William W. Lee

The Chemical Genomic Portrait of Yeast: Uncovering a Phenotype for All Genes

Genetics aims to understand the relation between genotype and phenotype. However, because complete deletion of most yeast genes (∼80%) has no obvious phenotypic consequence in rich medium, it is difficult to study their functions. To uncover phenotypes for this nonessential fraction of the genome, we performed 1144 chemical genomic assays on the yeast whole-genome heterozygous and homozygous deletion collections and quantified the growth fitness of each deletion strain in the presence of chemical or environmental stress conditions. We found that 97% of gene deletions exhibited a measurable growth phenotype, suggesting that nearly all genes are essential for optimal growth in at least one condition.

A high-resolution atlas of nucleosome occupancy in yeast

We present the first complete high-resolution map of nucleosome occupancy across the whole Saccharomyces cerevisiae genome, identifying over 70,000 positioned nucleosomes occupying 81% of the genome. On a genome-wide scale, the persistent nucleosome-depleted region identified previously in a subset of genes demarcates the transcription start site. Both nucleosome occupancy signatures and overall occupancy correlate with transcript abundance and transcription rate. In addition, functionally related genes can be clustered on the basis of the nucleosome occupancy patterns observed at their promoters. A quantitative model of nucleosome occupancy indicates that DNA structural features may account for much of the global nucleosome occupancy.

SR-2508: A 2-nitroimidazole amide which should be superior to misonidazole as a radiosensitizer for clinical use☆

Abstract Using a combination of toxicological, pharmacological and radiosensitization experiments on cells in vitro and tumors in vivo , we have selected two 2-nitroimidadole amidos which should prove superior to misoaidazole (MIS) for clinical use. The two drugs, SR-2508 and SR-2555, are considered to be close to the optimum for variants of misonidazole of the same electron affinity; their main difference is a lower lipophilicity than MIS. This lower lipophilicity leads to poorer penetration into neural tissues than that of MIS and this leads, as expected, to lower neurotoxicity of these drugs compared to MIS (as judged by an accelerating rotarod test following 4–5 weeks of daily injections). The in vitro radiosensitization tests on hypoxic Chinese hamster ovary HA-1 cells show that SR-2508 and MIS have the same radiosensitization efficiency but are slightly more potent as radiosensitizers than SR-2555. For the in vivo radiosensitization experiments we used the in vivo-in vitro cloning assay with the EMT6 tumor, the regrowth assay with RIF-1 and KHJJ tumors and the TCD 50 assay with the MDAH/MCa4 carcinoma. All show roughly equivalent levels of radiosensitization of both SR-2508 and SR-2555 to MIS although, as predicted by the in vitro results, SR-2555 is slightly inferior to SR-21.508. We conclude that, although SR-2555 appears to be slightly less neurotoxic than SR-2508, its lower radiosensitizing efficacy is likely to make it less attractive than SR-2508 as a replacement for MIS for clinical use. Extrapolating the neurotoxicity data obtained in the mouse together with pharmacological studies of SR-2508 in spontaneous tumors of the dog, we estimate that levels of SR-2508 of at least 7.5 times those of MIS will be able to be achieved in human tumors for the equivalent level of neurotoxicity. This should enable essentially maximum radlosensitization of the hypoxic cells in human tumors to be obtained in conventional daily fractionation.

Genome-Wide Requirements for Resistance to Functionally Distinct DNA-Damaging Agents

The mechanistic and therapeutic differences in the cellular response to DNA-damaging compounds are not completely understood, despite intense study. To expand our knowledge of DNA damage, we assayed the effects of 12 closely related DNA-damaging agents on the complete pool of ~4,700 barcoded homozygous deletion strains of Saccharomyces cerevisiae. In our protocol, deletion strains are pooled together and grown competitively in the presence of compound. Relative strain sensitivity is determined by hybridization of PCR-amplified barcodes to an oligonucleotide array carrying the barcode complements. These screens identified genes in well-characterized DNA-damage-response pathways as well as genes whose role in the DNA-damage response had not been previously established. High-throughput individual growth analysis was used to independently confirm microarray results. Each compound produced a unique genome-wide profile. Analysis of these data allowed us to determine the relative importance of DNA-repair modules for resistance to each of the 12 profiled compounds. Clustering the data for 12 distinct compounds uncovered both known and novel functional interactions that comprise the DNA-damage response and allowed us to define the genetic determinants required for repair of interstrand cross-links. Further genetic analysis allowed determination of epistasis for one of these functional groups.

Misonidazole neurotoxicity in mice decreased by administration with pyridoxine

A series of toxicological and pharmacological experiments was performed to test the hypothesis that alterations of pyridoxine (Vitamin B6) metabolism may play an important role in the development of misonidazole (MISO) neurotoxicity. The formation of a Schiffs base between the final reduction product of MISO, 2-amino MISO (NH2-MISO), and pyridoxal-HCl in ethanol was demonstrated. Mice receiving daily intraperitoneal injections of MISO suffered significantly less toxicity (as determined by survival, weight gain and neurological tests) when large doses of pyridoxine-HCl (PYR) were delivered concomitantly, and consequently were able to tolerate administration of more than twice as many MISO injections. PYR did not alter the pharmacokinetics of MISO, either when given simultaneously or when given by multiple repeated daily injections prior to MISO. The administration of PYR also did not alter the radiosensitization by MISO in an in vivo-in vitro cloning assay with the EMT6 tumor in BALB/c mice. If depletion or altered metabolism of pyridoxine by reduced metabolites is also responsible for the neurotoxic effects of nitroimidazoles in humans, then concomitant administration of pyridoxine (in doses greater than the molar quantity of NH2-MISO formed) should inhibit the development of such symptoms and allow administration of larger doses of MISO than are currently clinically employable.