Expression of plasmid R388-encoded type II dihydrofolate reductase as a dominant selective marker in Saccharomyces cerevisiae.

Abstract

The R388 plasmid-encoded drug-resistant type II dihydrofolate reductase gene (R * dhfr) was expressed in Saccharomyces cerevisiae by fusing the R * dhfr coding sequence to the yeast TRP5 promoter. Yeast cells harboring these recombinant plasmids grew in media with 10 ,ug of methotrexate per ml and 5 mg of sulfanilamide per ml, a condition which inhibits the growth of wild-type cells. Addition of a 390-base-pair fragment from the 3'-noncoding region of TRP5 downstream from R * dhfr increased expression. Presumably, the added segment promoted termination or polyadenylation or both of the R dhfr transcript. The activity of the plasmid-encoded dihydrofolate reductase and the copy number of the R dhfr plasmid in cells grown in drug-selective media were higher by one order of magnitude than those grown in nutritionselective media. Plasmid copy number, as well as the plasmid-encoded enzyme level, decreased when cells were selected for prototrophy. In drug-selective media, the plasmid-encoded enzyme level and the content of R * dhfr transcripts were nearly constant in cells harboring R * dhfr plasmids containing different yeast promoters. In contrast, the plasmid copy number and ,-lactamase activity encoded in cis by plasmids were much higher when R * dhfr was associated with the weak TRP5 promoter than when it was fused to the strong ADCJ promoter. These results indicate that plasmid copy number, i.e., gene dosage of R * dhfr, correlates inversely with the strength of the promoter associated with R * dhfr, and cells with a higher plasmid copy number were enriched in drug-selective media. The transformation efficiency of R * dhfr fused to the ADCJ promoter was almost the same on drug-selective plates as on nutrition-selective plates, indicating that R * dhfr is suitable as a dominant selective transformation marker in S. cerevisiae.

Identification of Cryptosporidium parvum Dihydrofolate Reductase Inhibitors by Complementation in Saccharomyces cerevisiae

“…Sulfanilamide had been used previously to inhibit yeast dihydropteroate synthase (26,33,34), and we included this drug at 1 mM in our DHFR inhibitor assays. A higher concentration of a drug is required to completely inhibit the growth of the yeast, so in the spoke assay, 10 l of a 5-g/ml solution of trimetrexate alone inhibited the growth of only the Pf-yeast strain (Fig.…”

Industrial production of heterologous proteins by fed-batch cultures of the yeast Saccharomyces cereuisiae

“…Jimenez and Davis [73] developed a sclectable marker for aminoglycoside G418 resistance encoded by the Tn601(903) aminoglycosidc phosphotransferase gene. Two other dominant selectable markers which can be used for vector copy number amplification by increased drug selection are the thymidine kinasc gene [74] and the dihydrofolate reductase [75].…”

Isolation and characterization of STI1, a stress-inducible gene from Saccharomyces cerevisiae.

“…Cells harboring pCN207 were transformed with the YEp24-derived Carlson-Botstein genomic library (6), and selection was simultaneously carried out for the marker on the library plasmid (uracil prototrophy) and for MTX resistance (resulting from enhanced expression of the SSA4 promoter). Special medium conditions for drug selection using MTX were exactly as described by Miyajima et al (27). Transformants recovered were subjected to a regimen to indicate that the gene on the library plasmid was specifically activating the SSA4 promoter in trans.…”