A Saccharomyces cerevisiae cell‐based quantitative β‐galactosidase assay compatible with robotic handling and high‐throughput screening

Abstract

Reporter-gene assays that employ the Escherichia coli lacZ gene are ubiquitously employed in biological research. However, we were not able to readily identify a quantitative method that worked reliably with yeast (Saccharomyces cerevisiae) cells and that was compatible with high-throughput screening and robotic liquid handling tools. We have therefore adapted a commercially available assay employing a 6-O-β-galactopyranosyl-luciferin substrate to provide the required sensitivity with minimal sample handling times. Our assay uses only one-tenth of the reagents suggested by the reagent manufacturer (Promega) for equivalent assays with mammalian cell cultures and produces rapid, sensitive and reproducible analysis with as little as 1 µl yeast cell culture and with <100 cells. We demonstrate that the assay is compatible with yeast strains generated by the systematic yeast deletion project and functions equally well with genomically integrated or plasmid-encoded lacZ reporters and with cells grown in complex or defined media. The high-sensitivity, miniaturized format reduced sample handling required will make this assay useful for a wide range of applications.

Discovery of Chemical Modulators of a Conserved Translational Control Pathway by Parallel Screening in Yeast

“…Measurement of β-galactosidase activity after overnight yeast growth showed that, as previously observed, 27 the mutant eIF2B strain gave at least twofold higher get uniform mixing in these plates. Therefore, we decided to grow cells in V-bottom polypropylene plates.…”

“…The assay was optimized for HTS by adaptation of a commercially available coupled β-galactosidase-fi refl y luciferase system for use with yeast cells. 27 Mutant and wild-type yeast strains were screened in parallel to identify compounds acting specifi cally on either strain. The parallel screening strategy was designed to eliminate nonspecifi c compounds directly following HTS and thereby minimize the number of cherry-picks required.…”

“…27 Absorbance assay to record yeast growth. For mutant yeast strain HTS, V-bottom polypropylene 384-well plates were fi lled with 25 μl of YPD medium using a Multidrop-384 reagent dispenser (Thermo Scientifi c, Franklin, MA), and an additional 25 μl of YPD medium was added to column 1.…”

“…and elevated activation of GCN4 target genes, including HIS4. 23,27 Thus any compound that restores mutant eIF2B activity should selectively reduce GCN4 activation in the mutant yeast strain. Conversely, compounds that activate GCN4 in an eIF2B-dependent manner should give selective enhancement of reporter gene activity in the wild-type strain.…”

“…Both mutant and wild-type strains contain a chromosomally integrated gene fusion of HIS4 to β-galactosidase, allowing GCN4 activation to be monitored using a coupled β-galactosidase-fi refl y luciferase readout adapted from commercially available reagents. 27 Constitutive down-regulation of eIF2B activity and hence elevated GCN4 activity are often accompanied by slow growth. 10,23 In this work, loss of eIF2B function in the mutant strain was observed to cause a modest reduction in overnight yeast growth ( Figs.…”