Killing Them

Mark Walberg walberg at simmons.swmed.edu
Wed Sep 13 14:14:36 EST 1995


Regarding yeast and methotrexate, look at this paper:


  Miyajima A.  Miyajima I.  Arai K.  Arai N.
  Expression of plasmid R388-encoded type II dihydrofolate reductase as a
  dominant selective marker in Saccharomyces cerevisiae.
  Molecular & Cellular Biology.  4(3):407-14, 1984 Mar.
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 micrograms 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 nutrition-selective 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 beta-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 ADC1 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.(ABSTRACT TRUNCATED AT
  250 WORDS)

walberg at simmons.swmed.edu





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