Kohji Miyahara

Feeding and food choice are crucial to the survival of an animal. The nematode Caenorhabditis elegans feeds on various microorganisms in nature, and is usually fed Escherichia coli in the laboratory. To elucidate the mechanisms of food/non-food discrimination in C. elegans, we examined the accumulation of various fluorescent polystyrene microspheres in the absence and presence of bacterial food. In the absence of food and on agar plates, C. elegans worms actively accumulated 0.5 and 1 μm diameter microspheres, whereas those microspheres <0.5 μm or >3 μm were rarely accumulated. Carboxylate microspheres were accumulated more than sulfate or amine microspheres. These results of accumulation in the absence of food probably well simulate uptake of or feeding on the microspheres. Presence of food bacteria even at bacteria:nematode ratios of 1:100 or 1:10 significantly reduced accumulation of 0.5 μm microspheres, and accumulation was reduced to approximately one-fourth of that observed in the absence of bacteria at a ratio of 1:1. When accumulation of microspheres was examined with the chemical sense mutants che-2, tax-2, odr-1 and odr-2, or the feeding mutant eat-1, all the mutants showed less accumulation than the wild type in the absence of food. In the presence of food, the che-2 mutant showed more accumulation than the wild type. It is possible that C. elegans discriminates food both physically, based on size, and chemically, based on taste and olfaction.

To dissect the action mechanism of reveromycin A (RM-A), a G(1)-specific inhibitor, a Saccharomyces cerevisiae dominant mutant specifically resistant to RM-A, was isolated from a strain in which the genes implicated in nonspecific multidrug resistance had been deleted. The mutant gene (YRR2-1) responsible for the resistance was identified as an allele of the ILS1 gene encoding tRNA(Ile) synthetase (IleRS). The activity of IleRS, but not several other aminoacyl-tRNA synthetases examined in wild type cell extract, was highly sensitive to RM-A (IC(50) = 8 ng/ml). The IleRS activity of the YRR2-1 mutant was 4-fold more resistant to the inhibitor compared with that of wild type. The mutation IleRS(N660D), near the KMSKS consensus sequence commonly found in the class I aminoacyl transferases, was found to be responsible for RM-A resistance. Moreover, overexpression of the ILS1 gene from a high-copy plasmid conferred RM-A resistance. These results indicated that IleRS is a target of RM-A in vivo. A defect of the GCN2 gene led to decreased RM-A resistance. IleRS inhibition by RM-A led to transcriptional activation of the ILS1 gene via the Gcn2-Gcn4 general amino acid control pathway, and this autoregulation seemed to contribute to RM-A resistance.