphase-Promoting Complex Is a Dual Integrator That Regulates Both MicroRNA-Mediated Transcriptional Regulation ofCyclin B1and Degradation of Cyclin B1 duringArabidopsisMale Gametophyte Development

“…For APC8 promoter analysis, a 2.4-kb fragment upstream of the ATG was amplified using primers APC8F1 and APC8R7 and was subcloned into pENTR-D/TOPO and then transferred into the plant expression vectors pGII-NLS3XGFP (Hellens et al, 2000) and pMDC164 (Curtis and Grossniklaus, 2003). For yeast complementation, full-length cDNAs of APC8 and APC8-1 were amplified from leaves of Col-0 and apc8-1 plants, respectively, using RT-PCR and primers APC8F3 and APC8R3, and then cloned into the pYPGE15 yeast expression vector (Brunelli and Pall, 1993). The amino acid Asp was changed to Glu by site-directed mutagenesis using primers APC8F11 and APC8R11.…”