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Neuropeptides and their precursors in the fruitfly, Drosophila melanogaster.

Neuropeptides form the most diverse class of chemical messenger molecules in metazoan nervous systems. They are usually generated from biosynthetic precursor polypeptides by enzymatic processing and modification. Many different peptides belonging to a number of distinct neuropeptide families have already been characterized from various insect species. The Drosophila Genome Sequencing Project has important implications for the future of neurobiological research. This paper describes the discovery of several new fruitfly neuropeptides by an in silico data mining approach. In addition, the state-of-the-art of Drosophila peptide research is reviewed.

Amino Acid Sequence↗

Genome and EST analyses and expression of a gene family with putative functions in insect chemoreception.

Odorant-binding proteins (OBPs) are thought to be responsible for the transport of semiochemicals across hydrophobic interfaces to olfactory receptors. In insects, a second class of OBPs with four conserved cysteines has been variously named as sensory appendage proteins, olfactory segment-D proteins, and chemosensory proteins (CSPs). The physiological functions of these proteins have remained elusive. Here we report a comprehensive survey of both genome and expressed sequence tags (EST) databases. This showed that CSPs are apparently only present in the phylum, Arthropoda, and in two subphyla, Crustacea and Uniramia. This is the first report of a putative CSP in Crustacea and suggests that the origin of these genes predates the divergence of Uniramia and Crustacea. For the Uniramia, we identified 74 new genes encoding putative CSPs of insect species from 10 different orders. Using tissue-specific EST libraries, we have examined the relative expression of putative CSP genes in many tissues from 22 insect species suggesting that the genes are expressed widely. One Drosophila CSPs is expressed sixfold higher in head than other CSPs. One Bombyx mori CSPs was found at a very high level in pheromone gland, and for the first time, six CSPs were identified in B. mori compound eyes. The different frequencies of CSP transcripts were observed between solitary and gregarious EST libraries of Locusta migratoria.

Animals↗

Isolation and expression of the eclosion hormone gene from the tobacco hornworm, Manduca sexta.

Eclosion hormone (EH) is a 62-amino acid neuropeptide that initiates the ecdysis behavior of insects. The EH-encoding gene of the tobacco hornworm, Manduca sexta, was isolated by using a designed 72-mer oligonucleotide probe. Sequence analysis of this gene and its corresponding cDNA showed that the EH gene is 7.8 kilobases and consists of three exons. Exon I is totally nontranslated; exon II contains a 26-amino acid signal peptide and amino acids 1-4 of the EH peptide, and exon III encodes the remainder of the peptide. The EH gene is present in a single copy per haploid genome and transcribes an 0.8-kb mRNA that is expressed in larval, diapausing pupal, and developing adult brains but not in the ventral nerve cord or in nonneural tissues. In situ hybridization showed that the EH gene is expressed in two pairs of ventromedial neurosecretory cells in brains of both larvae and developing adults.

Amino Acid Sequence↗

Rapid cloning of insect transposon insertion junctions using 'universal' PCR.

Very highly degenerate primers with short specific 3' anchor sequences and 5' adaptors were used in conjunction with nested specific primers to amplify large numbers of unknown insertion junctions of the insect retrotransposon Woot, using genomic DNA as template for the polymerase chain reaction (PCR). This technique, sometimes referred to as universal PCR, is a powerful method for molecular characterization of transposon insertions into genomes, and more generally for short-distance chromosome walking through unknown DNA. Twenty-four unique insertion junctions were cloned and sequenced from two strains of Tribolium castaneum and one strain of T. freemani. Inspection of these sequences revealed that integration of the Woot retrotransposon is cued by the insertion target motif, GTAC, in both species.

Animals↗

A genome-wide screen identifies a Bordetella type III secretion effector and candidate effectors in other species.

Bordetella bronchiseptica utilizes a type III secretion system (TTSS) for induction of non-apoptotic cytotoxicity in host cells and modulation of host immunity. The identity of Bordetella TTSS effectors, however, has remained elusive. Here we report a genome-wide screen for TTSS effectors based on shared biophysical and functional characteristics of class I chaperones and their frequent colocalization with TTSS effectors. When applied to B. bronchiseptica, the screen identified the first TTSS chaperone-effector locus, btcA-bteA, and we experimentally confirmed its function. Expression of bteA is co-ordinated with expression of TTSS apparatus genes, BteA is secreted through the TTSS of B. bronchiseptica, it is required for cytotoxicity towards mammalian cells, and it is highly conserved in the human-adapted subspecies B. pertussis and B. parapertussis. Transfection of bteA into epithlieal cells results in rapid cell death, indicating that BteA alone is sufficient to induce potent cytotoxicity. Finally, an in vitro interaction between BteA and BtcA was demonstrated. The search for TTSS chaperones and effectors was then expanded to other bacterial genomes, including mammalian and insect pathogens, where we identified a large number of novel candidate chaperones and effectors. Although the majority of putative effectors are proteins of unknown function, several have similarities to eukaryotic protein domains or previously identified effectors from other species.

Bacterial Proteins↗

Insect transmission of plant viruses: a constraint on virus variability.

Genetic diversity in viruses is shaped by high rates of recombination and is constrained by host defenses and the requirements of transmission. Recent studies of insect-transmitted plant viruses demonstrate highly conserved molecular motifs in viral genomes that regulate the specificity of insect transmission. In contrast, advances in our understanding of host plant response to virus infection reveal some generalized patterns of host defense to a diversity of viruses.

Animals↗

Baculovirus diversity and molecular biology.

With the identification and characterization of a number of structural and nonstructural protein genes, advances have been made in our understanding of baculovirus structure, regulation of gene expression, and replication. Since less than 30% of the AcMNPV genome has been sequenced and characterized, the continued identification and assignment of function to baculovirus genes is perhaps the most crucial of enterprises now facing baculovirologists and is critical to the development of our understanding of the baculovirus genome and its replication. The size and diversity of baculovirus genomes appears to be strongly influenced by mobile DNA from the insect host. Also, transposon-mediated mutations of baculoviruses provide examples of functional inactivation of viral genes (FP phenotype mutations) and transcriptional activation (TE-D insertion). Another role transposable elements may play is the introduction of insect promoters and enhancers to the baculovirus genome. Since early baculovirus genes are likely transcribed in a way similar to normal insect genes, transposons that insert strong constitutive promoters or cellular enhancers near early baculovirus genes may cause mutations that are subsequently selected for. If this does occur, baculovirus early gene promoters may exhibit a great deal of variability in sequence and may resemble host promoters. Given the overall similarity between the genomes of OpMNPV and AcMNPV and the apparent absence of a region, similar to the AcMNPV HindIII-K/EcoR1-S in OpMNPV, it is intriguing to speculate that this region which contains two ORFs and the hr5 enhancer, may have been inserted into the AcMNPV genome by transposition, possibly delivering several helpful genes (35k and 94k) and a powerful enhancer. The highly repeated enhancer may have been subsequently amplified by recombination. In such a model, the acquisition of general or species-specific enhancers might influence both virulence and host range. Acquisition of general enhancers could increase the level of early gene expression, thus accelerating the cellular infection cycle and making the virus more virulent. Similarly, the acquisition of species-specific enhancers might affect host range by accelerating the infection cycle, but only in a specific host or cell type. One might therefore postulate that diversity in baculoviruses may reflect not only different selection pressures but also the diversity of mobile DNA within host insect species. Although our understanding of baculovirus diversity and molecular biology is rapidly advancing, many of the fundamental characteristics that define the unique nature of baculoviruses remain poorly understood. One fundamental feature of baculoviruses is the production of the two virion phenotypes, PDV and BV.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Efficient transformation of the beetle Tribolium castaneum using the Minos transposable element: quantitative and qualitative analysis of genomic integration events.

Genetic transformation in insects holds great promise as a tool for genetic manipulation in species of particular scientific, economic, or medical interest. A number of transposable elements have been tested recently as potential vectors for transformation in a range of insects. Minos is one of the most promising elements because it appears to be active in diverse species and has the capacity to carry large inserts. We report here the use of the Minos element as a transformation vector in the red flour beetle Tribolium castaneum (Coleoptera), an important species for comparative developmental and pest management studies. Transgenic G(1) beetles were recovered from 32.4% of fertile G(0)'s injected with a plasmid carrying a 3xP3-EGFP-marked transposon and in vitro synthesized mRNA encoding the Minos transposase. This transformation efficiency is 2.8-fold higher than that observed when using a plasmid helper. Molecular and genetic analyses show that several independent insertions can be recovered from a single injected parent, but that the majority of transformed individuals carry single Minos insertions. These results establish Minos as one of the most efficient vectors for genetic transformation in insects. In combination with piggyBac-based transgenesis, our work allows the introduction of sophisticated multicomponent genetic tools in Tribolium.

Animals↗

Molecular cloning and genomic organization of an allatostatin preprohormone from Drosophila melanogaster.

The insect allatostatins are neurohormones, acting on the corpora allata (where they block the release of juvenile hormone) and on the insect gut (where they block smooth muscle contraction). We screened the "Drosophila Genome Project" database with electronic sequences corresponding to various insect allatostatins. This resulted in alignment with a DNA sequence coding for some Drosophila allatostatins (drostatins). Using PCR with oligonucleotide primers directed against the presumed exons of this Drosophila allatostatin gene and subsequent 3'- and 5'-RACE, we were able to clone its cDNA. The Drosophila allatostatin preprohormone contains four amino acid sequences that after processing would give rise to four Drosophila allatostatins: Val-Glu-Arg-Tyr-Ala-Phe-Gly-Leu-NH(2) (drostatin-1), Leu-Pro-Val-Tyr-Asn-Phe-Gly-Leu-NH(2) (drostatin-2), Ser-Arg-Pro-Tyr-Ser-Phe-Gly-Leu-NH(2) (drostatin-3), and Thr-Thr-Arg-Pro-Gln-Pro-Phe-Asn-Phe-Gly-Leu-NH(2) (drostatin-4). Drostatin-2 is identical to helicostatin-2 (11-18) and drostatin-3 to helicostatin-3, two neurohormones previously isolated from the moth Helicoverpa armigera. Furthermore, drostatin-3 has previously been isolated from Drosophila itself. Drostatins-1 and -4 are novel members of the insect allatostatin neuropeptide family. The Drosophila allatostatin preprohormone gene contains two introns and three exons. The gene is located on the right arm of the third chromosome, position 96A-B. The existence of at least four different Drosophila allatostatins opens the possibility of a differential action of some of these hormones on the two recently cloned Drosophila allatostatin receptors, DAR-1 and -2. This is the first report on an allatostatin preprohormone from Drosophila.

Amino Acid Sequence↗

Genome sequence of the endocellular obligate symbiont of tsetse flies, Wigglesworthia glossinidia.

Many insects that rely on a single food source throughout their developmental cycle harbor beneficial microbes that provide nutrients absent from their restricted diet. Tsetse flies, the vectors of African trypanosomes, feed exclusively on blood and rely on one such intracellular microbe for nutritional provisioning and fecundity. As a result of co-evolution with hosts over millions of years, these mutualists have lost the ability to survive outside the sheltered environment of their host insect cells. We present the complete annotated genome of Wigglesworthia glossinidia brevipalpis, which is composed of one chromosome of 697,724 base pairs (bp) and one small plasmid, called pWig1, of 5,200 bp. Genes involved in the biosynthesis of vitamin metabolites, apparently essential for host nutrition and fecundity, have been retained. Unexpectedly, this obligate's genome bears hallmarks of both parasitic and free-living microbes, and the gene encoding the important regulatory protein DnaA is absent.

Animals↗

Isolation and structure of the Drosophila corazonin gene.

A recombinant DNA clone containing the corazonin gene was isolated from a genomic library of Drosophila melanogaster. Its nucleotide sequence predicts a preprocorazonin consisting of an 19 amino acid putative signal peptide, the 11 amino acid corazonin sequence, a Gly used for amidation, a Lys-Arg proteolytic processing site, and a 39 amino acid corazonin-precursor-related peptide (CPRP). CPRP has an internal Arg-Arg sequence and thus could possibly be further processed into a tripeptide and a 34-mer. Neither CPRP or its possible products are structurally related to any known neuropeptide, and their physiological function is unknown. The structure of the predicted preprocorazonin is remarkably similar to the preprohormone of adipokinetic hormone, which suggests that corazonin and adipokinetic hormone have a common evolutionary origin.

Amino Acid Sequence↗

Structure and expression of the tenecin 3 gene in Tenebrio molitor.

A genomic DNA fragment encoding tenecin 3, an antifungal protein was cloned from the genomic DNA library of Tenebrio molitor. The DNA sequence analysis showed that the coding region is divided into two exons by an intron of 49 bp in the middle of the putative leader peptide coding region. Southern blot analysis suggests that the gene is present as a single copy. The transcription initiation site was determined by primer extension analysis and S1 mapping. The TATA box and CCAAT box sequences were found at -30, and -121, respectively, from the transcription initiation site. Tenecin 3 mRNA is abundantly expressed in larvae and adults, while little was detected in RNAs from pupae, suggesting that the expression of the tenecin 3 gene is developmentally regulated.

Amino Acid Sequence↗

The Drosophila CPEB homolog, orb, is required for oskar protein expression in oocytes.

The establishment of polarity axes in the Drosophila egg and embryo depends upon the localization and on-site expression of maternal mRNAs. The critical step in the targeting of posterior determinants is the localization of oskar (osk) mRNA to the pole and its on-site translation. Osk protein then recruits other posterior group gene products involved in the formation of pole plasm and in the localization and regulation of the posterior determinant, nanos. Here we have investigated the role of the Drosophila CPEB homolog, the orb gene, in the osk mRNA localization pathway. We demonstrate that the expression of Osk protein is dependent upon the orb gene. In strong orb mutants, Osk protein expression is undetectable, while in the hypomorphic mutant, orb(mel), little or no on-site expression of Osk protein at the posterior pole is observed. The defects in Osk protein accumulation in orb mutant ovaries are correlated with a reduction in the length of the osk poly(A) tails. We show that osk mRNA is in immunoprecipitable complexes with Orb protein in ovaries and that the osk 3' UTR can be UV cross-linked to Orb protein in ovarian extracts. These data suggest that Orb is required to activate the translation of osk mRNA and at that this may be accomplished by a mechanism similar to that used by the Xenopus CPEB protein to control translation of "masked" mRNAs.

3' Untranslated Regions↗

A gene (DLG2) located at 17q12-q21 encodes a new homologue of the Drosophila tumor suppressor dIg-A.

We have isolated a novel cDNA that maps distal to BRCA1 at 17q12-q21. The total sequence predicts a protein of 576 amino acids with three conserved regions: a 90-amino-acid repeat domain, a SH3 (src homology region 3) motif, and a guanylate kinase domain. These conserved regions are shared among members of the discs-large family of proteins that include human p55, a membrane protein expressed in erythrocytes, rat PSD-95/SAP90, a synapse protein expressed in brain, Drosophila dIg-A, a septate junction protein expressed in various epithelia, and human and mouse ZO-1 and canine ZO-2, two tight junction proteins. dIg-A has been shown to act as a tumor suppressor, and the other members may all be involved in signal transduction through specialized membrane domains with highly organized cytoskeletons and thus are potential tumor suppressors. Since allelic loss has been reported in the 17q12-q21 region in breast and ovarian cancer and it appears that BRCA1 is not the target of the losses, we looked for somatic alterations in DLG2 in sporadic breast tumors. No evidence for mutation was found, making it unlikely that DLG2 is involved in sporadic breast cancer.

Amino Acid Sequence↗

Complete genomic sequence of Powassan virus: evaluation of genetic elements in tick-borne versus mosquito-borne flaviviruses.

The complete nucleotide sequence of the positive-stranded RNA genome of the tick-borne flavivirus Powassan (10,839 nucleotides) was elucidated and the amino acid sequence of all viral proteins was derived. Based on this sequence as well as serological data, Powassan virus represents the most divergent member of the tick-borne serocomplex within the genus flaviviruses, family Flaviviridae. The primary nucleotide sequence and potential RNA secondary structures of the Powassan virus genome as well as the protein sequences and the reactivities of the virion with a panel of monoclonal antibodies were compared to other tick-borne and mosquito-borne flaviviruses. These analyses corroborated significant differences between tick-borne and mosquito-borne flaviviruses, but also emphasized structural elements that are conserved among both vector groups. The comparisons among tick-borne flaviviruses revealed conserved sequence elements that might represent important determinants of the tick-borne flavivirus phenotype.

Amino Acid Sequence↗

Nucleotide sequence and genomic organization of Acyrthosiphon pisum virus.

The nucleotide sequence of the genomic RNA of Acyrthosiphon pisum virus was determined. The APV genome is 10,016 nucleotides in length, excluding the 3'-end poly(A) track, and contains two large open reading frames (ORFs), encoding proteins of 296,340 and 63,279 Da. The ORF1 is preceded by an untranslated leader sequence of 267 nucleotides. The ORF1 product contains sequence motifs characteristic of RNA-dependent RNA polymerases, chymotrypsin-like proteases, and helicases. Interviral sequence comparison revealed significant similarities with viruses belonging to the so-called picornavirus superfamily. The ORF2 is most likely expressed by a -1 translational frameshift and is followed by an untranslated sequence of 222 nucleotides. Internal amino acid sequences of three capsid proteins (66K, 34K, 23/24K) were determined. Comparison of the obtained amino acid sequences with the APV sequence disclosed that the structural proteins are located in the 3'-terminal half of the genome. The 34K protein is encoded by the ORF1, while the 66K protein contains both ORF1-(34K) and ORF2-derived sequences and is probably expressed by a translational frameshift. The 23/24K proteins most likely arise by proteolytic breakdown of the 34K protein. Although the deduced APV genomic organization in some aspects resembles that of the picornaviruses, its overall genomic organization indicates that APV is a distinct species only distantly related to the Picornaviridae.

Amino Acid Sequence↗

Sequence of the genomic RNA of nudaurelia beta virus (Tetraviridae) defines a novel virus genome organization.

The monopartite genome of Nudaurelia beta virus, the type species of the Betatetravirus genus of the family Tetraviridae, consists of a single-stranded positive-sense RNA (ss+RNA) of 6625 nucleotides containing two open reading frames (ORFs). The 5' proximal ORF of 5778 nucleotides encodes a protein of 215 kDa containing three functional domains characteristic of RNA-dependent RNA polymerases of ss+RNA viruses. The 3' proximal ORF of 1836 nucleotides, which encodes the 66-kDa capsid precursor protein, overlaps the replicase gene by more than 99% (1827 nucleotides) and is in the +1 reading frame relative to the replicase reading frame. This capsid precursor is expressed via a 2656-nucleotide subgenomic RNA. The 3' terminus of the genome can be folded into a tRNA-like secondary structure that has a valine anticodon; the tRNA-like structure lacks a pseudoknot in the aminoacyl stem, a feature common to both genera of tetraviruses. Comparison of the sequences of Nudaurelia beta virus and another member of the Tetraviridae, Helicoverpa armigera stunt virus, which is in the genus Omegatetravirus, shows identities of 31.6% for the replicase and 24.5% for the capsid protein. The viruses in the genera Betatetravirus and Omegatetravirus of the Tetraviridae are clearly related but show significant differences in their genome organization. It is concluded that the ancestral virus with a bipartite genome, as found in the genus Omegatetravirus, likely evolved from a virus with an unsegmented genome, as found in the genus Betatetravirus, through evolution of the subgenomic RNA into a separate genomic component, with the accompanying loss of the capsid gene from the longer genomic RNA.

Amino Acid Sequence↗

Characterization and template properties of RNA dimers generated during flock house virus RNA replication.

Flock house virus (FHV) is the best studied member of the Nodaviridae, a family of small, nonenveloped, isometric RNA viruses of insects and fish. Nodavirus genomes comprise two single-stranded positive-sense RNA segments (RNAs 1 and 2) that encode the viral RNA-dependent RNA polymerase (RdRp) and capsid protein precursor, respectively. The RdRp replicates both genomic RNAs and also generates a subgenomic RNA (RNA3) that is not encapsidated. Although genomic RNAs replicate through negative-sense intermediates, little is known about these RNAs or the details of the replication mechanism. Negative-sense RNAs 1, 2, and 3, as well as putative dimers of RNAs 2 and 3, have been detected in previous studies. In this study we detected dimers of RNAs 1, 2, and 3 by Northern blot analyses of RNA samples from FHV-infected Drosophila cells, as well as from mammalian and yeast cells supporting FHV RNA replication. Characterization of these RNA species by RT-PCR and sequence determination showed that they contained head-to-tail junctions of FHV RNAs. RNAs containing the complete sequence of RNA2 joined to RNA3 were also detected during replication. To examine the template properties of these dimeric RNAs, we made corresponding cDNAs and transcribed them from a T7 promoter in mammalian cells constitutively expressing T7 RNA polymerase, together with RNA1 to provide the RdRp. Although heterologous terminal extensions inhibit FHV RNA replication, monomeric RNA2 was resolved and replicated from complete or partial homodimer templates and from an RNA2-RNA3 heterodimer.

Animals↗