High-throughput screening of the Saccharomyces cerevisiae genome for 2-amino-3-methylimidazo [4,5-f] quinoline resistance identifies colon cancer-associated genes.

Heterocyclic aromatic amines (HAAs) are potent carcinogenic agents found in charred meats and cigarette smoke. However, few eukaryotic resistance genes have been identified. We used Saccharomyces cerevisiae (budding yeast) to identify genes that confer resistance to 2-amino-3-methylimidazo[4,5-f] quinoline (IQ). CYP1A2 and NAT2 activate IQ to become a mutagenic nitrenium compound. Deletion libraries expressing human CYP1A2 and NAT2 or no human genes were exposed to either 400 or 800 µM IQ for 5 or 10 generations. DNA barcodes were sequenced using the Illumina HiSeq 2500 platform and statistical significance was determined for exactly matched barcodes. We identified 424 ORFs, including 337 genes of known function, in duplicate screens of the "humanized" collection for IQ resistance; resistance was further validated for a select group of 51 genes by growth curves, competitive growth, or trypan blue assays. Screens of the library not expressing human genes identified 143 ORFs conferring resistance to IQ per se. Ribosomal protein and protein modification genes were identified as IQ resistance genes in both the original and "humanized" libraries, while nitrogen metabolism, DNA repair, and growth control genes were also prominent in the "humanized" library. Protein complexes identified included the casein kinase 2 (CK2) and histone chaperone (HIR) complex. Among DNA Repair and checkpoint genes, we identified those that function in postreplication repair (RAD18, UBC13, REV7), base excision repair (NTG1), and checkpoint signaling (CHK1, PSY2). These studies underscore the role of ribosomal protein genes in conferring IQ resistance, and illuminate DNA repair pathways for conferring resistance to activated IQ.

Quorum Sensing Behavior in the Model Unicellular Eukaryote Chlamydomonas reinhardtii.

Microbial communities display behavioral changes in response to variable environmental conditions. In some bacteria, motility increases as a function of cell density, allowing for population dispersal before the onset of nutrient scarcity. Utilizing automated particle tracking, we now report on a population-dependent increase in the swimming speeds of the photosynthetic unicellular eukaryotes Chlamydomonas reinhardtii and C. moewussi. Our findings confirm that this acceleration in swimming speed arises as a function of culture density, rather than with age and/or nutrient availability. Furthermore, this phenomenon depends on the synthesis and detection of a low-molecular-weight compound which can be transferred between cultures and stimulates comparable effects across both species, supporting the existence of a conserved phenomenon, not unlike bacterial quorum sensing, among members of this genus. The potential expansion of density-dependent phenomena to a new group of unicellular eukaryotes provides important insight into how microbial populations evolve and regulate "social" behaviors.