4 A,B and ?and55 A,B). Cycloheximide (CYH), 4-nitroquinoline-oxide (4-NQO), sulfomethuron methyl (SMM), and 5-fluoro-orotic acid (5FOA) (BioMol, PA). Zeocin (Zeo) (Invitrogen, CA). X-Gal (Clontech, CA). 96-pin replicator (Nalge Nunc International Corp., IL). Methods The methods layed out below describe (1) the construction of expression plasmids bearing conditionally regulated yeast and hexose transporter genes, (2) integration of and chromosomal loci of yeast two-hybrid strains, (3) analysis of the permeability of newly developed yeast strains with selected known small-molecule inhibitors, and by screen of a large combinatorial library of compounds, and (4) selection of inhibitors of the conversation between human Ras and Raf-1 by screening the combinatorial library of compounds in the obtained hyperpermeable yeast two-hybrid strain. Expression Plasmids In order to WRG-28 enhance the permeability of the yeast to small-molecular-weight compounds, the two yeast hexose transporters and were subcloned under control of the galactose-inducible promoter and subsequently integrated by homologous recombination into the genetic loci for and gene is usually targeted to the yeast locus. (A) A plasmid vector made up of upstream (5) and downstream (3) flanking regions of the gene separated by a unique restriction site (BamHI). (B) An integrative cassette made up of is usually inserted at the unique BamHI site between the Ptgs1 gene fragments. (C) The structure of the recombination intermediate made up of a gene disrupted by the cassette is usually shown. (D) Recombination between and sequences of the cassette around the chromosome and the and sequences on separately prepared linear cassette is usually detected by growth on 5FOA (i.e., into the chromosomal location of WRG-28 the gene with loss of PDR1 and URA3. (E) The genomic structure of the locus of the altered yeast strains SKY51 and SKY194, derived from SKY48 and SKY191, respectively. Open in a separate windows Fig. 2. In a second step of the integrative transformation strategy used to construct the altered yeast strains SKY54 and SKY197 from strains SKY51 and SKY194, the gene is usually targeted to the yeast locus. (A) Plasmid vector was constructed made up of the upstream (5) and downstream (3) flanking regions of the gene separated by a unique restriction site (BamHI). (B) An integrative cassette made up of was inserted at the unique BamHI site between the gene fragments. This was utilized for integration by homologous replacement of as selected by a gene disrupted by the cassette is usually shown. (D) Next, homologous recombination between and sequences of the integrated cassette around the chromosome and a separately prepared, linear cassette results in insertion of the gene at the endogenous yeast 1ocus as selected by growth on 5FOA media. (E) The genomic structure of the locus of the final yeast strains, SKY54 and SKY197. Table 1 Oligonucleotide Primers Used in Plasmid and Strain Construction fragments were cloned into pGem-T/A (Promega, Madison, WI) to construct the pGem5-3-plasmid, with a unique BamHI site between the fragments. Next, the pHisCadA plasmid was constructed by replacing the DNA fragment in the pNKY51 plasmid with the gene operon. Next, the gene fragment from pHis-was isolated, purified, and ligated into the BamHI site of the pGem5-3-vector to construct the pPDR1-Hisplasmid (Fig. 3 A) (gene fragment is usually flanked by upstream (PDR1-5) and downstream (PDRl-3) sequences of cassette (Fig. 1). (B) Map of the pPDR3-HisInt plasmid. The gene fragment is usually flanked by upstream (PDR3-5) and downstream (PDR3-3) sequences of WRG-28 cassette (Fig. 2). Construction of the pPDR3-Hislnt Plasmid Primers VK07 and VKl0 were used to amplify an 839-bp 5 fragment of the yeast gene, and VK09 and VK11 were used to amplify a 743-bp 3 fragment of the yeast gene. The amplified fragments were then cloned into pGem-T/A to produce pGEM5-3-PDR3, which bears a unique BamHI site between the fragments. The pHisInt plasmid was then constructed by replacing the 3 fragment of the pNKY51 plasmid with a fragment from your human immunodeficiency computer virus (HIV) integrase gene (fragment was then WRG-28 isolated, purified, and ligated into the BamHI site of WRG-28 the pGem5-3-PDR3 vector to produce the pPDR3-HisInt plasmid (Fig. 3 B). Construction of the pHisCadA-HXT9 Plasmid Primers VK05 and VK06 were used to.