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Mutations affecting ion channels and neuronal membrane excitability have been identified in Drosophila as well as in other organisms and characterized for their acute effects on behavior and neuronal function. However, the long-term effect of these perturbations on the maintenance of neuronal viability has not been studied in detail. Here we perform an initial survey of mutations affecting Na+ channels and K+ channels in Drosophila to investigate their effects on life span and neuronal viability as a function of age. We find that mutations that decrease membrane excitability as well as those that increase excitability can trigger neurodegeneration to varying degrees. Results of double-mutant interactions with dominant Na+/K+ ATPase mutations, which themselves cause severe neurodegeneration, suggest that excitotoxicity owing to hyperexcitability is insufficient to explain the resultant phenotype. Although the exact mechanisms remain unclear, our results suggest that there is an important link between maintenance of proper neuronal signaling and maintenance of long-term neuronal viability. Disruption of these signaling mechanisms in any of a variety of ways increases the incidence of neurodegeneration.

The N-ethylmaleimide-sensitive fusion protein (NSF) is a cytoplasmic protein implicated in the fusion of intracellular transport vesicles with their target membranes. NSF is thought to function in the fusion of essentially all types of vesicles, including endoplasmic reticulum, Golgi, and endocytic vesicles, as well as secretory vesicles undergoing regulated fusion (for review see Rothman, J. E.(1994) Nature 372, 55-63). However, little [Medline] experimental evidence exists to address the possibility that organisms might have multiple NSF proteins serving distinct functions in the same or different cells. We previously cloned a neurally expressed Drosophila homolog, dNSF-1 (Ordway, R. W., Pallanck, L., and Ganetzky, B.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5715-5719), and have subsequently identified mutations in this gene that confer an apparent failure of synaptic transmission at elevated temperature (Pallanck, L., Ordway, R. W., and Ganetzky, B.(1995) Nature, 376, 25; Siddiqi, O., and Benzer, S.(1976) Proc. Natl. Acad. Sci. U. S. A. 73, 3253-3257). Here we report that 1) Drosophila contains a second NSF homolog, termed dNSF-2, that exhibits 84% amino acid identity to dNSF-1, 2) dNSF-1 and dNSF-2 display overlapping but different temporal expression, and 3) multiple transcripts are derived from the dNSF-2 gene. These findings raise the possibility that different NSF gene products serve distinct or overlapping functions within the organism.

This summary article presents an overview of the molecular relationships among the voltage-gated potassium channels and a standard nomenclature for them, which is derived from the IUPHAR Compendium of Voltage-Gated Ion Channels.1 The complete Compendium, including data tables for each member of the potassium channel family can be found at http://www.iuphar-db.org/iuphar-ic/.

A very large number of evolutionarily conserved potassium channels have been identified but very little is known about their function or modulation in vivo. Metamorphosis of the tobacco hornworm, Manduca sexta, is a compelling model system for such studies because it permits analysis to be conducted at the level of identified neurons whose roles in simple behaviors and endocrine regulation are known. We present here the characterization of the first ion channel to be cloned from this animal. Partial genomic sequence for Manduca sexta ether à-go-go (Mseag) and a cDNA clone encoding the Mseag open reading frame were obtained. Genomic Southern analysis indicates that Manduca contains a single member of the eag subfamily per haploid genome. When expressed in Xenopus oocytes, MsEag channels conduct a voltage-dependent, K+ selective outward current with an inactivating component that closely resembles the Drosophila eag current. Mseag transcripts were restricted to the nervous system, adult antenna, and one set of larval skeletal muscles. Steroid hormonal regulation of Mseag expression is suggested by the temporal correlation of developmental changes in transcript expression with the changing steroid titers that promote metamorphosis. These results provide the foundation for functional and modulatory studies of the Eag family of K+ channels in Manduca, which will complement the genetic analysis in Drosophila.

Segregation Distorter (SD) is a meiotic drive system in Drosophila that causes preferential transmission of the SD chromosome from SD/SD(+) males owing to induced dysfunction of SD(+) spermatids. Since its discovery in 1956, SD and its mode of action have baffled biologists. Recently, substantial progress has been made in elucidating this puzzle. Sd, the primary gene responsible for distortion encodes a mutant RanGAP, a key protein in the Ran signaling pathway required for nuclear transport and other nuclear functions. The mutant protein is enzymatically active but mislocalized to nuclei, which apparently disrupts Ran signaling by reducing intranuclear Ran-GTP levels. Some evidence suggests that a defect in nuclear transport may be the main cause of sperm dysfunction. Although important questions remain, the basic mechanism of distortion is now understood sufficiently well that specific hypotheses can be formulated and tested. This previously mysterious genetic system may now offer unique insights into novel aspects of regulation by Ran.

Multidisciplinary studies have led to the discovery and characterization of cysteine string proteins (csps) in both Drosophila and Torpedo. Phenotypic analysis of csp mutants in Drosophila demonstrates a crucial role for csp in synaptic transmission. Expression studies of Torpedo csp (Tcsp) in Xenopus oocytes suggests that the protein has some role in the function of presynaptic Ca2+ channels. However, biochemical purification of Tcsp indicates that is associated with synaptic vesicles rather than with the plasma membrane of presynaptic terminals where Ca2+ channels reside. These results suggest a model in which csps serve as a link by which docked synaptic vesicles could modulate the activity of presynaptic Ca2+ channels.

Age-dependent neurodegeneration is a pathological condition found in many metazoans. Despite the biological and medical significance of this condition, the cellular and molecular mechanisms underlying neurodegeneration are poorly understood. The availability of a large collection of mutants exhibiting neurodegeneration will provide a valuable resource to elucidate these mechanisms. We have developed an effective screen for isolating neurodegeneration mutants in Drosophila. This screen is based on the observation that neuronal dysfunction, which leads to observable behavioral phenotypes, is often associated with neurodegeneration. Thus, we used a secondary histological screen to examine a collection of mutants originally isolated on the basis of conditional paralytic phenotypes. Using this strategy, we have identified 15 mutations affecting at least nine loci that cause gross neurodegenerative pathology. Here, we present a genetic, behavioral, and anatomical analysis of vacuous (vacu), the first of these mutants to be characterized, and an overview of other mutants isolated in the screen. vacu is a recessive mutation located cytologically at 85D-E that causes locomotor defects in both larvae and adults as well as neuronal hyperactivity. In addition, vacu exhibits extensive age-dependent neurodegeneration throughout the central nervous system. We also identified mutations in at least eight other loci that showed significant levels of neurodegeneration with a diverse array of neuropathological phenotypes. These results demonstrate the effectiveness of our screen in identifying mutations causing neurodegeneration. Further studies of vacu and the other neurodegenerative mutants isolated should ultimately help dissect the biochemical pathways leading to neurodegeneration.

Rabphilin, a putative rab effector, interacts specifically with the GTP-bound form of the synaptic vesicle-associated protein rab3a. In this study, we define in vivo functions for rabphilin through the characterization of mutants that disrupt the Caenorhabditis elegans rabphilin homolog. The mutants do not display the general synaptic defects associated with rab3 lesions, as assayed at the pharmacological, physiological, and ultrastructural level. However, rabphilin mutants exhibit severe lethargy in the absence of mechanical stimulation. Furthermore, rabphilin mutations display strong synergistic interactions with hypomorphic lesions in the syntaxin, synaptosomal-associated protein of 25 kDa, and synaptobrevin soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) genes; double mutants were nonresponsive to mechanical stimulation. These synergistic interactions were independent of rab3 function and were not observed in rab3-SNARE double mutants. Our data reveal rab3-independent functions for rabphilin in the potentiation of SNARE function.

Segregation Distorter (SD) is a meiotic drive system in Drosophila that causes preferential transmission of the SD chromosome from SD/SD+ males owing to dysfunction of SD+ spermatids. The Sd locus, which is essential for distortion, encodes a truncated RanGAP (Ran GTPase activating protein), a key nuclear transport factor. Here, we show that Sd-RanGAP retains normal enzyme activity but is mislocalized to nuclei. Distortion is abolished when enzymatic activity or nuclear localization of Sd-RanGAP is perturbed. Overexpression of Ran or RanGEF (Ran GTPase exchange factor) in the male germline fully suppresses distortion. We conclude that mislocalization of Sd-RanGAP causes distortion by reducing nuclear RanGTP, thereby disrupting the Ran signaling pathway. Nuclear transport of a GFP reporter in salivary glands is impaired by SD, suggesting that a defect in nuclear transport may underlie sperm dysfunction.

Post-transcriptional editing of pre-mRNAs through the action of dsRNA adenosine deaminases results in the modification of particular adenosine (A) residues to inosine (I), which can alter the coding potential of the modified transcripts. We describe here three sites in the para transcript, which encodes the major voltage-activated Na+ channel polypeptide in Drosophila, where RNA editing occurs. The occurrence of RNA editing at the three sites was found to be developmentally regulated. Editing at two of these sites was also conserved across species between the D. melanogaster and D. virilis. In each case, a highly conserved region was found in the intron downstream of the editing site and this region was shown to be complementary to the region of the exonic editing site. Thus, editing at these sites would appear to involve a mechanism whereby the edited exon forms a base-paired secondary structure with the distant conserved noncoding sequences located in adjacent downstream introns, similar to the mechanism shown for A-to-I RNA editing of mammalian glutamate receptor subunits (GluRs). For the third site, neither RNA editing nor the predicted RNA secondary structures were evolutionarily conserved. Transcripts from transgenic Drosophila expressing a minimal editing site construct for this site were shown to faithfully undergo RNA editing. These results demonstrate that Na+ channel diversity in Drosophila is increased by RNA editing via a mechanism analogous to that described for transcripts encoding mammalian GluRs.

Many of the signaling properties of neurons and other electrically excitable cells are determined by a diverse family of potassium channels. A number of genes that encode potassium channel polypeptides have been cloned from various organisms on the basis of their sequence similarity to the Drosophila Shaker (Sh) locus. As an alternative strategy, a molecular analysis of other Drosophila genes that were defined by mutations that perturb potassium channel function was undertaken. Sequence analysis of complementary DNA from the ether à go-go (eag) locus revealed that it encodes a structural component of potassium channels that is related to but is distinct from all identified potassium channel polypeptides.

The newly identified axotactin (AXO) protein in Drosophila, a member of the neurexin superfamily, appears to be a key element that mediates the effects of glial cells on neuronal membrane excitability and synaptic plasticity.

Recent genetic studies have significantly advanced our understanding of the role of endocytosis in SV recycling. It is increasingly apparent that the regulation of vesicle size and composition is coupled during clathrin-dependent reassembly at the plasma membrane. Further, SV proteins, such as synaptotagmin, can no longer be considered as exocytic proteins only, but also have an active role in the recycling process. These studies leave many important questions to be addressed in the future. An immediate goal will be to dissect the mechanistic details by which the CLASS proteins affect clathrin cage assembly. A systematic in vitro and in vivo structure–function analysis should afford a better understanding of the origin of the uniformity of coated vesicles, including those at synapses. Another interesting direction is to examine whether SV size can be regulated under physiological conditions and if such modulations contribute to synaptic plasticity.§To whom correspondence should be addressed (e-mail: bxz@bcm.tmc.edu [B. Z.] and vnmurthy@fas.harvard.edu [V. N. M.]).

Segregation Distorter ( SD ) in Drosophila melanogaster is a naturally occurring meiotic drive system in which the SD chromosome is transmitted from SD/SD + males in vast excess over its homolog owing to the induced dysfunction of SD + -bearing spermatids. The Sd locus is the key distorting gene responsible for this phenotype. A genomic fragment from the Sd region conferred full distorting activity when introduced into the appropriate genetic background by germline transformation. The only functional product encoded by this fragment is a truncated version of the RanGAP nuclear transport protein. These results demonstrate that this mutant RanGAP is the functional Sd product.

In the nervous system, glial cells greatly outnumber neurons but the full extent of their role in determining neural activity remains unknown. Here the axotactin ( axo ) gene of Drosophila was shown to encode a member of the neurexin protein superfamily secreted by glia and subsequently localized to axonal tracts. Null mutations of axo caused temperature-sensitive paralysis and a corresponding blockade of axonal conduction. Thus, the AXO protein appears to be a component of a glial-neuronal signaling mechanism that helps to determine the membrane electrical properties of target axons.

Theeagfamily of K+channels contains three known subtypes:eag,elk, anderg. Genes representing the first two subtypes have been identified in flies and mammals, whereas the third subtype has been defined only by the human HERG gene, which encodes an inwardly rectifying channel that is mutated in some cardiac arrhythmias. To establish the predicted existence of aDrosophilagene in theergsubfamily and to learn more about the structure and biological function of channels within this subfamily, we undertook a search for theDrosophilacounterpart of HERG. Here we report the isolation and characterization of theDrosophila erggene. We show that it corresponds with the previously identified seizure (sei) locus, mutations of which cause a temperature-sensitive paralytic phenotype associated with hyperactivity in the flight motor pathway. These results yield new insights into the structure and evolution of theeagfamily of channels, provide a molecular explanation for theseimutant phenotype, and demonstrate the important physiological roles oferg-type channels from invertebrates to mammals.

Mammalian high conductance, calcium-activated potassium (maxi-K) channels are composed of two dissimilar subunits, alpha and beta. We have examined the functional contribution of the beta subunit to the properties of maxi-K channels expressed heterologously in Xenopus oocytes. Channels from oocytes injected with cRNAs encoding both alpha and beta subunits were much more sensitive to activation by voltage and calcium than channels composed of the alpha subunit alone, while expression levels, single-channel conductance, and ionic selectivity appeared unaffected. Channels from oocytes expressing both subunits were sensitive to DHS-I, a potent agonist of native maxi-K channels, whereas channels composed of the alpha subunit alone were insensitive. Thus, alpha and beta subunits together contribute to the functional properties of expressed maxi-K channels. Regulation of co-assembly might contribute to the functional diversity noted among members of this family of potassium channels.

Several lines of investigation have now converged to indicate that the neurotransmitter release apparatus is formed by assembly of cytosolic proteins with proteins of the synaptic vesicle and presynaptic terminal membranes. We are undertaking a genetic approach in Drosophila melanogaster to investigate the functions of two types of cytosolic proteins thought to function in this complex: N-ethylmaleimide-sensitive fusion protein (NSF) and the soluble NSF attachment proteins (SNAPs). We have identified Drosophila homologs of the vertebrate and yeast NSF and SNAP genes. Both Drosophila genes encode polypeptides that closely resemble their vertebrate counterparts and are expressed in the nervous system; neither appears to be in a family of closely related Drosophila genes. These results indicate that the Drosophila NSF and SNAP genes are excellent candidates for mutational analysis of neurotransmitter release.

The enormous information handling capacity of nervous systems depends on the proper function of a rich variety of neurons and other excitable cells that display distinctive signal processing characteristics. This physiological complexity reflects cellular differences in the expression and regulation of ion channels. Genetic dissection has been successfully applied in elucidation of mechanisms underlying many biological processes in model systems. Drosophila has been a favored organism for such an approach to complex problems in higher organisms, such as function and development of the nervous system (Benzer, 1973; Pak and Pinto, 1976).

We investigate the molecular and cellular etiologies that underlie the deletion of the six amino acid residues (DeltaF323-Y328; DeltaFY) in human torsin A (HtorA). The most common and severe mutation involved with early-onset torsion dystonia is a glutamic acid deletion (DeltaE 302/303; DeltaE) in HtorA which induces protein aggregates in neurons and cells. Even though DeltaFY HtorA forms no protein clusters, flies expressing DeltaFY HtorA in neurons or muscles manifested a similar but delayed onset of adult locomotor disability compared with flies expressing DeltaE in HtorA. In addition, flies expressing DeltaFY HtorA had fewer aberrant ultrastructures at synapses compared with flies expressing DeltaE HtorA. Taken together, the DeltaFY mutation in HtorA may be responsible for behavioral and anatomical aberrations in gDrosophila.

nap ts is a recessive mutation that affects the level of sodium channel activity and, at high temperature, causes paralysis associated with a loss of action potentials. We show, by genetic complementation tests, germline transformation, and analysis of mutations, that nap ts is a gain-of-function mutation of mle a gene required for X chromosome dosage compensation and male viability. Molecular analyses of nap and mle mutations indicate that mle + , nap +, and nap ts activities are encoded by the same open reading frame and suggest that nap ts is due to a single amino acid substitution. Although nap ts is known to act via para +, an X-linked sodium channel structural gene, its effect is not due to a simple defect in para + dosage compensation.

Calcium-activated potassium channels mediate many biologically important functions in electrically excitable cells. Despite recent progress in the molecular analysis of voltage-activated K+ channels, Ca(2+)-activated K+ channels have not been similarly characterized. The Drosophila slowpoke (slo) locus, mutations of which specifically abolish a Ca(2+)-activated K+ current in muscles and neurons, provides an opportunity for molecular characterization of these channels. Genomic and complementary DNA clones from the slo locus were isolated and sequenced. The polypeptide predicted by slo is similar to voltage-activated K+ channel polypeptides in discrete domains known to be essential for function. Thus, these results indicate that slo encodes a structural component of Ca(2+)-activated K+ channels.

Since our 7 July 1995 report “HERG, a human inward rectifier in the voltage-gated potassium channel family” was published ([1][1]), two previously undetected differences between our expression clone and the published nucleotide sequence ([2][2]) have been identified: T593A, yielding amino acid change V198E; and C605T, yielding P202L. These differences are thought to be the result of artifacts generated by use of the polymerase chain reaction technique. The fundamental consequence of the two mutations is a variable reduction of inward rectification. Full characterizations of the mutant and the wild type phenotypes will appear in a subsequent report. 1. 1.[↵][3] 1. M. C. Trudeau 2. et al. , Science 269, 92 (1995). [OpenUrl][4][Abstract/FREE Full Text][5] 2. 2.[↵][6] 1. J. W. Warmke, 2. B. Ganetzky , Proc. Natl. Acad. Sci. U.S.A. 91, 3438 (1994). [OpenUrl][7][Abstract/FREE Full Text][8] [1]: #ref-1 [2]: #ref-2 [3]: #xref-ref-1-1 "View reference 1. in text" [4]: {openurl}?query=rft.jtitle%253DScience%26rft.stitle%253DScience%26rft.issn%253D0036-8075%26rft.aulast%253DTrudeau%26rft.auinit1%253DM.%26rft.volume%253D269%26rft.issue%253D5220%26rft.spage%253D92%26rft.epage%253D95%26rft.atitle%253DHERG%252C%2Ba%2Bhuman%2Binward%2Brectifier%2Bin%2Bthe%2Bvoltage-gated%2Bpotassium%2Bchannel%2Bfamily%26rft_id%253Dinfo%253Adoi%252F10.1126%252Fscience.7604285%26rft_id%253Dinfo%253Apmid%252F7604285%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [5]: /lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6Mzoic2NpIjtzOjU6InJlc2lkIjtzOjExOiIyNjkvNTIyMC85MiI7czo0OiJhdG9tIjtzOjI2OiIvc2NpLzI3Mi81MjY1LzEwODMuMTEuYXRvbSI7fXM6ODoiZnJhZ21lbnQiO3M6MDoiIjt9 [6]: #xref-ref-2-1 "View reference 2. in text" [7]: {openurl}?query=rft.jtitle%253DPNAS%26rft.stitle%253DProc.%2BNatl.%2BAcad.%2BSci.%2BUSA%26rft.issn%253D0027-8424%26rft.aulast%253DWarmke%26rft.auinit1%253DJ.%2BW.%26rft.volume%253D91%26rft.issue%253D8%26rft.spage%253D3438%26rft.epage%253D3442%26rft.atitle%253DA%2Bfamily%2Bof%2Bpotassium%2Bchannel%2Bgenes%2Brelated%2Bto%2Beag%2Bin%2BDrosophila%2Band%2Bmammals.%26rft_id%253Dinfo%253Adoi%252F10.1073%252Fpnas.91.8.3438%26rft_id%253Dinfo%253Apmid%252F8159766%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [8]: /lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6NDoicG5hcyI7czo1OiJyZXNpZCI7czo5OiI5MS84LzM0MzgiO3M6NDoiYXRvbSI7czoyNjoiL3NjaS8yNzIvNTI2NS8xMDgzLjExLmF0b20iO31zOjg6ImZyYWdtZW50IjtzOjA6IiI7fQ==

The segregation distorter (SD) complex is a naturally occurring meiotic drive system with the property that males heterozygous for an SD-bearing chromosome 2 and an SD +-bearing homolog transmit the SD-bearing chromosome almost exclusively. This distorted segregation is the consequence of an induced dysfunction of those sperm that receive the SD + homolog. From previous studies, two loci have been implicated in this phenomenon: the Sd locus which is required to produce distortion, and the Responder (Rsp) locus that is the site at which Sd acts. There are two allelic alternatives of Rsp—sensitive (Rspsens) and insensitive (Rspins); a chromosome carrying Rspins is not distorted by SD. In the present study, the function and location of each of these elements was examined by a genetic and cytological characterization of X-ray-induced mutations at each locus. The results indicate the following: (1) the Rsp locus is located in the proximal heterochromatin of 2R; (2) a deletion for the Rsp locus renders a chromosome insensitive to distortion; (3) the Sd locus is located to the left of pr(2-54.5), in the region from 37D2-D7 to 38A6-B2 of the salivary chromosome map; (4) an SD chromosome deleted for Sd loses its ability to distort; (5) there is another important component of the SD system, E(SD), in or near the proximal heterochromatin of 2L, that behaves as a strong enhancer of distortion. The results of these studies allow a reinterpretation of results from earlier analyses of the SD system and serve to limit the possible mechanisms to account for segregation distortion.

A collection of behavioral mutations perturbing various aspects of neuronal membrane excitability has been obtained in Drosophila . These mutations are the subjects of a multidisciplinary analysis using genetic, electrophysiological, and molecular techniques to learn how and why they perturb excitable membranes. Results from these studies can help elucidate how the signalling capabilities of different neurons are genetically specified and how changes in the activity of these cells modifies behavior.

Recombinational dissection of the ShrK0120 strain of Drosophila melanogaster demonstrated that its extremely prolonged neuromuscular transmission, a defect qualitatively different from other Sh alleles, results from a synergistic interaction with a second site mutation. This new mutation caused spontaneous repetitive firing in motor axons and increased transmitter release. Complementation tests showed that it is allelic to eag. Combining either eag allele with various Sh alleles reproduced the same extreme defect observed in the original ShrK0120 strain. Thus, from their effects on neuromuscular transmission no qualitative difference among Sh alleles is apparent while Sh, eag, and eag Sh double mutant individuals can be uniquely distinguished. Pharmacological experiments indicated that Sh affects a membrane component sensitive to the potassium channel blockers 4-AP and TEA while eag affects a component sensitive to TEA but not 4-AP. We suggest that eag and Sh preferentially disrupt different potassium currents explaining their synergistic interaction.

A mutation of Drosophila, slowpoke (slo), specifically abolishes a Ca2+-dependent K+ current, IC, from dorsal longitudinal flight muscles of adult flies. Other K+ currents remain normal, providing evidence that IC is mediated by a molecularly distinguishable set of channels. The pharmacological properties of IC are similar to those of Ca2+-dependent currents in some vertebrate cells. The muscle action potential was significantly lengthened in slo flies, indicating that IC plays the major role in its repolarization.

We have isolated and sequenced a cDNA from Drosophila melanogaster that is homologous to the mouse Tcp-1 gene encoding the t complex polypeptide 1, TCP-1. The Drosophila gene maps by in situ hybridization to bands 94B 1–2 of the polytene chromosomes. It shares 66% nucleotide sequence identity with the mouse gene. The predicted Drosophila protein consists of 557 amino acids and shares 72% identity with the mouse polypeptide. The TCP-1 polypeptide appears to be highly conserved in evolution from mammals to simple eukaryotes because the Drosophila gene probe also detects related sequences in DNA from the yeast, Saccharomyces cerevisiae . The presence of TCP-1-related polypeptides in organisms such as Drosophila and yeast should facilitate biochemical and genetic analysis of its function.

Ion channels play key roles in the generation and propagation of nerve impulses by mediating ionic fluxes across excitable membranes. Elucidation of the structure and function of these proteins is a major goal in understanding the molecular mechanisms of membrane excitability. Much work has focused on sodium channels, whose activity is of primary importance in producing nerve impulses. Despite the recent cloning and sequencing of a sodium channel structural gene, many unanswered questions remain. To address some of these question genetically, Drosophila mutants with altered sodium channels are being isolated and subjected to multidisciplinary analyses. Ultimately, these can provide novel approaches for understanding the function and regulation of sodium channels at a molecular level.

Two classes of X-linked behavioral mutants of Drosophila melanogaster, leg-shaking mutants and bang-sensitive mutants, are suppressed by napts (no action potential, temperature-sensitive), an autosomal temperature-sensitive paralytic mutation. So far, napts is found to suppress thirteen mutations at seven loci, two of which produce leg shaking and five bang-sensitivity. Suppression is recessive, occurs at temperatures permissive for napts, and is indirect and function-specific rather than allele-specific. At restrictive temperatures, napts is known to completely block all nerve activity. Several of the mutants suppressed by napts are shown by neurophysiological experiments to have increased nerve excitability. The physiological defect of these mutants as well as their behavioral defect is suppressed by napts. Thus, suppression occurs within individual neurons at the level of excitable membranes and apparently depends on the reduction in membrane excitability caused by napts even under permissive conditions. We suggest that all mutants suppressed by napts may have related defects leading to enhanced nerve excitability. Genetic interactions of this type help reveal functional relationships between different behavioral mutants and suggest ways of isolating new mutants with altered excitable membranes.

The growth of ovaries of a wild type strain of Drosophila melanogaster has been studied from eclosion to 7 days later. Wet weight, protein, DNA, RNA, and carbohydrate have been determined at successive intervals. All experiments were performed on flies grown axenically on a killed yeast medium. The results indicate that wet weight increases rapidly from day 1 to day 4 and reaches a maximum at day 6. This increase is associated with the appearance of vitellogenic and post-vitellogenic stages of oogenesis. The increase in protein and RNA generally follows the wet weight curve and remains more or less constant after reaching a maximum at day 6. The DNA content reaches a maximum at day 3 and falls rapidly thereafter. The carbohydrate content increases slowly, reaching a maximum at day 7. The data are compared with available evidence from other strains and genotypes of D. melanogaster .

We describe the isolation and characterization of nwk (nervous wreck), a temperature-sensitive paralytic mutant that causes excessive growth of larval neuromuscular junctions (NMJs), resulting in increased synaptic bouton number and branch formation. Ultrastructurally, mutant boutons have reduced size and fewer active zones, associated with a reduction in synaptic transmission. nwk encodes an FCH and SH3 domain-containing adaptor protein that localizes to the periactive zone of presynaptic terminals and binds to the Drosophila ortholog of Wasp (Wsp), a key regulator of actin polymerization. wsp null mutants display synaptic overgrowth similar to nwk and enhance the nwk morphological phenotype in a dose-dependent manner. Evolutionarily, Nwk belongs to a previously undescribed family of adaptor proteins that includes the human srGAPs, which regulate Rho activity downstream of Robo receptors. We propose that Nwk controls synapse morphology by regulating actin dynamics downstream of growth signals in presynaptic terminals.

The mle napts mutation causes temperature-dependent blockade of action potentials resulting from decreased abundance of para -encoded Na + channels. Although maleless ( mle ) encodes a double-stranded RNA (dsRNA) helicase, exactly how mle napts affects para expression remained uncertain. Here, we show that para transcripts undergo adenosine-to-inosine (A-to-I) RNA editing via a mechanism that apparently requires dsRNA secondary structure formation encompassing the edited exon and the downstream intron. In an mle napts background, >80% of para transcripts are aberrant, owing to internal deletions that include the edited exon. We propose that the Mle helicase is required to resolve the dsRNA structure and that failure to do so in an mle napts background causes exon skipping because the normal splice donor is occluded. These results explain how mle napts affects Na + channel expression and provide new insights into the mechanism of RNA editing.

Single mutations that disrupt basic physiological processes such as axonal conduction and synaptic transmission provide one means to study the membrane components and mechanisms that underlie these processes1,8. Temperature-sensitive paralytic mutants of Drosophila melanogaster which behave normally at a permissive temperature (23–25 °C) but become paralysed at a restrictive temperature (usually 29–37 °C) have been utilized in such studies. An example is napts (no action potential, temperature-sensitive), a recessive second-chromosome mutation that causes paralysis of larvae and adults at 37 °C. Electrophysiological recordings have demonstrated that napts specifically affects a component of axonal membranes5. Here we show that axonal conduction in napts larvae is abnormally sensitive to tetrodotoxin (TTX), a specific blocker of voltage-sensitive sodium channels9, and that the refractory period of action potentials differs from wild type, consistent with the idea that napts alters the function of sodium channels. It is further shown that parats (paralysed, temperature-sensitive), a recessive X-linked mutation1, also specifically disrupts axonal conduction and that the two mutations affect functionally related membrane components. Identification of other mutants defective in associated components of nerve membranes is made feasible by using techniques described here.

Electrophysiological analysis of the Drosophila behavioral mutants Eag and Sh and the double mutant Eag Sh indicates that the products of both genes take part in the control of potassium currents in the membranes of both nerve and muscle. In voltage-clamped larval muscle fibers, Sh affects the transient A current, whereas Eag reduces the delayed rectification and, to a lesser extent, the A current.

Male Drosophila heterozygous for an SD-bearing second chromosome and a normal homolog preferentially transmit the SD chromosome to their offspring. The distorted transmission involves the induced dysfunction of the sperm that receive the SD+ chromosome. The loci on the SD chromosome responsible for causing distortion are the Sd locus the the E(SD) locus. Their target of action on the SD+ chromosome is the Rsps locus. Previous studies of Rsps indicated that deletion of this locus rendered a chromosome insensitive to the action of SD and mapped Rsps physically within the centric heterochromatin of 2R. In this study we have constructed a collection of marked free duplications for the centromeric region of a second chromosome that carried Rsps. The heterochromatic extent of each duplication as well as its sensitivity to distortion was determined. We found that Rsps is the most proximal known locus within the 2R heterochromatin. Furthermore, our results demonstrate that the presence of Rsps is not only necessary but sufficient to confer sensitivity to distortion irrespective of its association with an intact second chromosome or one that pairs meiotically with an SD chromosome. By use of these duplications we increased the usual dosage of Rsps relative to SD to determine whether there was any competition for limited amounts of SD [and/or E(SD)] product. When two Rsps-bearing chromosomes are present within the same spermatocyte nucleus an SD chromosome is capable of causing efficient distortion of both. However, at least in some cases the degree of distortion against a given Rsps was reduced by the presence of an extra dose of Rsps indicating that there was some competition between them. The bearing of these results on present models of segregation distortion are discussed.

Analysis of X-ray-induced deletions in the Segregation Distorter (SD) chromosome, SD-5, revealed that this chromosome had a gene proximal to lt in the centric heterochromatin of 2L that strongly enhanced the meiotic drive caused by the SD chromosome. This Enhancer of Segregation Distortion [E(SD)] locus had not been characterized in earlier studies of SD chromosomes because it cannot be readily separated by recombination from the Responder (Rsp) locus in the proximal heterochromatin of 2R.—To determine whether E(SD) is a general component of all SD chromosomes and to examine further its effects on distortion, we produced deletions of E(SD) in three additional SD chromosomes. Analysis of these deletions leads to the following conclusions: (1) along with Sd and Rsp, E(SD) is common to all SD chromosomes; (2) the E(SD) allele on each SD chromosome enhances distortion by the same amount, which indicates that allelic variation at the E(SD) locus is not responsible for the different drive strengths seen among SD chromosomes; (3) E(SD) causes very little or no distortion by itself in the absence of Sd; (4) E(SD), like Sd, acts in a dosage-dependent manner; (5) E(SD) exerts its effect in cis or trans to Sd; and (6) if E(SD) + exists, its function is not related to SD.

Previous behavioral, electrophysiological, pharmacological, and genetic studies of mutations of the para locus in Drosophila melanogaster suggested that these mutations alter the structure or function of sodium channels. To identify the protein encoded by this gene and to elucidate the molecular basis of the mutant phenotypes, genomic DNA from the para locus was cloned. Mutational lesions in nine different para alleles were mapped and found to be distributed over a region of 45 kb. Analysis of cDNAs revealed that the para locus comprises a minimum of 26 exons distributed over more than 60 kb of genomic DNA. The nucleotide sequence of the complementary DNA predicts a protein whose structure and amino acid sequence are extremely similar to those of vertebrate sodium channels. The results support the conclusion that para encodes a functionally predominant class of sodium channels in Drosophila neurons. Furthermore, the para transcript appears to undergo alternative splicing to produce several distinct subtypes of this channel.

Two mutants of Drosophila melanogaster, parats 1 (1-53.9) and napts (2-56.2) both display similar temperature-sensitive paralysis associated with blockage in the conduction of nerve action potentials, suggesting that the two gene products have a similar function. This idea is supported by the observation that the double mutant is unconditionally lethal. Genetic analysis of this synergistic interaction has revealed the following: 1) it specifically involves the para and nap loci; (2) all para alleles interact with napts, but the strength of the interaction varies in an allele-dependent fashion; (3) lethality of the double mutant occurs during the first larval instar with parats 1 but differs with other para alleles; (4) hypodosage of para + causes lethality in a napts background. These results together with previous electrophysiological, behavioral and pharmacological studies of these mutants suggest that both para and nap affect sodium channels and possibly encode different subunits.

A combined molecular and genetic analysis of the 87E actin gene (Act87E) in Drosophila melanogaster was undertaken. A clone of Act87E was isolated and characterized. The Act87E transcription unit is 1.57 kb and includes a 556-base intervening sequence in the 5' leader of the gene. The protein-coding region is contiguous and encodes a protein that is greater than 93% identical to the other Drosophila actins. By in situ hybridization with a series of deficiencies that break in 87E, Act87E was localized to a region encompassing one to three faint, polytene chromosome bands. The region between the deficiency endpoints that flank the actin gene was isolated and measures approximately 24-30 kb. The closest proximal deficiency endpoint lies 8-10 kb 5' to the actin gene; the closest distal deficiency endpoint lies 16-20 kb 3' to the actin gene. A single, recessive lethal complementation group lies between the deficiency endpoints that flank the actin gene. An EMS mutagenesis screen produced four additional members of this recessive lethal complementation group. Molecular analysis of the members of this complementation group indicated that two of the newly induced mutations have deletions of approximately 1 kb in a transcribed region 4-5 kb 3' (distal) to the actin gene. This result suggests that the recessive lethal complementation group represents a gene separate from and distal to the actin gene. The mutagenesis screen failed to identify additional recessive lethal complementation groups in the actin gene-containing region. The implications of the failure to identify recessive lethal mutations in the actin gene are discussed in reference to studies of other conserved multigene families and other muscle protein mutations.

Identification of allelic variability at the two major loci (Sd and Rsp) that interact to cause sperm dysfunction in Segregation distorter (SD) males of D. melanogaster has been hampered by the difficulty in separating the elements recombinationally. In addition, small differences in the strength of Sd alleles or sensitivities of Rsp alleles to Sd are difficult to measure against background genetic or environmental variation. Viability effects of the markers used to score progeny classes may also introduce a bias. Removal of Sd and E(SD) from their second chromosome location to create a Dp(2;Y)Sd E(SD) chromosome eliminates these problems, since any combination of Rsp alleles can be easily tested without resorting to recombinational techniques. Further, since these pairs of Rsp alleles are compared in their response to Dp Sd E(SD) in the same individual males, background variation and viability effects can be easily removed to allow finescale resolution of Rsp differences. Tests of all possible pairwise combination of six laboratory chromosomes in this way revealed at least three and possibly four different Rsp allelic classes. In addition, the hierarchical nature of the tests further allowed for determination of the presence of linked suppressors or enhancers of Sd activity. A sample of 11 second chromosomes selected from a group recently isolated from a natural population was also unambiguously ordered as to Rsp allelic status using this approach. The resultant pattern was similar to that obtained for the laboratory chromosomes, except for the not unexpected observation that the natural population apparently harbored more drive suppressors. The pattern of results obtained from these pairwise combinations of Rsp alleles supports the notion that there are no dominance interactions within the group, but that each responds more or less independently to Sd in giving sperm dysfunction.

Article de synthese sur les progres dans le domaine des proteines des canaux ioniques transmembranaires et de la genetique de ces proteines chez D. melanogaster et C. elegans

Synaptic transmission in Drosophila can be altered by mutations in specific genes. For example, mutations in the Shaker (Sh) gene, which encodes the rapidly inactivating A-type potassium channel, cause repetitive nerve firing and prolonged transmitter release at the neuromuscular junction. Here we show that mutations in the Hyperkinetic (Hk) gene also affect the properties of synaptic transmission at the neuromuscular junction. In particular, we find that whereas single or low frequency nerve stimulation evokes a wild type postsynaptic response, at higher frequencies of nerve stimulation, each stimulus results in repetitive nerve firing and increased postsynaptic response, which is similar to that observed in Sh mutants. Various experiments suggest that this increased postsynaptic response results from prolonged depolarization of the nerve terminal, leading to increased transmitter release at the neuromuscular junction. The similarity in phenotypes between Sh and Hk mutants, along with the observation that Sh is epistatic to Hk in its effects on synaptic transmission, suggest that Hk acts on synaptic transmission by an effect on A-type potassium channels.