Processing of Drosophila endo-siRNAs depends on a specific Loquacious isoform

Drosophila melanogaster expresses three classes of small RNAs, which are classified according to their mechanisms of biogenesis. MicroRNAs are ∼22–23 nucleotides (nt), ubiquitously expressed small RNAs that are sequentially processed from hairpin-like precursors by Drosha/Pasha and Dcr-1/Loquacious complexes. MicroRNAs usually associate with AGO1 and regulate the expression of protein-coding genes. Piwi-interacting RNAs (piRNAs) of ∼24–28 nt associate with Piwi-family proteins and can arise from single-stranded precursors. piRNAs function in transposon silencing and are mainly restricted to gonadal tissues. Endo-siRNAs are found in both germline and somatic tissues. These ∼21-nt RNAs are produced by a distinct Dicer, Dcr-2, and do not depend on Drosha/Pasha complexes. They predominantly bind to AGO2 and target both mobile elements and protein-coding genes. Surprisingly, a subset of endo-siRNAs strongly depend for their production on the dsRNA-binding protein Loquacious (Loqs), thought generally to be a partner for Dcr-1 and a cofactor for miRNA biogenesis. Endo-siRNA production depends on a specific Loqs isoform, Loqs-PD, which is distinct from the one, Loqs-PB, required for the production of microRNAs. Paralleling their roles in the biogenesis of distinct small RNA classes, Loqs-PD and Loqs-PB bind to different Dicer proteins, with Dcr-1/Loqs-PB complexes and Dcr-2/Loqs-PD complexes driving microRNA and endo-siRNA biogenesis, respectively.     

Central complex substructures are required for the maintenance of locomotor activity in Drosophila melanogaster

In Drosophila melanogaster, former studies based on structural brain mutants have suggested that the central complex is a higher control center of locomotor behavior. Continuing this investigation we studied the effect of the central complex on the temporal structure of spontaneous locomotor activity in the time domain of a few hours. In an attempt to dissect the internal circuitry of the central complex we perturbed a putative local neuronal network connecting the four neuropil regions of the central complex, the protocerebral bridge, the fan-shape body, the noduli and the ellipsoid body. Two independent and non-invasive methods were applied: mutations affecting the neuroarchitecture of the protocerebral bridge, and the targeted expression of tetanus toxin in small subsets of central complex neurons using the binary enhancer trap P[GAL4] system. All groups of flies with a disturbed component of this network exhibited a common phenotype: a drastic decrease in locomotor activity. While locomotor activity was still clustered in bouts and these were initiated at the normal rate, their duration was reduced. This finding suggests that the bridge and some of its neural connections to the other neuropil regions of the central complex are required for the maintenance but not the initiation of walking. Accepted: 21 June 1999     

Circadian regulation of egg-laying behavior in fruit flies Drosophila melanogaster

Significant progress has been made in our understanding of the neurogenetics of circadian clocks in fruit flies Drosophila melanogaster. Several pacemaker neurons and clock genes have now been identified and their roles in the cellular and molecular clockwork established. Some recent findings suggest that the basic architecture of the clock is multi-oscillatory; the clock mechanisms in the ventral lateral neurons (LN(v)s) of the fly brain govern locomotor activity and adult emergence rhythms, while the peripheral oscillators located in antennal cells regulate olfactory rhythm. Among circadian phenomena exhibited by Drosophila, the egg-laying rhythm is unique in many ways: (i) this rhythm persists under constant light (LL), while locomotor activity and adult emergence become arrhythmic, (ii) its circadian periodicity is much longer than 24h, and (iii) while egg-laying is rhythmic under constant darkness, the expression of two core clock genes period (per) and timeless (tim), is non-oscillatory in the ovaries. In this paper, we review our current knowledge of the circadian regulation of egg-laying behavior in Drosophila, and provide some possible explanations for its self-sustained nature. We conclude by discussing the existing limitations in our understanding of the regulatory mechanisms and propose few approaches to address them.     

The effect of neurospecific knockdown of candidate genes for locomotor behavior and sound production in Drosophila melanogaster

Molecular mechanisms underlying the functioning of central pattern generators (CPGs) are poorly understood. Investigations using genetic approaches in the model organism Drosophila may help to identify unknown molecular players participating in the formation or control of motor patterns. Here we report Drosophila genes as candidates for involvement in the neural mechanisms responsible for motor functions, such as locomotion and courtship song. Twenty-two Drosophila lines, used for gene identification, were isolated from a previously created collection of 1064 lines, each carrying a P element insertion in one of the autosomes. The lines displayed extreme deviations in locomotor and/or courtship song parameters compared with the whole collection. The behavioral consequences of CNS-specific RNAi-mediated knockdowns for 10 identified genes were estimated. The most prominent changes in the courtship song interpulse interval (IPI) were seen in flies with Sps2 or CG15630 knockdown. Glia-specific knockdown of these genes produced no effect on the IPI. Estrogen-induced knockdown of CG15630 in adults reduced the IPI. The product of the CNS-specific gene, CG15630 (a predicted cell surface receptor), is likely to be directly involved in the functioning of the CPG generating the pulse song pattern. Future studies should ascertain its functional role in the neurons that constitute the song CPG. Other genes (Sps2, CG34460), whose CNS-specific knockdown resulted in IPI reduction, are also worthy of detailed examination.     

Genetic study on emigration behavior of Drosophila melanogaster in a natural population

Genetic study on emigration behavior of Drosophila melanogaster in the Ishinomaki population was conducted with 140 2nd chromosome lines. Fourteen sets of 5X5 partial diallel cross experiments were made to examine the emigration activity of F1 progeny. Emigration activity was scored using the method of Sakai et al. (1958). Additive genetic variance was 0.0377 +/- 0.0069 and dominance variance 0.0076 +/- 0.0032. The average degree of dominance of mildly deleterious genes for emigration activity in an equilibrium population was 0.069 +/- 0.042. The estimated degree of dominance at a gene locus affecting emigration activity was 0.067, which revealed nearly complete dominance for the tendency of heterozygote flies to move from their original place to another. Average degree of dominance of lethal gene for emigration activity was 0.012.     

Comprehensively surveying structure and function of RING domains from Drosophila melanogaster

Using a complete set of RING domains from Drosophila melanogaster, all the solved RING domains and cocrystal structures of RING-containing ubiquitin-ligases (RING-E3) and ubiquitin-conjugating enzyme (E2) pairs, we analyzed RING domains structures from their primary to quarternary structures. The results showed that: i) putative orthologs of RING domains between Drosophila melanogaster and the human largely occur (118/139, 84.9%); ii) of the 118 orthologous pairs from Drosophila melanogaster and the human, 117 pairs (117/118, 99.2%) were found to retain entirely uniform domain architectures, only Iap2/Diap2 experienced evolutionary expansion of domain architecture; iii) 4 evolutionary structurally conserved regions (SCRs) are responsible for homologous folding of RING domains at the superfamily level; iv) besides the conserved Cys/His chelating zinc ions, 6 equivalent residues (4 hydrophobic and 2 polar residues) in the SCRs possess good-consensus and conservation- these 4 SCRs function in the structural positioning of 6 equivalent residues as determinants for RING-E3 catalysis; v) members of these RING proteins located nucleus, multiple subcellular compartments, membrane protein and mitochondrion are respectively 42 (42/139, 30.2%), 71 (71/139, 51.1%), 22 (22/139, 15.8%) and 4 (4/139, 2.9%); vi) CG15104 (Topors) and CG1134 (Mul1) in C3HC4, and CG3929 (Deltex) in C3H2C3 seem to display broader E2s binding profiles than other RING-E3s; vii) analyzing intermolecular interfaces of E2/RING-E3 complexes indicate that residues directly interacting with E2s are all from the SCRs in RING domains. Of the 6 residues, 2 hydrophobic ones contribute to constructing the conserved hydrophobic core, while the 2 hydrophobic and 2 polar residues directly participate in E2/RING-E3 interactions. Based on sequence and structural data, SCRs, conserved equivalent residues and features of intermolecular interfaces were extracted, highlighting the presence of a nucleus for RING domain fold and formation of catalytic core in which related residues and regions exhibit preferential evolutionary conservation.     

An investigation of heterochromatin domains on the fourth chromosome of Drosophila melanogaster

The banded portion of Drosophila melanogaster chromosome 4 exhibits euchromatic and heterochromatic characteristics. Reminiscent of heterochromatin, it contains a high percentage of repetitive elements, does not undergo recombination, and exhibits high levels of HP1 and histone-3 lysine-9 dimethylation. However, in the distal 1.2 Mb, the gene density is typical of euchromatin, and this region is polytene in salivary gland nuclei. Using P-element reporters carrying a copy of hsp70-white, alternative chromatin packaging domains can be distinguished by the eye color phenotype. Mapping studies identified the repetitive element 1360 as a candidate for heterochromatin targeting in the fourth chromosome Hcf region. We report here two new screens using this reporter to look for additional heterochromatin target sites. We confirm that reporter elements within 10 kb of 1360 are usually packaged as heterochromatin; however, heterochromatin packaging occurs in the sv region in the absence of 1360. Analyses of the sequences adjacent to P-element reporters show no simple association between specific repeated elements and transgene expression phenotype on a whole chromosome level. The data require that heterochromatin formation as a whole depends on a more complex pattern of sequence organization rather than the presence of a single sequence element.     

Effects of female behavior on wing display and courtship of male Drosophila simulans and Drosophila melanogaster

Corrolations between female rejection behaviors and male wing display were calculated for both Drosophila simulans and Drosophila melanogaster intraspicific pair-matings. No significant correlations were found for D. melanogaster, but in D. simulans flicking by the female appeared to be associated with a shift in male wing display pattern resulting in higher levels of vibration. Flicking did not appear to discourage courtship by males in either species.     

Identification and characterization of the nuclear isoform of Drosophila melanogaster CTP:phosphocholine cytidylyltransferase

CTP:phosphocholine cytidylyltransferase (CCT) catalyzes the conversion of phosphocholine and cytidine 5'-triphosphate (CTP) to CDP-choline for the eventual synthesis of phosphatidylcholine (PC). The enzyme is regulated by reversible association with cellular membranes, with the rate of catalysis increasing following membrane association. Two isoforms of CCT appear to be present in higher eukaryotes, including Drosophila melanogaster, which contains the tandem genes Cct1 and Cct2. Before this study, the CCT1 isoform had not been characterized and the cellular location of each enzyme was unknown. In this investigation, the cDNA encoding the CCT1 isoform from D. melanogaster has been cloned and the recombinant enzyme purified and characterized to determine catalytic properties and the effect of lipid vesicles on activity. CCT1 exhibited a V max of 23904 nmol of CDP-choline min (-1) mg (-1) and apparent K m values for phosphocholine and CTP of 2.29 and 1.21 mM, respectively, in the presence of 20 muM PC/oleate vesicles. Cytidylyltransferases require a divalent cation for catalysis, and the cation preference of CCT1 was found to be as follows: Mg (2+) > Mn (2+) = Co (2+) > Ca (2+) = Ni (2+) > Zn (2+). The activity of the enzyme is stimulated by a variety of lipids, including phosphatidylcholine, phosphatidylinositol, phosphatidylglycerol, phosphatidylserine, diphosphatidylglycerol, and the fatty acid oleate. Phosphatidylethanolamine and phosphatidic acid, however, did not have a significant effect on CCT1 activity. The cellular location of both CCT1 and CCT2 isoforms was elucidated by expressing green fluorescent fusion proteins in cultured D. melanogaster Schneider 2 cells. CCT1 was identified as the nuclear isoform, while CCT2 is cytoplasmic.     

Isolation of a cloned DNA segment containing a ribosomal protein gene of Drosophila melanogaster

A Drosophila genomic DNA library in the vector Charon 4 was screened using cDNA derived from the small (6S-12S) poly(A)+ mRNA of 2-6-h-old Drosophila embryos. This fraction of mRNA is enriched for ribosomal protein-coding sequences. The selected recombinants were hybridized to total mRNA under conditions which allowed for isolation of homologous mRNAs. The mRNA from these RNA/DNA hybrids was eluted and translated in vitro. The translation products were analyzed by one- and two-dimensional electrophoresis with authentic ribosomal proteins as standards. One cloned DNA segment was found to contain a ribosomal protein gene, and a sequence which hybridizes strongly to at least 5 other ribosomal protein mRNAs.     

Thermosensory and nonthermosensory isoforms of Drosophila melanogaster TRPA1 reveal heat-sensor domains of a thermoTRP Channel

Specialized somatosensory neurons detect temperatures ranging from pleasantly cool or warm to burning hot and painful (nociceptive). The precise temperature ranges sensed by thermally sensitive neurons is determined by tissue-specific expression of ion channels of the transient receptor potential(TRP) family.We show here that in Drosophila, TRPA1 is required for the sensing of nociceptive heat. We identify two previously unidentified protein isoforms of dTRPA1, named dTRPA1-C and dTRPA1-D, that explain this requirement. A dTRPA1-C/D reporter was exclusively expressed in nociceptors, and dTRPA1-C rescued thermal nociception phenotypes when restored to mutant nociceptors. However,surprisingly, we find that dTRPA1-C is not a direct heat sensor. Alternative splicing generates at least four isoforms of dTRPA1. Our analysis of these isoforms reveals a 37-amino-acid-long intracellular region (encoded by a single exon) that is critical for dTRPA1 temperature responses. The identification of these amino acids opens the door to a biophysical understanding of a molecular thermosensor.     

Observations on the regulation of uricase activity during development of Drosophila melanogaster

To elucidate the mechanisms involved in the regulation of uricase activity in Drosophila melanogaster, a comparative analysis of the patterns of uricase activity during development was undertaken for the wild type, Ore-R, and the mutants ry ² and ma-1. Uricase activity in ry ² and ma-l, unlike that in Ore-R, increased rapidly following emergence of the adult. This study indicates that uricase in Drosophila, in contrast to that in several microorganisms, is not induced by uric acid, since ry ² and ma-l with no detectable uric acid have higher activity than the wild type.This study was supported by grants from NIH, PHS 5-TO 1 GM 0071-13 and 1F02 HD 50,527-01     

Functional characterization of a second porin isoform in Drosophila melanogaster. DmPorin2 forms voltage-independent cation-selective pores

Mitochondrial porins or voltage-dependent anion-selective channels are channel-forming proteins mainly found in the mitochondrial outer membrane. Genome sequencing of the fruit fly Drosophila melanogaster revealed the presence of three additional porin-like genes. No functional information was available for the different gene products. In this work we have studied the function of the gene product closest to the known Porin gene (CG17137 coding for DmPorin2). Its coding sequence was expressed in Escherichia coli. The recombinant DmPorin2 protein is able to form channels similar to those formed by DmPorin1 reconstituted in artificial membranes. Furthermore, DmPorin2 is clearly voltage-independent and cation-selective, whereas its counterpart isoform 1 is voltage-dependent and anion-selective. Sequence comparison of the two porin isoforms indicates the exchange of four lysines in DmPorin1 for four glutamic acids in DmPorin2. We have mutated two of them (Glu-66 and Glu-163) to lysines to investigate their role in the functional features of the pore. The mutants E163K and E66K/E163K are endowed with an almost full inversion of the ion selectivity. Both single mutations partially restore the voltage dependence of the pore. We found that an additional effect with the double mutant E66K/E163K was the restoration of voltage dependence. Protein structure predictions highlight a 16 beta-strand pattern, typical for porins. In a three-dimensional model of DmPorin2, Glu-66 and Glu-163 are close to the rim of the channel, on two opposite sides. DmPorin2 is expressed in all the fly tissues and in all the developmental stages tested. Our main conclusions are as follows. 1) The CG17137 gene may express a porin with a functional role in D. melanogaster. 2) We have identified two amino acids of major relevance for the voltage dependence of the porin pore.     

Turning males on: activation of male courtship behavior in Drosophila melanogaster

The innate sexual behaviors of Drosophila melanogaster males are an attractive system for elucidating how complex behavior patterns are generated. The potential for male sexual behavior in D. melanogaster is specified by the fruitless (fru) and doublesex (dsx) sex regulatory genes. We used the temperature-sensitive activator dTRPA1 to probe the roles of fru(M)- and dsx-expressing neurons in male courtship behaviors. Almost all steps of courtship, from courtship song to ejaculation, can be induced at very high levels through activation of either all fru(M) or all dsx neurons in solitary males. Detailed characterizations reveal different roles for fru(M) and dsx in male courtship. Surprisingly, the system for mate discrimination still works well when all dsx neurons are activated, but is impaired when all fru(M) neurons are activated. Most strikingly, we provide evidence for a fru(M)-independent courtship pathway that is primarily vision dependent.