Genomic analysis of severe hypersensitivity to hygromycin B reveals linkage to vacuolar defects and new vacuolar gene functions in Saccharomyces cerevisiae.

The vacuole of Saccharomyces cerevisiae has been a seminal model for studies of lysosomal trafficking, biogenesis, and function. Several yeast mutants defective in such vacuolar events have been unable to grow at low levels of hygromycin B, an aminoglycoside antibiotic. We hypothesized that such severe hypersensitivity to hygromycin B (hhy) is linked to vacuolar defects and performed a genomic screen for the phenotype using a haploid deletion strain library of non-essential genes. Fourteen HHY genes were initially identified and were subjected to bioinformatics analyses. The uncovered hhy mutants were experimentally characterized with respect to vesicular trafficking, vacuole morphology, and growth under various stress and drug conditions. The combination of bioinformatics analyses and phenotypic characterizations implicate defects in vesicular trafficking, vacuole fusion/fission, or vacuole function in all hhy mutants. The collection was enriched for sensitivity to monensin, indicative of vacuolar trafficking defects. Additionally, all hhy mutants showed severe sensitivities to rapamycin and caffeine, suggestive of TOR kinase pathway defects. Our experimental results also establish a new role in vacuolar and vesicular functions for two genes: PAF1, encoding a RNAP II-associated protein required for expression of cell cycle-regulated genes, and TPD3, encoding the regulatory subunit of protein phosphatase 2A. Thus, our results support linkage between severe hypersensitivity to hygromycin B and vacuolar defects.

Identification of the axon pathways which mediate functional recovery of a paralyzed hemidiaphragm following spinal cord hemisection in the adult rat.

Despite extensive neurophysiological work carried out to characterize the crossed phrenic phenomenon, relatively little is known about the morphological substrate of this reflex which restores function to a hemidiaphragm paralyzed by spinal cord injury. In the present study WGA-HRP was injected into normal and functionally recovered hemidiaphragm muscle in rats during the crossed phrenic phenomenon. The retrograde transynaptic transport characteristics of WGA-HRP was utilized to delineate the source of the neurons which mediate the crossed phrenic phenomenon. The results indicated that the neurons which drive phrenic motoneurons in spinal hemisected rats during the crossed phrenic phenomenon are located bilaterally in the rostral ventral respiratory group (rVRG) of the medulla. No transneuronal labeling of propriospinal neurons was noted in either normal or spinal-hemisected rats. Thus, propriospinal neurons do not relay respiratory drive to phrenic motoneurons. The neurons of the rVRG project monosynaptically to phrenic motoneurons. The present results suggest that both crossed and uncrossed bulbospinal pathways from the rVRG collateralize to both the left and right phrenic nucleic and functional recovery of a hemidiaphragm paralyzed by ipsilateral spinal cord hemisection is mediated by supraspinal neurons from both sides of the brain stem. These results are important to our complete understanding of the mechanisms which govern motor recovery in mammals following spinal cord injury.