Generation and characterization of new alleles of quiver (qvr) that encodes an extracellular modulator of the Shaker potassium channel.

Our earlier genetic screen uncovered a paraquat-sensitive leg-shaking mutant quiver1 (qvr1), whose gene product interacts with the Shaker (Sh) K+ channel. We also mapped the qvr locus to EY04063 and noticed altered day-night activity patterns in these mutants. Such circadian behavioral defects were independently reported by another group, who employed the qvr1 allele we supplied them, and attributed the extreme restless phenotype of EY04063 to the qvr gene. However, their report adopted a new noncanonical gene name sleepless (sss) for qvr. In addition to qvr1 and qvrEY, our continuous effort since the early 2000s generated a number of novel recessive qvr alleles, including ethyl methanesulfonate (EMS)-induced mutations qvr2 and qvr3, and P-element excision lines qvrip6 (imprecise jumpout), qvrrv7, and qvrrv9 (revertants) derived from qvrEY. Distinct from the original intron-located qvr1 allele that generates abnormal-sized mRNAs, qvr2, and qvr3 had their lesion sites in exons 6 and 7, respectively, producing nearly normal-sized mRNA products. A set of RNA-editing sites are nearby the lesion sites of qvr3 and qvrEY on exon 7. Except for the revertants, all qvr alleles display a clear ether-induced leg-shaking phenotype just like Sh, and weakened climbing abilities to varying degrees. Unlike Sh, all shaking qvr alleles (except for qvrf01257) displayed a unique activity-dependent enhancement in excitatory junction potentials (EJPs) at larval neuromuscular junctions (NMJs) at very low stimulus frequencies, with qvrEY displaying the largest EJP and more significant NMJ overgrowth than other alleles. Our detailed characterization of a collection of qvr alleles helps to establish links between novel molecular lesions and different behavioral and physiological consequences, revealing how modifications of the qvr gene lead to a wide spectrum of phenotypes, including neuromuscular hyperexcitability, defective motor ability and activity-rest cycles.

Mutations of the withered (whd) gene in Drosophila melanogaster confer hypersensitivity to oxidative stress and are lesions of the carnitine palmitoyltransferase I (CPT I) gene.

Since some oxygen defense mutants of Drosophila melanogaster exhibit a crinkled wing phenotype, a screen was performed on strains bearing mutant alleles conferring a visible wing phenotype to determine whether any were hypersensitive to oxidative stress. One mutant, withered (whd), was found to be sensitive to both dietary paraquat and hyperoxia. New alleles of whd were induced on a defined genetic background and strains carrying these alleles were also found to be sensitive to oxidative stress. To identify the product of the whd gene we used a sequence-based positional candidate approach and by this method we determined that whd encodes carnitine palmitoyltransferase I (CPT I), an enzyme of the outer mitochondrial membrane that is required for the import of long-chain fatty acids into the mitochondria for beta-oxidation. Although this function is not vital under laboratory conditions, whd adults were found to be highly sensitive to starvation and to heavy metal toxicity relative to controls. This work uncovers a novel relationship between fatty acid metabolism and reactive oxygen metabolism. Further, these results in conjunction with past research on whd and on mammalian CPT I support the hypothesis that CPT I serves a vital function in the response to thymine supplementation.

Utp8p is a nucleolar tRNA-binding protein that forms a complex with components of the nuclear tRNA export machinery in Saccharomyces cerevisiae.

Utp8p is an essential nucleolar component of the nuclear tRNA export machinery in Saccharomyces cerevisiae. It is thought to act at a step between tRNA maturation/aminoacylation and translocation of the tRNA across the nuclear pore complex. To understand the function of Utp8p in nuclear tRNA export, a comprehensive affinity purification analysis was conducted to identify proteins that interact with Utp8p in vivo. In addition to finding proteins that have been shown previously to copurify with Utp8p, a number of new interactions were identified. These interactions include aminoacyl-tRNA synthetases, the RanGTPase Gsp1p, and nuclear tRNA export receptors such as Los1p and Msn5p. Characterization of the interaction of Utp8p with a subset of the newly identified proteins suggests that Utp8p most likely transfer tRNAs to the nuclear tRNA export receptors by using a channeling mechanism.

The Pseudomonas aeruginosa initiation factor IF-2 is responsible for formylation-independent protein initiation in P. aeruginosa.

Formylation of the initiator methionyl-tRNA (Met-tRNAfMet) was generally thought to be essential for initiation of protein synthesis in all eubacteria based on studies conducted primarily in Escherichia coli. However, this view of eubacterial protein initiation has changed because some bacteria have been demonstrated to have the capacity to initiate protein synthesis with the unformylated Met-tRNAfMet. Here we show that the Pseudomonas aeruginosa initiation factor IF-2 is required for formylation-independent protein initiation in P. aeruginosa, the first bacterium shown to have the ability to initiate protein synthesis with both the initiator formyl-methionyl-tRNA (fMet-tRNAfMet) and Met-tRNAfMet. The E. coli IF-2, which participates exclusively in formylation-dependent protein initiation in E. coli, was unable to facilitate utilization of Met-tRNAfMet in initiation in P. aeruginosa. However, the E. coli IF-2 was made to function in formylation-independent protein initiation in P. aeruginosa by decreasing the positive charge potential of the cleft that binds the amino end of the amino acid attached to the tRNA. Furthermore increasing the positive charge potential of this cleft in the P. aeruginosa IF-2 prevented the protein from participating in formylation-independent protein initiation. Thus, this is the first demonstration of a eubacterial IF-2 with an inherent capacity to facilitate utilization of Met-tRNAfMet in protein initiation, discounting the dogma that eubacterial IF-2 can only allow the use of fMet-tRNAfMet in protein initiation. Furthermore these findings give important clues to the basis for discriminating the initiator Met-tRNA by IF-2 and for the evolution of alternative mechanisms for discrimination.