Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis.

The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.

Genomic profiling of drug sensitivities via induced haploinsufficiency.

Lowering the dosage of a single gene from two copies to one copy in diploid yeast results in a heterozygote that is sensitized to any drug that acts on the product of this gene. This haploinsufficient phenotype thereby identifies the gene product of the heterozygous locus as the likely drug target. We exploited this finding in a genomic approach to drug-target identification. Genome sequence information was used to generate molecularly tagged heterozygous yeast strains that were pooled, grown competitively in drug and analysed for drug sensitivity using high-density oligonucleotide arrays. Individual heterozygous strain analysis verified six known drug targets. Parallel analysis identified the known target and two hypersensitive loci in a mixed culture of 233 strains in the presence of the drug tunicamycin. Our discovery that both drug target and hypersensitive loci exhibit drug-induced haploinsufficiency may have important consequences in pharmacogenomics and variable drug toxicity observed in human populations.

A variant of lambda repressor with an altered pattern of cooperative binding to DNA sites.

The bacteriophage lambda repressor binds cooperatively to pairs of adjacent sites in the lambda chromosome, one repressor dimer binding to each site. The repressor's amino domain (that which mediates DNA binding) is connected to its carboxyl domain (that which mediates dimerization and the interaction between dimers) by a protease-sensitive linker region. We have generated a variant lambda repressor that lacks this linker region. We show that dimers of the variant protein are deficient in cooperative binding to sites at certain, but not all, distances. The linker region thus extends the range over which carboxyl domains of DNA-bound dimers can interact. In particular, the linker is required for cooperative binding to a pair of sites as found in the lambda chromosome, and thus is essential for the repressor's physiological function.

Cellular glutathione, thermal sensitivity, and thermotolerance in Chinese hamster fibroblasts and their heat-resistant variants.

HA-1 Chinese hamster fibroblasts and two heat-resistant variants, designated 2242 and 3012. have been investigated to determine the role that glutathione (GSH) plays in intrinsic cellular resistance to heat and in the development of thermotolerance. The constitutive levels of GSH did not correlate with intrinsic heat sensitivities, but depletion of GSH sensitized all three cell lines to thermal stress. After heating (43.5 degrees C/2 h), surviving fractions were 1 X 10(-3), 1 X 10(-2), and 8 X 10(-3) for HA-1, 2242, and 3012 cells, respectively. Depletion of cellular GSH with L-buthionine-S, R-sulfoximine to less than 10% of control values sensitized such that the thermal responses of these three cell lines were nearly indistinguishable at 43.5 degrees C. Surviving fractions were 2 X 10(-4), 1 X 10(-4), and 1 X 10(-4) for L-buthionine-S,R-sulfoximine-treated HA-1, 2242, and 3012 cells, respectively, following heating at 43.5 degrees C for 2 h. The development of thermotolerance in HA-1 cells following heat shock (45 degrees C/15 min) was unaffected by the inhibition of GSH synthesis. On the other hand, when GSH levels were maintained at extremely low levels, the development of thermotolerance was inhibited. In addition, following heat shock, cellular GSH was decreased and remained below control levels during the development of thermotolerance.