Regulation of Drosophila FMRFamide neuropeptide gene expression.

Physiologically important peptides are often encoded in precursors that contain several gene products; thus, regulation of expression of polypeptide proteins is crucial to transduction pathways. Differential processing of precursors by cell- or tissue-specific proteolytic enzymes can yield messengers with diverse distributions and dissimilar activities. FMRFamide-related peptides (FaRPs) are present throughout the animal kingdom and affect both neural and gastrointestinal functions. Organisms have several genes encoding numerous FaRPs with a common C-terminal structure but different N-terminal amino acid extensions. We have isolated SDNFMRFamide, DPKQDFMRFamide, and TPAEDFMRFamide contained in the Drosophila FMRFamide gene. To investigate the regulation of expression of FMRFamide peptides, we generated antisera to distinguish among the three neuropeptides. We have previously reported the distribution of SDNFMRFamide and DPKQDFMRFamide. In this article, we describe TPAEDFMRFamide expression. TPAEDFMRFamide antisera stain cells in embryonic, larval, pupal, and adult thoracic and abdominal ganglia. In addition, TPAEDFMRFamide-immunoreactive material is present in a lateral protocerebrum cell in adult. Thus, TPAEDFMRFamide antisera staining of neural tissue is different from SDNFMRFamide or DPKQDFMRFamide. In addition, TPAEDFMRFamide antisera stain larval, pupal, and adult gut, while SDNFMRFamide and DPKQDFMRFamide do not. TPAEDFMRFamide immunoreactivity is present in cells stained by FMRFamide antisera. Taken together, these data support the conclusion that TPAEDFMRFamide is differentially processed from the FMRFamide polypeptide protein precursor and may act in both neural and gastrointestinal tissue.     

Regulation of Drosophila FMRFamide neuropeptide gene expression

Physiologically important peptides are often encoded in precursors that contain several gene products; thus, regulation of expression of polypeptide proteins is crucial to transduction pathways. Differential processing of precursors by cell‐ or tissue‐specific proteolytic enzymes can yield messengers with diverse distributions and dissimilar activities. FMRFamide‐related peptides (FaRPs) are present throughout the animal kingdom and affect both neural and gastrointestinal functions. Organisms have several genes encoding numerous FaRPs with a common C‐terminal structure but different N‐terminal amino acid extensions. We have isolated SDNFMRFamide, DPKQDFMRFamide, and TPAEDFMRFamide contained in the Drosophila FMRFamide gene. To investigate the regulation of expression of FMRFamide peptides, we generated antisera to distinguish among the three neuropeptides. We have previously reported the distribution of SDNFMRFamide and DPKQDFMRFamide. In this article, we describe TPAEDFMRFamide expression. TPAEDFMRFamide antisera stain cells in embryonic, larval, pupal, and adult thoracic and abdominal ganglia. In addition, TPAEDFMRFamide‐immunoreactive material is present in a lateral protocerebrum cell in adult. Thus, TPAEDFMRFamide antisera staining of neural tissue is different from SDNFMRFamide or DPKQDFMRFamide. In addition, TPAEDFMRFamide antisera stain larval, pupal, and adult gut, while SDNFMRFamide and DPKQDFMRFamide do not. TPAEDFMRFamide immunoreactivity is present in cells stained by FMRFamide antisera. Taken together, these data support the conclusion that TPAEDFMRFamide is differentially processed from the FMRFamide polypeptide protein precursor and may act in both neural and gastrointestinal tissue. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 347–358, 1999     

Circular DNA Molecules in the Genus Drosophila

The satellite DNA's from the embryos of five species of Drosophila (D. melanogaster, D. simulans, D. nasuta, D. virilis and D. hydei) have been analyzed for the presence of closed circular duplex DNA molecules, as determined by CsCl-EBr gradients. Circular DNA molecules were found in every species but D. melanogaster. Analyses of cell fractions from adult Drosophila and organ fractions from Drosophila larvae show that fractions containing mitochondria are highly enriched in these molecules.     

Drosophila virilis has long and highly polymorphic microsatellites

Comparative genomics is a powerful approach to inference of the dynamics of genome evolution. Most information about the evolution of microsatellites in the genus Drosophila has been obtained from Drosophila melanogaster. For comparison, we collected microsatellite data for the distantly related species Drosophila virilis. Screening about 0.5 Mb of nonredundant genomic sequence from GenBank, we identified 239 dinucleotide microsatellites. On average, D. virilis dinucleotides were significantly longer than D. melanogaster microsatellites (7.69 repeats vs. 6.75 repeats). Similarly, direct cloning of microsatellites resulted in a higher mean repeat number in D. virilis than in D. melanogaster (12.7 repeats vs. 12.2 repeats). Characterization of 11 microsatellite loci mapping to division 40-49 on the fourth chromosome of D. virilis indicated that D. virilis microsatellites are more variable than those of D. melanogaster.     

Effects of octopamine on reproduction, juvenile hormone metabolism, dopamine, and 20-hydroxyecdysone contents in Drosophila

The effect of an experimentally increased octopamine content (feeding flies with OA) on the levels of juvenile hormone (JH) degradation, dopamine (DA), and 20-hydroxyecdysone (20E) contents, oogenesis, and fecundity of wild type Drosophila flies has been studied. OA feeding of the flies was found to (1) cause a considerable decrease in JH degradation in females, but not males, of D. melanogaster and D. virilis; (2) have no effect on DA content in D. melanogaster and D. virilis; (3) increase 20E contents in D. virilis females; (4) decrease to a large extent the number of vitellogenic (stages 8-10) and mature (stage 14) oocytes in D. virilis; and (5) decrease the fecundity of D. melanogaster and D. virilis. A possible mechanism of action of OA as a neurohormone on the reproductive function of Drosophila is discussed.     

Immunocytochemical staining of central neurones in Periplaneta americana using monoclonal antibodies to choline acetyltransferase

Immunocytochemical mapping of cholinergic neurones in the CNS of the cockroach Periplaneta americana has been attempted using monoclonal antibodies to choline acetyltransferase (ChAT, acetyl-CoA: choline O-acetyltransferase, EC 2.3.1.6). Monoclonal antibodies 11 255 and 1E6 raised against rat brain ChAT and 1C8 raised against Drosophila melanogaster ChAT were ineffective in staining Periplaneta neurones. However, the cytoplasm of certain neuronal cell bodies was stained by monoclonal antibody 4D7 prepared against rat ChAT. Staining of cell bodies by 4D7 was enhanced following in vivo pre-treatment with colchicine. The staining of specific neurones by monoclonal antibody 4D7 indicates that these cockroach cells are rich in a protein with antigenic determinants resembling those of vertebrate ChAT. For some unidentified neurones, 4D7 staining is associated with the presence of acetylcholinesterase indicating that this monoclonal antibody offers a probe for mapping cholinergic neurones in the CNS of Periplaneta americana. The fast coxal depressor motoneurone (D(f)) was not stained by monoclonal antibody 4D7. Some neuronal processes in the sixth abdominal ganglion, and sensory cell bodies in the cerci were lightly stained by monoclonal antibody 4D7 following pre-injection of animals for 36 hr with colchicine.     

Nucleotide variation at the no-on-transient A gene in Drosophila littoralis

The no-on-transient A (nonA) gene encodes a putative RNA-binding protein, and mutations in this gene are known to affect vision, male courtship song and viability in Drosophila melanogaster. Here we have sequenced the coding region of the nonA gene of Drosophila littoralis and compared it with those of Drosophila virilis and D. melanogaster. All portions of nonA appeared to be conserved between D. littoralis and D. virilis, while the 5' region of the gene of these two species showed high divergence from that of a more distantly-related species, D. melanogaster. The same was true for the glycine repeat regions. No significant deviation from neutrality was observed in the analysis of intraspecific nucleotide variation in 5' or 3' region of the nonA gene in D. littoralis population. Also, comparison of D. littoralis sequences with homologous sequence of D. virilis suggests that the gene is evolving neutrally in D. virilis group. Divergence of the 5' regions between D. virilis group species and D. melanogaster could be a result of positive selection, but this finding is obscured by the long divergence time of the species groups.     

Isolation and characterization of a Drosophila neuropeptide gene

We have purified a 9 amino acid amidated neuropeptide, DPKQDFMRFamide, from whole adult D. melanogaster. This peptide exhibits sequence homology to the molluscan bioactive tetrapeptide FMRFamide and is a novel member of the FMRFamide peptide family. The gene encoding DPKQDFMRFamide has been cloned and characterized. It is present in a single copy per haploid genome, is expressed as a unique 1.7 kb mRNA species, and cytologically maps to 46C on the right arm of chromosome 2. Characterization of a cDNA clone indicates that the precursor protein is 347 amino acids in length and contains 5 copies of DPKQDFMRFamide, as well as 10 additional amidated peptides exhibiting varying degrees of structural relatedness. The Drosophila DPKQDFMRFamide gene and the Aplysia FMRFamide gene are ancestrally related; however, peptides display a higher degree of homology within a species than between species, suggesting intragenic concerted evolution of these neuropeptides.     

A 9.6 kb intervening sequence in D. virilis rDNA, and sequence homology in rDNA interruptions of diverse species of Drosophila and other diptera.

A large proportion of the 28S ribosomal RNA genes in Drosophila virilis are interrupted by a DNA sequence 9.6 kilobase pairs long. As regards both its presence and its position in the 28S gene (about two thirds of the way in), the D. virilis rDNA intervening sequence is similar to that found in D. melanogaster rDNA, but lengths differ markedly between the two species. Degrees of nucleotide sequence homology have been detected bewteen rDNA interruptions of the two species. This homology extends to putative rDNA intervening sequences in diverse higher diptera (other Drosophila species, the house fly and the flesh fly), but hybridization of cloned D. melanogaster and D. virilis rDNA interruption segments to DNA of several lower diptera has been negative. As is the case with melanogaster rDNA interruptions, segments of the virilis rDNA intervening sequence hybridize with non-rDNA components of the virilis genome, and interspecific homology may involve these non-rDNA sequences as well as rDNA interruptions. There is, however, evidence from buoyant density fractionation of DNA that the distributions of interruption-related sequences are distinct in D. melanogaster and D. virilis genomes. Moreover, thermal denaturation studies have indicated differing extents of homology between hybridizable sequences in D. virilis DNA and different segments of the D. melanogaster rDNA intervening sequence. We infer from our studies that rDNA intervening sequences are prevalent among higher diptera; that in the course of the evolution of these organisms, elements of the intervening sequences have been moderately to highly conserved; and that this conservation extends in at least two distantly related species of Drosophila to similar sequences found elsewhere in the genomes.     

Components Acting in Localization of Bicoid mRNA Are Conserved among Drosophila Species

Substantial insights into basic strategies for embryonic body patterning have been obtained from genetic analyses of Drosophila melanogaster. This knowledge has been used in evolutionary comparisons to ask if genes and functions are conserved. To begin to ask how highly conserved are the mechanisms of mRNA localization, a process crucial to Drosophila body patterning, we have focused on the localization of bcd mRNA to the anterior pole of the embryo. Here we consider two components involved in that process: the exuperantia (exu) gene, required for an early step in localization; and the cis-acting signal that directs bcd mRNA localization. First, we use the cloned D. melanogaster exu gene to identify the exu genes from Drosophila virilis and Drosophila pseudoobscura and to isolate them for comparisons at the structural and functional levels. Surprisingly, D. pseudoobscura has two closely related exu genes, while D. melanogaster and D. virilis have only one each. When expressed in D. melanogaster ovaries, the D. virilis exu gene and one of the D. pseudoobscura exu genes can substitute for the endogenous exu gene in supporting localization of bcd mRNA, demonstrating that function is conserved. Second, we reevaluate the ability of the D. pseudoobscura bcd mRNA localization signal to function in D. melanogaster. In contrast to a previous report, we find that function is retained. Thus, among these Drosophila species there is substantial conservation of components acting in mRNA localization, and presumably the mechanisms underlying this process.     

Regulative interactions between cells of wing discs from different dipteran species

The mechanism by which patterns are produced appears to be repeated in each segment of an animal, and it has been proposed that it may even have been conserved in evolution so that different species would have the same system of positional information. This idea has been tested by mixing cells of a defined fragment of the wing disc of Drosophila melanogaster with wing disc fragments of five other dipteran species to assay the ability of these disc fragments to stimulate intercalary regeneration of the D. melanogaster cells. The genetically marked (y; mwh) D. melanogaster fragment was mechanically mixed with wing discs or wing disc fragments of four drosophilids (D. melanogaster as a control, D. virilis, D. hydei, Zaprionus vittiger), of Musca domestica, and of Piophila casei. The mixed aggregates were cultured in vivo for 7 days, then metamorphosed in D. melanogaster larval hosts. The D. melanogaster fragments were only stimulated to regenerate when combined with complementary fragments from D. melanogaster or D. virilis wing discs. In the combination between D. melanogaster and D. hydei, the tissue formed integrated mosaic patterns, but no regeneration ensued. The one positive result (D. melanogaster mixed with D. virilis) shows that positional cues can be exchanged and correctly interpreted between cells of different species. The negative results do not prove that the mechanism for establishing patterns is different in the tested species, but may be due to incompatibilities that are not related to pattern formation.     

The interaction of bromodeoxyuridine with ribosomal RNA cistron redundancy in Drosophila

We demonstrate that Bromodeoxyuridine (BrdU)-induced reductions of the ribosomal RNA cistrons (rDNAs) are observed in Drosophila virilis, and Drosophila busckii, but not in karyotypically normal adults or larvae of Drosophila melanogaster. However, BrdU does reduce the redundancy of the rDNAs of XO D melanogaster males, in which a compensatory response is evidenced in the untreated XO sibling controls. These results suggest that the BrdU-rDNA interaction is specific to events which modulate rDNA redundancy. Further, both thymidine and deoxycytidine “reverse” the BrdU effect in D virilis, an observation which is inconsistent with current working hypotheses describing the mechanisms of BrdUs effects.     

In situ digestion of Drosophila virilis polytene chromosomes by AluI and HaeIII restriction endonucleases

Polytene chromosomes of Drosophila virilis were treated with AluI and HaeIII restriction endonucleases. Both enzymes were capable of extensively digesting chromosomal DNA, with the exception of some regions that contain repetitive DNAs. Moreover, a comparison was made between our data and the data already obtained with the same enzymes in D. melanogaster. On this basis, AluI digestion showed that the 5S RNA genes of D. virilis and D. melanogaster have different base composition, while digestion with HaeIII revealed resistance of the histone genes in D. virilis, contrary to what was previously found in D. melanogaster.     

Characterization of the dCaMKII-GAL4 driver line whose expression is controlled by the <Emphasis Type="Italic">Drosophila</Emphasis> Ca<Superscript>2+</Superscript>/calmodulin-dependent protein kinase II promoter

Transgenic flies that can drive GAL4 expression under the control of the 7 kb 5'-region of the Drosophila Ca(2+)/calmodulin-dependent protein kinase II (dCaMKII) gene (dCaMKII-GAL4) were established. Characteristic features of this dCaMKII-GAL4 driven reporter expression were compatible with the endogenous dCaMKII expression pattern: The dCaMKII-GAL4 driven reporter gene was expressed preferentially in the central nervous system of the embryo and larvae. Reporter expression was also observed in the brain, thoracic ganglion, and gut of the adult. The whole-brain distribution and projections of dCaMKII-GAL4-expressing cells in the adults were visualized three-dimensionally by using UAS-linked reporter genes. Prominent signals of nuclear-localized beta-Gal reporter gene expression were found in extensive brain regions, especially in the Kenyon cells of the mushroom body (MB), cells in the pars intercerebralis, and subesophageal ganglion (SOG). tau reporter gene expression highlighting neurite projections was detected in the MB lobes, median bundle, antennal lobe glomeruli, and fibers of clusters in the SOG, ventrolateral protocerebrum and superior lateral protocerebrum. These observations agree with those of a previous study mapping the dCaMKII-dependent memory circuits in courtship conditioning. Interestingly, green fluorescent protein reporter gene expression in adult MB lobes was predominantly observed in the alpha and beta lobes with a core-deficient pattern, but not in the alpha' and beta' lobes, similar to Fasciclin II immunoreactivity.