PCR-generated conspecific sodium channel gene probe for the house fly.

A segment of the house fly (Musca domestica) homologue of the para (paralytic) sodium channel gene of Drosophila melanogaster was isolated by using mixed sequence oligonucleotide primers in the polymerase chain reaction (PCR). The specificity of the procedure was demonstrated by genomic Southern analysis using the housefly PCR amplification product as a probe and by DNA sequence analysis. The latter showed structural homology to the para gene, but not to the corresponding region of DSC1, another D. melanogaster gene with structural similarity to vertebrate sodium channel genes.     

The DSC1 channel,encoded by the smi60E locus,contributes to odor-guided behavior in Drosophila melanogaster

Previously, we generated P-element insert lines in Drosophila melanogaster with impaired olfactory behavior. One of these smell-impaired (smi) mutants, smi60E, contains a P[lArB] transposon in the second intron of the dsc1 gene near a nested gene encoding the L41 ribosomal protein. The dsc1 gene encodes an ion channel of unknown function homologous to the paralytic (para) sodium channel, which mediates neuronal excitability. Complementation tests between the smi60E mutant and several EP insert lines map the smell-impaired phenotype to the P[lArB] insertion site. Wild-type behavior is restored upon P-element excision. Evidence that reduction in DSC1 rather than in L41 expression is responsible for the smell-impaired phenotype comes from a phenotypic revertant in which imprecise P-element excision restores the DSC1 message while further reducing L41 expression. Behavioral assays show that a threefold decrease in DSC1 mRNA is accompanied by a threefold shift in the dose response for avoidance of the repellent odorant, benzaldehyde, toward higher odorant concentrations. In situ hybridization reveals widespread expression of the dsc1 gene in the major olfactory organs, the third antennal segment and the maxillary palps, and in the CNS. These results indicate that the DSC1 channel contributes to processing of olfactory information during the olfactory avoidance response.     

Genetic organization of the ci-M-pan region on chromosome IV in Drosophila melanogaster.

The genes cubitus interruptus (ci), ribosomal protein S3A (RpS3A), and pangolin (pan) are localized within 73 kb in the cytological region 101F-102A on chromosome IV in Drosophila melanogaster. A region of 13 kb harbours the regulatory regions of both ci and pan, transcribed in opposite directions, and a 1.1-kb gene encoding RpS3A. This dense clustering gives rise to very complicated complementation patterns between different alleles in these loci. We investigated this region genetically and molecularly by use of an enhancer trap line (IA5), where the P-element was found to be inserted into the first intron of pan. Screens for imprecise excisions of the P-element were performed, and complementations between new and old established mutant lines were investigated. We found that when mutated or deleted the RpS3A gene gives rise to a Minute phenotype, and we conclude that M(4)101 encodes the ribosomal protein S3A.     

Dosage Effects of a Drosophila Sodium Channel Gene on Behavior and Axonal Excitability

The effects of para mutations on behavior and axonal excitability in Drosophila suggested that para specifically affects sodium channels. This hypothesis was confirmed by molecular analysis of the para locus, which demonstrates that the encoded para product is a sodium channel polypeptide. Here we characterize the effects of altered para+ dosage on behavior and axonal excitability, both in an otherwise wild-type background and in combination with two other mutations: napts, which also affects sodium channels, and ShKS133, which specifically affects potassium channels. Whereas it was previously shown that decreased dosage of para+ is unconditionally lethal in a napts background, we find that increased dosage of para+ suppresses napts. Similarly, we find that para hypomorphs or decreased dosage of para+ suppresses ShKS133, whereas increased dosage of para+ enhances ShKS133). The electrophysiological basis for these effects is investigated. Other genes in Drosophila that have sequence homology to sodium channels do not show such dosage effects, which suggests that the para+ product has a function distinct from that of other putative Drosophila sodium channel genes. We conclude that the number of sodium channels present in at least some Drosophila neurons can be affected by changes in para+ gene dosage, and that the level of para+ expression can strongly influence neuronal excitability.     

The pharmacological flexibility of the insect voltage gated sodium channel: toxicity of AaIT to knockdown resistant (kdr) flies.

AaIT is an insect selective neurotoxic polypeptide shown to affect insect neuronal sodium conductance by binding to excitable sodium channels. In the present study the paralytic potency of AaIT to wild type and various mutant strains of houseflies (Musca domestica) and fruitflies (Drosophila melanogaster) was examined and it has been shown that: On the basis of body weight when compared to published data on Sarcophaga falculata blowflies, the Musca and Drosophila flies reveal at least two orders of magnitude decreased susceptibility to the AaIT. When compared to wild type flies the toxicity of AaIT is greatly altered in knockdown resistant fly strains which are mutated in their para gene encoding the voltage gated sodium channel. Several strains, with genetically mapped para mutations conferring pyrethroid resistance, exhibited opposing response to AaIT. The para ts2 Drosophila strain, with a point of mutation in domain I of the para gene conferring a 6-fold resistance to deltamethrin also showed about 15-fold tolerance to AaIT. On the other hand the Musca kdr and super-kdr flies, with a single or a double point mutation, respectively in domain II of the para gene, are about 9- and 14-fold more susceptible to AaIT, respectively. The above data are interpreted in terms of the pharmacological diversity and flexibility ("allosteric coupling") of voltage gated sodium channels and their implications for the management of pesticide resistance are discussed.     

Alterations in the expression and gating of Drosophila sodium channels by mutations in the para gene

Mutations in the para gene specifically affect the expression of sodium currents in Drosophila. While 65% of wild-type embryonic neurons in culture express sodium currents, three distinct mutations in the para locus resulted in a decrease in the fraction of cells from which sodium currents could be recorded. This reduction was allele-dependent: macroscopic sodium currents were exhibited in 49% of the neurons in parats1 cultures, 35% in parats2, and only 2% in paraST76. Voltage-clamp experiments demonstrated that the parats2 mutation also affected the gating properties of sodium channels. These results provide convincing evidence that para, a gene recently shown to exhibit sequence similarity to vertebrate sodium channels alpha subunits, encodes functional sodium channels in Drosophila. The finding that one para allele (paraST76) can virtually eliminate the expression of sodium currents strongly argues that the para gene codes for the majority of sodium channels in cultured embryonic neurons.     

Analysis of DNA interband regions 3A5/A6, 3C5-6/c7 and 60E8-9/E10 of Drosophila melanogaster polytene chromosomes]

Using electron microscopic (EM) data on the formation of a novel band from the P-element material after its insertion in the interband and the procedure of P-target rescue, DNA interband regions 3A5/A6, and 60E8-9/E10 of Drosophila melanogaster polytene chromosomes were cloned and sequenced. EM analysis of the 3C region have shown that the formation of the full-size 3C5-6/C7 interband requires a 880-bp DNA sequences removed by deletion Df(1)faswb. A comparison of DNA sequences of six bands, two of which were obtained in the present work and four were described earlier, demonstrated the uniqueness of each of them in the Drosophila genome and heterogeneity of their molecular organization. Interband 60E8-9/E10 contains gene rpl19 transcribed throughout the development, in particular in salivary glands. In the other interbands examined 5' and 3' nontranslated gene regions are located. These results suggest that Drosophila interbands may contain both housekeeping genes and regulatory sequences of currently inactive genes from adjacent bands.     

Molecular characterization of the Rpt1/p48B ATPase subunit of the <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> 26S proteasome

The function and the molecular properties of the Rpt1/p48B ATPase subunit of the regulatory particle of the Drosophila melanogaster 26S proteasome have been studied by analyzing three mutant Drosophila stocks in which P-element insertions occurred in the 5′-non-translated region of the Rpt1/p48B gene. These P-element insertions resulted in larval lethality during the second instar larval phase. Since the Rpt1/p48B gene resides within a long intron of an annotated, but uncharacterized Drosophila gene (CG17985), the second instar larval lethality may be a consequence of a combined damage to two independent genes. To analyze the phenotypic effect of the mutations affecting the Rpt1/p48B gene alone, imprecise P-element excision mutants were selected. One of them, the pupal lethal P1 mutation, is a hypomorphic allele of the Rpt1/p48B gene, in which the displacement of two essential regulatory sequences of the gene occurred due to the insertion of a 32 bp residual P-element sequence. This mutation caused a 30-fold drop in the cellular concentration of the Rpt1/p48B mRNA. The decline in the cellular Rpt1/p48B protein concentration induced serious damage in the assembly of the 26S proteasomes, the accumulation of multiubiquitinated proteins, a change in the phosphorylation pattern of the subunit and depletion of this ATPase protein from the chromatin.