Behavioral and Cytogenetic Analysis of the cacophony Courtship Song Mutant and Interacting Genetic Variants in Drosophila melanogaster

The courtship song of a Drosophila melanogaster male consists of tone pulses interspersed with humming sounds. An X chromosomal mutation, cacophony (cac), causes the production of polycyclic pulses readily distinguishable from those in wild type, which are mono- or bicyclic. Yet, courtship hums and flight wing beats are normal in this mutant, suggesting a specific role of the cac gene in the neural program underlying one particular feature of the fly's wing vibrations. A precise cytogenetic localization of cac is presented; this was obtained by uncovering the song abnormality with deletions that are missing all or the distal part of region 11A; the flies tested were diplo-X adults that had been turned into males by the transformer mutation. Duplications including distal 11A covered cac. The possibility of behavioral specificity for cac's effects was examined by screening a variety of sexual and nonsexual behaviors; these experiments included tests of flies in which the mutation was uncovered by a small deletion. We conclude that cac causes only a limited array of well-defined defects: longer and louder tone pulses in the song and depressed locomotor activity. Further complementation tests involving cac and other closely linked genetic variants--the night-blind-A (nbA) visual mutation, l(1)L13 lethal mutations, and a series of X chromosomal breakpoints--suggested complex interactions among these factors: the breakpoints uncover all three types of mutations; cac and nbA appear to be alleles of l(1)L13, whereas the two behavioral mutations complement each other.     

Progressive ataxia due to a missense mutation in a calcium-channel gene.

We describe a family with severe progressive cerebellar ataxia involving the trunk, the extremities, and speech. The proband, who has prominent atrophy of the cerebellum, shown by magnetic resonance imaging, was confined to a wheelchair at the age of 44 years. Two sons have episodes of vertigo and ataxia that are not responsive to acetazolamide. Quantitative eye-movement testing showed a consistent pattern of abnormalities localizing to the cerebellum. Genotyping suggested linkage to chromosome 19p, and SSCP showed an aberrant migrating fragment in exon 6 of the calcium-channel gene CACNA1A, which cosegregated with the disease. Sequencing of exon 6 identified a G-->A transposition in one allele, at nucleotide 1152, resulting in a predicted glycine-to-arginine substitution at codon 293. The CAG-repeat expansion associated with spinocerebellar ataxia 6 was not present in any family members. This family is unique in having a non-CAG-repeat mutation that leads to severe progressive ataxia. Since a great deal is known about the function of calcium channels, we speculate on how this missense mutation leads to the combination of clinical symptoms and signs.     

Courtship, aggression and avoidance: pheromones, receptors and neurons for social behaviors in Drosophila

Chemical communication between individual Drosophila is extremely important for social behaviors required for survival and reproduction, such as con-specific recognition, courtship, aggression and avoidance of odor from "stressed" flies. Characterization of the receptors and neural circuits that detect pheromone cues and an understanding of how these circuits are modulated by the social interactions are fundamental questions about the neurobiology of social behaviors. Recent years have seen important advances in the identification of chemoreceptors and sensory neurons that are involved in sensing pheromones. Here we present a brief review of the current understanding of the peripheral chemosensory systems that are involved in social behaviors.     

Synthetic lethality of retinoblastoma mutant cells in the Drosophila eye by mutation of a novel peptidyl prolyl isomerase gene

Mutations that inactivate the retinoblastoma (Rb) pathway are common in human tumors. Such mutations promote tumor growth by deregulating the G1 cell cycle checkpoint. However, uncontrolled cell cycle progression can also produce new liabilities for cell survival. To uncover such liabilities in Rb mutant cells, we performed a clonal screen in the Drosophila eye to identify second-site mutations that eliminate Rbf(-) cells, but allow Rbf(+) cells to survive. Here we report the identification of a mutation in a novel highly conserved peptidyl prolyl isomerase (PPIase) that selectively eliminates Rbf(-) cells from the Drosophila eye.     

Genetic modifiers of the Drosophila NSF mutant, comatose, include a temperature-sensitive paralytic allele of the calcium channel alpha1-subunit gene, cacophony

The N-ethylmaleimide-sensitive fusion protein (NSF) has been implicated in vesicle trafficking in perhaps all eukaryotic cells. The Drosophila comatose (comt) gene encodes an NSF homolog, dNSF1. Our previous work with temperature-sensitive (TS) paralytic alleles of comt has revealed a function for dNSF1 at synapses, where it appears to prime synaptic vesicles for neurotransmitter release. To further examine the molecular basis of dNSF1 function and to broaden our analysis of synaptic transmission to other gene products, we have performed a genetic screen for mutations that interact with comt. Here we report the isolation and analysis of four mutations that modify TS paralysis in comt, including two intragenic modifiers (one enhancer and one suppressor) and two extragenic modifiers (both enhancers). The intragenic mutations will contribute to structure-function analysis of dNSF1 and the extragenic mutations identify gene products with related functions in synaptic transmission. Both extragenic enhancers result in TS behavioral phenotypes when separated from comt, and both map to loci not previously identified in screens for TS mutants. One of these mutations is a TS paralytic allele of the calcium channel alpha1-subunit gene, cacophony (cac). Analysis of synaptic function in these mutants alone and in combination will further define the in vivo functions and interactions of specific gene products in synaptic transmission.     

Identification of a novel mutation in WFS1 in a family affected by low-frequency hearing impairment

Previously we confirmed linkage of autosomal dominantly inherited low-frequency sensorineural hearing impairment (LFSNHI) in a German family to the genetic locus DFNA6/DFNA14 on chromosome 4p16.3 close to the markers D4S432 and D4S431. Analysis of data from the Human Genome Project, showed that WFS1 is located in this region. Mutations in WFS1 are known to be responsible for Wolfram syndrome (DIDMOAD, MIM #606201), which follows an autosomal recessive trait. Studies in low-frequency hearing loss families showed that mutations in WFS1 were responsible for the phenotype. In all affected family members analysed, we detected a missense mutation in WFS1 (K705N) and therefore confirm the finding that the majority of mutations responsible for LFSNHI are missense mutations which localise to the C-terminal domain of the protein.     

Mutations of the Calcium Channel Gene cacophony Suppress Seizures in Drosophila

Bang sensitive (BS) Drosophila mutants display characteristic seizure-like phenotypes resembling, in some aspects, those of human seizure disorders such as epilepsy. The BS mutant para^bss1, caused by a gain-of-function mutation of the voltage-gated Na⁺ channel gene, is extremely seizure-sensitive with phenotypes that have proven difficult to ameliorate by anti-epileptic drug feeding or by seizure-suppressor mutation. It has been presented as a model for intractable human epilepsy. Here we show that cacophony (cac^TS2), a mutation of the Drosophila presynaptic Ca⁺⁺ channel α₁ subunit gene, is a particularly potent seizure-suppressor mutation, reverting seizure-like phenotypes for para^bss1 and other BS mutants. Seizure-like phenotypes for para^bss1 may be suppressed by as much as 90% in double mutant combinations with cac^TS2. Unexpectedly, we find that para^bss1 also reciprocally suppresses cac^TS2 seizure-like phenotypes. The cac^TS2 mutant displays these seizure-like behaviors and spontaneous high-frequency action potential firing transiently after exposure to high temperature. We find that this seizure-like behavior in cac^TS2 is ameliorated by 85% in double mutant combinations with para^bss1.     

Overexpression of a novel gene toothrin in Drosophila

A newly found locus of the Drosophila melanogaster genome, named toothrin (tth) has been used to study the role of the conserved domain 2/3 of genes from the d4 family. In contrast to the 2/3 domain of all vertebrates studied (including humans), which is always accompanied by the d4 domain, the tth gene contains the sequence encoding the 2/3 domain but lacks that encoding the d4 domain. The tth gene overexpression has been studied using the two-component system UAS-GAL4. It has been demonstrated that the tth overexpression at the third-instar larval (prepupal) stage decreases survival rate, simultaneously causing a substantial deceleration of development in Drosophila. It is known that the change of developmental stages in Drosophila is controlled by the rates of the expression of ecdysteroid and juvenile hormones (JHs). It is supposed that the overexpression of the tth gene causes either a shift in the ecdysterone-to-JH ratio (through a decreased JH decay rate or a delayed initiation of ecdysone synthesis) or a deceleration of the release of ecdysterones synthesized.     

Enhancer of terminal gene conversion,a new mutation in Drosophila melanogaster that induces telomere elongation by gene conversion

Telomeres of Drosophila melanogaster contain arrays of the retrotransposon-like elements HeT-A and TART. Terminally deleted chromosomes can be maintained for many generations. Thus, broken chromosome ends behave as real telomeres. It was previously shown that gene conversion may extend the broken ends. Here we found that the frequency of terminal DNA elongation by gene conversion strongly depends on the genotype. A dominant E(tc) (Enhancer of terminal gene conversion) mutation markedly increases the frequency of this event but does not significantly influence the frequency of HeT-A and TART attachment to the broken chromosome end and recombination between directly repeated sequences at the end of the truncated chromosome. The E(tc) mutation was mapped to the 91-93 region on chromosome 3. Drosophila lines that bear the E(tc) mutation for many generations have telomeres, consisting of HeT-A and TART elements, that are longer than those found in wild-type lines. Thus, the E(tc) mutation plays a significant role in the control of telomere elongation in D. melanogaster.     

Homeobrain, a novel paired-like homeobox gene is expressed in the Drosophila brain

The homeobrain (hbn) gene is a new paired-like homeobox gene which is expressed in the embryonic brain and the ventral nerve cord. Expression of homeobrain initiates during the blastoderm stage in the anterior dorsal head primordia and the gene is persistently expressed in these cells which form parts of the brain during later embryonic stages. An additional weaker expression pattern is detected in cells of the ventral nerve cord from stage 11 on. The homeodomain in the Homeobrain protein is most similar to the Drosophila proteins DRx, Aristaless and Munster. In addition, the localized brain expression patterns of homeobrain and DRx resemble each other. Two other homeobox genes, orthopedia and DRx are clustered in the 57B region along with homeobrain. The current evidence indicates that homeobrain, DRx and orthopedia form a homeobox gene cluster in which all the members are expressed in specific embryonic brain subregions.     

Interactions between fused, a segment-polarity gene in Drosophila, and other segmentation genes.

Fused (fu) is a segment polarity gene whose product is maternally required in the posterior part of each segment. To define further the role of fused and determine how it interacts with other segmentation genes, we examined the phenotypes obtained by combining fused with mutations of pair rule, homeotic and other segment polarity loci. When it was possible, we also looked at the distribution of corresponding proteins in fused mutant embryos. We observed that fused-naked (fu;nkd) double mutant embryos display a phenotypic suppression of simple mutant phenotypes: both naked cuticle and denticle belts, which would normally have been deleted by one of the two mutants alone, were restored. In fused mutant embryos, engrailed (en) and wingless (wg) expression was normal until germ band extension, but partially and completely disappeared respectively during germ band retraction. In the fu;nkd double mutant embryo, en was expressed as in nkd mutant at germ band extension, but later this expression was restricted and became normal at germ band retraction. On the contrary, wg expression disappeared as in fu simple mutant embryos. We conclude that the requirements for fused, naked and wingless activities for normal segmental patterning are not absolute, and propose mechanisms by which these genes interact to specify anterior and posterior cell fates.     

Courtship in Drosophila quadrilineata with a unique male behavioral element, abdomen bending

This paper describes courtship of Drosophila quadrilineata of the immigrans group and reports a species-specific element, abdomen bending, that has not been described in Drosophila. The male bends his abdomen right and left as a display to the female. During courtship the male circles around the female, bends his abdomen, and taps her. The male licks the female ovipositor repeatedly, often after the females extrusion. Although the females ovipositor extrusion is thought to indicate rejection of a courting male in many species, the male of D. quadrilineata continues courtship. It is possible that the extrusion is a females mating signal to the courting male. The male of D. quadrilineata mounts the female in a rearward position and the genitalia of both sexes couple after mounting. This mounting position is shared by the species belonging to the immigrans group. We never observed behavior in which it appeared as if the male displayed the black stripes to the female. Males court one another and a courtship chain is frequently formed even in the absence of females. The males abdomen bending and the females extrusion followed by the males licking are discussed.     

Grey, a novel mutation in the murine Lyst gene, causes the beige phenotype by skipping of exon 25

The murine beige mutant phenotype and the human Chediak-Higashi syndrome are caused by mutations in the murine Lyst (lysosomal trafficking regulator) gene and the human CHS gene, respectively. In this report we have analyzed a novel murine mutant Lyst allele, called Lyst(bg-grey), that had been found in an ENU mutation screen and named grey because of the grey coat color of affected mice. The phenotype caused by the Lyst(bg-grey) mutation was inherited in a recessive fashion. Melanosomes of melanocytes associated with hair follicles and the choroid layer of the eye, as well as melanosomes in the neural tube-derived pigment epithelium of the retina, were larger and irregularly shaped in homozygous mutants compared with those of wild-type controls. Secretory vesicles in dermal mast cells of the mutant skin were enlarged as well. Test crosses with beige homozygous mutant mice (Lyst(bg)) showed that double heterozygotes (Lyst(bg)/Lyst(bg-grey)) were phenotypically indistinguishable from either homozygous parent, demonstrating that the ENU mutation was an allele of the murine Lyst gene. RT-PCR analyses revealed the skipping of exon 25 in Lyst(bg-grey) mutants, which is predicted to cause a missense D2399E mutation and the loss of the following 77 amino acids encoded by exon 25 but leave the C-terminal end of the protein intact. Analysis of the genomic Lyst locus around exon 25 showed that the splice donor at the end of exon 25 showed a T-to-C transition point mutation. Western blot analysis suggests that the Lyst(bg-grey) mutation causes instability of the LYST protein. Because the phenotype of Lyst(bg) and Lyst(bg-grey) mutants is indistinguishable, at least with respect to melanosomes and secretory granules in mast cells, the Lyst(bg-grey) mutation defines a critical region for the stability of the murine LYST protein.     

Possible temperature-dependent blockage of synaptic vesicle recycling induced by a single gene mutation in Drosophila

The temperature-sensitive Drosophila mutant, shibirets1 (shi), has been shown to exhibit a reversible block in synaptic transmission at 30 degrees C. Various synaptic terminals (neuromuscular, sensory, central) of this mutant were observed by electron microscopy. At 19 degrees C, all terminals of shi showed essentially the same structural features as those of wild-type (Oregon-R) flies, while at 30 degrees C (5 or 10 min of exposure) shi terminals exhibited various structural changes not seen in the wild type. The major structural change observed in all of the various types of terminals was the accumulation of many pitlike structures on the plasma membrane near presynaptic sites. These structures consisted of a spherical head portion, about 50-100 nm in diameter, and a cylindrical neck portion, about 20 nm long and 20-25 nm in diameter. The neck portion was surrounded by a kind of cytoplasmic dense material, about 10 nm thick, reminiscent of a "collar." Thus, these pits are referred to as "collared pits." Similar kinds of pits were observed, although very rarely, in wild-type flies at 19 and 30 degrees C and in shi flies at 19 degrees C. In addition, various degrees of vesicle depletion, and an increase in membranous structures (infoldings and cisternalike or tubulelike structures) often accompanied pit formation. The possibility that these pits are the result of a blocked step in the endocytotic process, which in turn causes vesicle depletion as exocytosis proceeds, is discussed.