Drosophila melanogaster flatline encodes a myotropin orthologue to Manduca sexta allatostatin

We identified a Drosophila melanogaster gene encoding a peptide that dramatically decreases spontaneous muscle contractions and, correspondingly, named the peptide flatline (FLT). This gene consisted of 4 exons and was cytologically localized to 32D2-3. Processing of a predicted 122 amino acid precursor would release pEVRYRQCYFNPISCF that differs from Manduca sexta allatostatin (Mas-AST) by one amino acid, Y4-->F4. FLT does not act as an allatostatin. In situ tissue hybridization further suggests FLT is a novel brain-gut peptide and specifically, the measured activity indicates that it is a potent myotropin. Despite its profound myotropic effect, pupae injected with FLT eclosed.     

Evolution of color vision in pierid butterflies: blue opsin duplication,ommatidial heterogeneity and eye regionalization in <Emphasis Type="Italic">Colias erate</Emphasis>

This paper documents the molecular organization of the eye of the Eastern Pale Clouded Yellow butterfly, Colias erate (Pieridae). We cloned four cDNAs encoding visual pigment opsins, corresponding to one ultraviolet, two blue and one long wavelength-absorbing visual pigments. Duplication of the blue visual pigment class occurs also in another pierid species, Pieris rapae, suggesting that blue duplication is a general feature in the family Pieridae. We localized the opsin mRNAs in the Colias retina by in situ hybridization. Among the nine photoreceptor cells in an ommatidium, R1-9, we found that R3-8 expressed the long wavelength class mRNA in all ommatidia. R1 and R2 expressed mRNAs of the short wavelength opsins in three fixed combinations, corresponding to three types of ommatidia. While the duplicated blue opsins in Pieris are separately expressed in two subsets of R1-2 photoreceptors, one blue sensitive and another violet sensitive, those of Colias appear to be always coexpressed.     

The optic lobe of Drosophila melanogaster

Summary We present a quantitative evaluation of Golgiimpregnated columnar neurons in the optic lobe of wildtype Drosophila melanogaster. This analysis reveals the overall connectivity pattern between the 10 neuropil layers of the medulla and demonstrates the existence of at least three major visual pathways. Pathway 1 connects medulla layer M10 to the lobula plate. Input layers of this pathway are M1 and M5. Pathway 2 connects M9 to shallow layers of the lobula, which in turn are tightly linked to the lobula plate. This pathway gets major input via M2. Pathways 1 and 2 receive input from retinula cells R1-6, either via the lamina monopolar cell L1 (terminating in M1 and M5) or via L2 and T1 (terminating in M2). Neurons of these pathways typically have small dendritic fields. We discuss evidence that pathways 1 and 2 may play a major role in motion detection. Pathway 3 connects M8 to deep layers of the lobula. In M8 information converges that is derived either from M3 (pathway 3a) or from M4 and M6 (pathway 3b), layers that get their major input from L3 and R8 or L4 and R7, respectively. Some neurons of pathway 3 have large dendritic fields. We suggest that they may be involved in the computation of form and colour. Possible analogies to the organization of pathways in the visual system of vertebrates are discussed.During the final editing of this work our friend A.P.M. Dittrich was tragically killed in an accident. Without him this and the previous work would never have been completed     

Characterization of a novel Drosophila melanogaster acylphosphatase

Analysis of the Drosophila melanogaster EST database led to the characterization of a novel acylphosphatase (AcPDro2). This is coded by the CG18505 (Acyp2) gene and is clearly distinct from a previously described AcPDro coded by the CG16870 (Acyp) gene from D. melanogaster. The two proteins show a 60% homology with both vertebrate isoenzymes. All the residues involved in the catalytic mechanism are conserved. AcPDro2 is a stable enzyme with a correct globular folded structure. Its activity on benzoylphosphate shows higher K(cat) but lower K(m) with respect to AcPDro. It is possible that AcPDro and AcPDro2 genes are not the direct ancestor of MT and CT vertebrate isoenzymes.     

Characterization of Drosophila melanogaster rhodopsin

A polypeptide present in Drosophila eye homogenates was identified as opsin. This polypeptide pI 7.8, with Mr 39,000 is a retina-specific protein. It has the spectral characteristics of rhodopsin contained in the R1-6 photoreceptors and decreases in amount with vitamin A deprivation. It contains a chromophore derived from vitamin A and linked to the protein moiety by a Schiff base. Moreover, the polypeptide identified corresponds to a retina-specific polypeptide that was shown previously to undergo light-dependent phosphorylation in living flies. These results indicate that many properties of Drosophila rhodopsin do not differ significantly from those reported for rhodopsins of other organisms. However, the isoelectric point of Drosophila opsin is considerably more basic than those reported for vertebrate rhodopsins.     

Characterization of acetylcholinesterase activity from Drosophila melanogaster

The acetylcholinesterase activity of the fruit fly, Drosophila melanogaster, was characterized biochemically. The activity is associated with a glycoprotein which is divided between a detergent-extractable membrane-bound fraction and a soluble fraction. The acetylcholinesterase activity is concentrated in the head of the insect. Through pharmacological methods, greater than 95% of the cholinesterase is judged to be true acetylcholinesterase, and not pseudocholinesterase. As expected for an acetylcholinesterase, the enzyme has a high affinity for acetylthiocholine and is inhibited by excess concentrations of acetylthiocholine. The soluble enzyme is found predominantly as a 7.8 S form; a smaller amount of an approximately 6 S form is also present, and a greater than or equal to 14 S form may exist. The detergent-solubilized acetylcholinesterase has a sedimentation coefficient of 7.5 S in the presence of detergent. The thermal inactivation rates for the soluble and the membrane bound enzymes are markedly different.     

Characterization of the Drosophila melanogaster ribosomal proteome

We have combined high-resolution two-dimensional (2-D) gel electrophoresis with mass spectrometry to identifying proteins represented in a 2-D gel database of Drosophila melanogaster ribosomes. First, we purified ribosomes from third instar Drosophila larvae and constructed a high-resolution 2-D gel database containing 58 Coomassie blue stained polypeptides. Next, we carried out preparative 2-D PAGE to isolate some of the polypeptides and characterize them by MALDI-TOF. Using this strategy we identified 52 ribosomal spots in the database, and in each case confirmed their identity by MALDI-TOF/TOF. The database can be used to analyze Minute mutants of Drosophila.     

Characterization of Lethal Drosophila melanogaster alpha-Actinin Mutants

We have partially characterized four Drosophilamelanogaster alpha-actinin gene mutants,I(1)2Cb¹, I(1)2Cb²,I(1)2Cb⁴, and I(1)2Cb⁵. Wedemonstrate that in each case the mutation is caused bya chromosomal rearrangement that precludes normal proteinsynthesis. In the absence of alpha-actinin, fliescomplete embryogenesis and develop into flaccid larvaethat die within approximately 24 hr. These larvae have noticeable muscle dysfunction at hatching,although they, nevertheless, are capable of escapingfrom the egg membranes and of subsequent crawlingmovements. During larval development muscles degenerate, progressively limiting mobility and ultimatelycausing death. Electron microscopy of mutant musclefibers reveals that myofibrils are grossly disrupted inone day old larvae and that electron-dense structures reminiscent of those seen in human nemalinemyopathies are present throughout larval life. Our workrigorously demonstrates that alpha-actinin deficienciesare the cause of I(1)2Cb muscle defects. We anticipate that the alpha-actinin mutants described hereinwill facilitate in vivo tests of spectrin superfamilyprotein domain functions using a combination of directedmutagenesis and germline transformation.     

Characterization of the Drosophila melanogaster mitochondrial proteome

We have combined high-resolution two-dimensional (2-D) gel electrophoresis with mass spectrometry with the aim of identifying proteins represented in the 2-D gel database of Drosophila melanogaster mitochondria. First, we purified mitochondria from third instar Drosophila larvae and constructed a high-resolution 2-D gel database containing 231 silver-stained polypeptides. Next, we carried out preparative 2-D PAGE to isolate some of the polypeptides and characterize them by MALDI-TOF analysis. Using this strategy, we identified 66 mitochondrial spots in the database, and in each case confirmed their identity by MALDI-TOF/TOF analysis. In addition, we generated antibodies against two of the mitochondrial proteins as tools for characterizing the organelle.     

Isolation and Characterization of a Photorepair-Deficient Mutant in Drosophila melanogaster

A mutation abolishing photorepair has been localized to map position 56.8 centimorgans on the second chromosome of Drosophila melanogaster. Strains homozygous for the phr allele are totally devoid of photorepair and partially deficient in excision repair. Both defects map to the chromosomal region between pr and c. Since a homozygous phr stock exhibits reduced photoreactivation, the corresponding wild-type allele plays a significant role in UV resistance.     

Characterization of a functional neuropeptide F receptor from Drosophila melanogaster

Potential receptors for Drosophila neuropeptide F (DmNPF) were identified in the genome database. One receptor (DmNPFR1) sequence resembled the Lymnaea NPY receptor, an invertebrate homolog of the vertebrate Y-receptor family. DmNPFR1 was cloned and tested for functionality in stably transfected mammalian CHO cells. In whole cell binding assays, DmNPF displaced 125I-NPF in a concentration-dependent manner (IC(50) = 65 nM). DmNPF inhibited forskolin-stimulated adenylyl cyclase activity similarly (IC(50) = 51 nM). Whole-mount in situ hybridization revealed that DmNPFR1 RNA is expressed in CNS and midgut of Drosophila larvae. DmNPFR1, a new invertebrate Y-receptor homolog, apparently is a functional receptor for DmNPF.     

Visual control of straight flight in Drosophila melanogaster

Summary The optomotor system of Drosophila is investigated in a flight simulator in which the fly's yaw torque controls the angular velocity of the panorama (striped drum, negative feedback). Flies in the flight simulator maintain a stable orientation even in a homogeneously textured panorama without landmarks. During straight flight, torque is not zero. It consists of small pulses mostly alternating in polarity. The course is controlled by the duration (and possibly amplitude) of the pulses. The system operates under reafference control. By comparing the pulses with the visual input the system continuously measures and adjusts the efficacy of the torque output. The comparison, however, is not between angular velocity and yaw torque but, instead, between visual acceleration and pretorque, the first time derivative of torque. For comparison, the system first computes a cross-correlation. If the correlation coefficient is above a certain threshold the system calculates the external gain and adjusts its internal gain so as to keep the total gain constant. With the correlation coefficient below threshold, however, the system keeps the internal gain low despite the infinitely small external gain. We propose that for a reafferent optomotor system the coupling coefficient and the correlation coefficient of pretorque and visual acceleration are more relevant than the distinction between exafference and reafference.Abbreviation EMD elementary movement detector     

Characterization of the mitochondrial porin from Drosophila melanogaster

Mitochondrial porin was isolated from the fruit fly Drosophila melanogaster at different developmental stages, starting from whole mitochondria. The porin from adults' mitochondria was fully characterized. The protein had a molecular mass of 31 kDa as judged from sodium dodecylsulfate electrophoretograms. It was very resistive against digestion with V8 proteinase of Staphylococcus aureus and a larger number of fragments were only obtained after digestion with papain. Drosophila porin showed little interaction with antibodies raised against mitochondrial porins from mammalia and Neurospora crassa, but a strong reactivity with antibodies raised against yeast porin. Reconstitution experiments with planar lipid bilayer membranes showed that the protein was able to form ion-permeable pores with a single-channel conductance of 0.41 nS in 0.1 M KCl. At low transmembrane voltages Drosophila porin had the properties of a general diffusion pore with an estimated effective diameter of about 1.7 nm and a small selectivity for anions over cations. Voltages larger than 20 to 30 mV resulted in a closure of the pore. The closed states of the pore were found to be cation-selective. The addition of a synthetic polyanion to the aqueous phase on one side of the membrane resulted in an asymmetric shift of the voltage dependence and the pore became already closed at very small voltages negative at the cis-side (the side of the addition of the polyanion).     

Proliferation pattern and early differentiation of the optic lobes in Drosophila melanogaster

Summary The larval and early pupal development of the optic lobes in Drosophila is described qualitatively and quantitatively using [³H]thymidine autoradiography on 2-m plastic sections. The optic lobes develop from 30–40 precursor cells present in each hemisphere of the freshly hatched larva. During the first and second larval instars, these cells develop to neuroblasts arranged in two epithelial optic anlagen. In the third larval instar and in the early pupa these neuroblasts generate the cells of the imaginal optic lobes at discrete proliferation zones, which can be correlated with individual visual neuropils.The different neuropils as well as the repetitive elements of each neuropil are generated in a defined temporal sequence. Cells of the medulla are the first to become postmitotic with the onset of the third larval instar, followed by cells of the lobula complex and finally of the lamina at about the middle of the third instar. The elements of each neuropil connected to the most posterior part of the retina are generated first, elements corresponding to the most anterior retina are generated last.The proliferation pattern of neuroblasts into ganglion mother cells and ganglion cells is likely to include equal as well as unequal divisions of neuroblasts, followed by one or two generations of ganglion mother cells. For the lamina the proliferation pattern and its temporal coordination with the differentiation of the retina are shown.     

Utilization of a Photosystem by Drosophila melanogaster Larvae (Diptera: Drosophilidae)

Foraging-stage third-instar larvae from most wild-type (normal) Drosophila melanogaster stocks are generally repelled by light. To identify factors that affect the larval photoresponse, we elucidated the effects of age, temperature, and time on the photoresponse of larvae from a wild-type Canton-S stock. In addition, we analyzed the larvae from the LI2 isofemale line, which are unresponsive to light in a photoassay. To determine whether LI2 larvae behave abnormally on other behavioral paradigms, in comparison to Canton-S controls, we tested larvae in taste and olfactory assays and observed them to determine whether they dispersed in a food source. Like Canton-S larvae, LI2 larvae and other isofemale lines whose progenitors were collected from the same natural population are responsive to taste and olfactory stimuli. Moreover, LI2 larvae disperse in the food source, as do Canton-S larvae tested in the dark. Larvae expressing para^sbl mutations, which respond normally to light but not to chemical stimuli, do not disperse normally in the food source, suggesting that dispersal may be mediated by perception of chemical cues.     

Characterization of the female-sterile mutant Almondex of Drosophila melanogaster

Drosophila melanogaster females homozygous for the sex-linked recessive mutation almondex (amx) are completely sterile when mated to amx males. Matings of amx X+ yield low numbers of heterozygous female offspring, which frequently show abnormalities of the thorax and abdominal sternites, and an occasional non-disjunctional, non-mutant (XO) male offspring. The results of mating experiments reported here can be explained by assuming that the cytoplasm of eggs produced by amx females is deficient for some material that is necessary for normal development.Homozygous amx females have apparently normal reporductive organs and a high egg yield. Eggs are usually fertilized. In matings to amx males, all zygotes die as embryos; in matings to non-amx males, all ordinary (XY) male zygotes and most female zygotes die as embryos. Survival to the adult stage is more frequent at higher temperatures and, surprisingly, increases also with maternal age.