The Escherichia coli R factor-derived chloramphenicol resistance (camr) nificant that the expression of the camr gene in yeast results in the acquisition by transformed cells of an easily detectable phenotype-namely, resistance to chloramphenicol. The possible use of the latter observation for the investigation of the mechanisms of transcription, RNA processing, and translation in eukaryotes will be discussed.
MATERIALS AND METHODSBacterial and Yeast Strains. The following strains were used in this work: E. coli, RR101 (F-pro leu thi lacY str' rk m-) harboring plasmid pBR325; and S. cerevisiae 21D (a leu 2-3 leu 2-112 his 4 lys 1), AH22 (a leu 2-3 leu 2-112 his 4), lGC (a leu 2-3 leu 2-112), and 2A (a leu 2-3 leu 2-112).Media. L medium (11) The supernatant resulting from centrifugation at 27,000 X g for 30 min was used for enzyme assays. Yeast cell-free extracts were prepared from 7-ml cultures grown in appropriately supplemented YNBD medium. The cells in early stationary phase (z108 cells per ml) were harvested by centrifugation, washed with water, suspended in 0.4 ml of 100 mM Tris-HCl at pH 7.8, and homogenized in the presence of glass beads for 2 min in a Braun homogenizer. After centrifugation at 7800 X g, the supernatant was used for enzyme assays. The enzyme reaction mixture (total volume, 0.25 ml) contained 23 nmol of Tris-HCl (pH 7.8), 40 nmol of acetyl-CoA, 5-10 nmol of [14C]chloramphenicol (43.8 mCi/mmol; 1 Ci = 3.7 X 1010 becquerels; New England Nuclear), and cell-free extract containing 0.2-1 mg of protein. Incubation of the reaction mixture was at room temperature for the yeast extracts and at 37°C for bacterial extracts. The reaction was terminated by adding 1 ml of ice-cold 2 M Tris base and extracting with ice-cold ethyl acetate when the activity was to be assayed by thin-layer chromatography or with ice-cold benzene when selective extraction of the acetylated derivatives of chloramphenicol was