Courtship and other behaviors affected by a heat-sensitive,molecularly novel mutation in the cacophony calcium-channel gene of Drosophila

The cacophony (cac) locus of Drosophila melanogaster, which encodes a calcium-channel subunit, has been mutated to cause courtship-song defects or abnormal responses to visual stimuli. However, the most recently isolated cac mutant was identified as an enhancer of a comatose mutation's effects on general locomotion. We analyzed the cac(TS2) mutation in terms of its intragenic molecular change and its effects on behaviors more complex than the fly's elementary ability to move. The molecular etiology of this mutation is a nucleotide substitution that causes a proline-to-serine change in a region of the polypeptide near its EF hand. Given that this motif is involved in channel inactivation, it was intriguing that cac(TS2) males generate song pulses containing larger-than-normal numbers of cycles--provided that such males are exposed to an elevated temperature. Similar treatments caused only mild visual-response abnormalities and generic locomotor sluggishness. These results are discussed in the context of calcium-channel functions that subserve certain behaviors and of defects exhibited by the original cacophony mutant. Despite its different kind of amino-acid substitution, compared with that of cac(TS2), cac(S) males sing abnormally in a manner that mimics the new mutant's heat-sensitive song anomaly.     

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.     

straightjacket is required for the synaptic stabilization of cacophony, a voltage-gated calcium channel alpha1 subunit

In a screen to identify genes involved in synaptic function, we isolated mutations in Drosophila melanogaster straightjacket (stj), an alpha(2)delta subunit of the voltage-gated calcium channel. stj mutant photoreceptors develop normal synaptic connections but display reduced "on-off" transients in electroretinogram recordings, indicating a failure to evoke postsynaptic responses and, thus, a defect in neurotransmission. stj is expressed in neurons but excluded from glia. Mutants exhibit endogenous seizure-like activity, indicating altered neuronal excitability. However, at the synaptic level, stj larval neuromuscular junctions exhibit approximately fourfold reduction in synaptic release compared with controls stemming from a reduced release probability at these synapses. These defects likely stem from destabilization of Cacophony (Cac), the primary presynaptic alpha(1) subunit in D. melanogaster. Interestingly, neuronal overexpression of cac partially rescues the viability and physiological defects in stj mutants, indicating a role for the alpha(2)delta Ca(2+) channel subunit in mediating the proper localization of an alpha(1) subunit at synapses.     

The meiotic defects of mutants in the Drosophila mps1 gene reveal a critical role of Mps1 in the segregation of achiasmate homologs

The conserved kinase Mps1 is necessary for the proper functioning of the mitotic and meiotic spindle checkpoints (MSCs), which monitor the integrity of the spindle apparatus and prevent cells from progressing into anaphase until chromosomes are properly aligned on the metaphase plate. In Drosophila melanogaster, a null allele of the gene encoding Mps1 was recently shown to be required for the proper functioning of the MSC, but it did not appear to exhibit a defect in female meiosis. We demonstrate here that the meiotic mutant ald1 is a hypomorphic allele of the mps1 gene. Both ald1 and a P-insertion allele of mps1 exhibit defects in female meiotic chromosome segregation. The observed segregational defects are substantially more severe for pairs of achiasmate homologs, which are normally segregated by the achiasmate (or distributive) segregation system, than they are for chiasmate bivalents. Furthermore, cytological analysis of ald1 mutant oocytes reveals both a failure in the coorientation of achiasmate homologs at metaphase I and a defect in the maintenance of the chiasmate homolog associations that are normally observed at metaphase I. We conclude that Mps1 plays an important role in Drosophila female meiosis by regulating processes that are especially critical for ensuring the proper segregation of nonexchange chromosomes.     

Genetic and developmental characterization of Dmca1D,a calcium channel alpha1 subunit gene in Drosophila melanogaster.

To begin unraveling the functional significance of calcium channel diversity, we identified mutations in Dmca1D, a Drosophila calcium channel alpha1 subunit cDNA that we recently cloned. These mutations constitute the l(2)35Fa lethal locus, which we rename Dmca1D. A severe allele, Dmca1D(X10), truncates the channel after the IV-S4 transmembrane domain. These mutants die as late embryos because they lack vigorous hatching movements. In the weaker allele, Dmca1D(AR66), a cysteine in transmembrane domain I-S1 is changed to tyrosine. Dmca1D(AR66) embryos hatch but pharate adults have difficulty eclosing. Those that do eclose have difficulty in fluid-filling of the wings. These studies show that this member of the calcium channel alpha1 subunit gene family plays a nonredundant, vital role in larvae and adults.     

Postreplication repair defects in mutants of Drosophila melanogaster

Summary Mutants of Drosophila melanogaster which are defective in DNA synthesis have been identified among mutagen-sensitive stocks through analysis of both organ and cell cultures. A new procedure employing larval brain ganglia allows poorly fertile or sterile mutants to be analyzed for the first time. Parallel studies were performed in both tissues to establish the sensitivity of the new assay relative to that of the proven cell-culture assay. Damage was induced in the DNA of cultured cells with UV irradiation and in that of ganglial cells with the carcinogen N-acetoxy-2-acetylaminofluorene. Cultures were then pulse-labeled with ³H-thymidine, incubated in the absence of thymidine, and the newly synthesized DNA was analyzed by alkaline sucrose gradient centrifugation. The molecular weight of labeled DNA from mutant cells was compared with that from control cells to assess the effect of the mutant on DNA synthesis. Among 16 mutant stocks that were scanned in either or both tissues, seven show reductions in DNA synthesis using an undamaged template. Mutants at five different genetic loci [mus(2)205, mus(3)304, mus(3)308, mus(3)310 and mus(3)311] possess a reduced capacity to synthesize DNA on a UV-damaged template in primary cell cultures. Four of these five defects can also be detected in carcinogen-treated organ cultures. Two additional defects in postreplication repair were observed with the brainganglia assay in strains that cannot be assayed in cell culture [mus(1)108, mus(2)206].Abbreviations MMS methyl methanesulfonate - HN2 nitrogen mustard - AAF 2-acetylaminofluorene - AAAF N-acetoxy-2-acetylaminofluorene - DMSO dimethyl sulfoxide     

Identification of the site of interaction of the dihydropyridine channel blockers nitrendipine and azidopine with the calcium-channel alpha 1 subunit.

The dihydropyridine binding site of the rabbit skeletal muscle calcium channel alpha 1 subunit was identified using tritiated azidopine and nitrendipine as ligands. The purified receptor complex was incubated either with azidopine or nitrenidpine at an alpha 1 subunit to ligand ratio of 1:1. The samples were then irradiated by a 200 W UV lamp. The ligands were only incorporated into the alpha 1 subunit, which was isolated by size exclusion chromatography and digested either by trypsin (azidopine) or endoproteinase Asp-N (nitrendipine). Each digest contained two radioactive peptides, which were isolated and sequenced. The azidopine peptides were identical with amino acids 13-18 (minor peak) and 1428-1437 (major peak) of the primary sequence of the skeletal muscle alpha 1 subunit. The nitrendipine peptides were identical with amino acids 1390-1399 (major peak) and 1410-1420 (minor peak). The sequence from amino acids 1390 to 1437 is identical in the alpha 1 subunits of skeletal, cardiac and smooth muscle and follows directly repeat IVS6. These results indicate that dihydropyridines bind to an area that is located at the putative cytosolic domain of the calcium channel.     

Loss-of-function mutations in the Arabidopsis heterotrimeric G-protein alpha subunit enhance the developmental defects of brassinosteroid signaling and biosynthesis mutants

Loss-of-function alleles of the sole heterotrimeric G-protein alpha subunit in Arabidopsis, GPA1, display defects in cell proliferation throughout plant development. Previous studies indicated that GPA1 is involved in brassinosteroid (BR) response. Here we provide genetic evidence that loss-of-function mutations in GPA1, gpa1-2 and gpa1-4, enhance the developmental defects of bri1-5, a weak allele of a BR receptor mutant, and det2-1, a BR-deficient mutant in Arabidopsis. gpa1-2 bri1-5 and gpa1-4 det2-1 double mutants had shorter hypocotyls, shorter roots and fewer lateral roots, and displayed more severe dwarfism than bri1-5 and det2-1 single mutants, respectively. By using the Arabidopsis hypocotyl as a model system where the parameters of cell division and cell elongation can be simultaneously measured, we found that gpa1 can specifically enhance the cell division defects of bri1-5 and det2-1 mutants. Similarly, gpa1 specifically enhances cell division defects in the primary roots of bri1-5 and det2-1 mutants. Furthermore, an additive effect on cell division between gpa1 and bri1-5 or det2-1 mutations was observed in the hypocotyls, whereas a synergistic effect was observed in the roots. Taken together, these results provided the first genetic evidence that G-protein- and BR-mediated pathways may be converged to modulate cell proliferation in a cell/tissue-specific manner.     

Cell shape and interaction defects in alpha-spectrin mutants of Drosophila melanogaster

We show that the alpha-spectrin gene is essential for larval survival and development by characterizing several alpha-spectrin mutations in Drosophila. P-element minigene rescue and sequence analysis were used to identify the alpha-spectrin gene as the l(3)dre3 complementation group of the Dras-Roughened-ecdysoneless region of chromosome 3 (Sliter et al., 1988). Germ line transformants carrying an alpha-spectrin cDNA, whose expression is driven by the ubiquitin promoter, fully rescued the first to second instar lethality characteristic of the l(3)dre3 alleles. The molecular defects in two gamma-ray-induced alleles were identified. One of these mutations, which resulted in second instar lethality, contained a 73-bp deletion in alpha-spectrin segment 22 (starting at amino acid residue 2312), producing a premature stop codon between the two EF hands found in this segment. The second mutation, which resulted in first instar lethality, contained a 20 base pair deletion in the middle of segment 1 (at amino acid residue 92), resulting in a premature stop codon. Examination of the spectrin- deficient larvae revealed a loss of contact between epithelial cells of the gut and disruption of cell-substratum interactions. The most pronounced morphological change was seen in tissues of complex cellular architecture such as the middle midgut where a loss of cell contact between cup-shaped cuprophilic cells and neighboring interstitial cells was accompanied by disorganization of the cuprophilic cell brush borders. Our examination of spectrin deficient larvae suggests that an important role of non-erythroid spectrin is to stabilize cell to cell interactions that are critical for the maintenance of cell shape and subcellular organization within tissues.     

Neuronal expression of a newly identified Drosophila melanogaster G protein alpha 0 subunit.

Guanine nucleotide-binding proteins (G proteins) mediate signals between activated cell-surface receptors and cellular effectors. A bovine G-protein alpha-subunit cDNA has been used to isolate similar sequences from Drosophila genomic and cDNA libraries. One class, which we call DG alpha 0, hybridized to position 47A on the second chromosome of Drosophila. The nucleotide sequence of the protein coding region of one cDNA has been determined, revealing an alpha subunit that is 81% identical with rat alpha 0. The cDNA hybridizes strongly to a 3.8 kb mRNA and weakly with a 5.3 kb message. Antibodies raised against a trp-E-DG alpha 0 fusion protein recognized a 39,000 Da protein in Drosophila extracts. In situ hybridization to adult Drosophila sections combined with immunohistochemical studies revealed expression throughout the optic lobes and central brain and in the thoracic and abdominal ganglia. DG alpha 0 message and protein were also detected in the antennae, oocytes, and ovarian nurse cells. The neuronal expression of this gene is similar to mammalian alpha 0, which is most abundantly expressed in the brain.     

Characterization of Saccharomyces cerevisiae mutants lacking the E1 alpha subunit of the pyruvate dehydrogenase complex.

Pyruvate dehydrogenase mutants of Saccharomyces cerevisiae were isolated by disruption of the PDA1 gene. To this end, the PDA1 gene encoding the E1 alpha subunit of the pyruvate dehydrogenase complex was replaced by the dominant Tn5ble marker. Disruption of the PDA1 gene abolished production of the E1 alpha subunit and pyruvate dehydrogenase activity. Two additional phenotypes were observed in the Pdh-mutants: (a) a reduced growth rate in glucose medium which was partially complemented by the amino acid leucine; (b) an increase in formation of petites which lack mitochondrial DNA [rho0], during growth on glucose. Both phenotypes were shown to be a result of inactivation of the PDA1 gene. Explanations for these phenotypes are discussed.     

Selection and analysis of non-interactive mutants in the Escherichia coli tryptophan synthase alpha subunit.

Summary The inherent infidelity of Taq DNA polymerase in the polymerase chain reaction was exploited to produce random mutations in thetrp A gene. Screening of the resulting clones allowed selection of non-interactive mutant subunits retaining their intrinsic catalytic activity. Two single changes responsible for this phenotype were identified by DNA sequencing as: 126 valine (GTG)glutamic acid (GAG) and 128 valine (GTT)aspartic acid (GAT). Three single changes giving a non-interactive phenotype with an impaired intrinsic catalytic activity were identified by DNA sequencing as a66 asparagine (AAC)aspartic acid (GAC); 109lysine (AAA) arginine (AGA); 118 cysteine (TGC)arginine (CGC). Where possible, we individually assessed the importance of these residues in interaction in light of structural information from X-ray crystallography and by intergeneric protein sequence comparison.     

Localized defects of blastoderm formation in maternal effect mutants of Drosophila

In the three maternal effect lethal mutant strains of D. melanogaster described in this report, the homozygous mutant females produce defective eggs that cannot support normal embryonic development. The embryos from these eggs begin to develop for the first 2 hr after fertilization in an apparently normal way, forming a blastula containing a cluster of pole cells at the posterior end and a layer of syncytial blastoderm nuclei. During the subsequent transition from a syncytial to a cellular blastoderm, cell formation in the blastoderm is either partially or totally blocked. In mutant mat(3)1 no blastoderm cells are formed, indicating that there are separate genetic controls for pole cells and blastoderm cells. The other two mutants form an incomplete cellular blastoderm in which certain regions of the blastoderm remain noncellular. The noncellular region in mutant mat(3)3 is on the posterior-dorsal surface, covering about 30% of the total blastoderm. In mutant mat(3)6 blastoderm cells are formed only at the anterior and posterior ends, separated by a noncellular region that covers about 70% of the total blastoderm. The selective effects on blastoderm cell formation in the three mutants emphasize the importance of components present in the egg before fertilization for the transition from a syncytial to a cellular blastoderm.The genes defective in the three mutants are essential only for oogenesis and not for any other period of development, as indicated by a strict dependence of the lethal phenotypes on the maternal genotypes. Heterozygous embryos from the eggs of homozygous mutant females die, whereas homozygous mutant embryos from the eggs of heterozygous females develop into viable adults.One of the mutants, mat(3)3, has a temperature-sensitive phenotype. Homozygous mat(3)3 females maintained at a restrictive temperature of 29°C show the lethal maternal effect. However, at a permissive temperature of 20°C the females produce viable adult progeny. The temperature-sensitive period in mat(3)3 females occurs during the last 12 hr of oogenesis, consistent with the maternal effect phenotype of the mutant.     

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.