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Detection of a gypsy-like sequence in the genome of the cat flea Ctenocephalides felis (Bouché 1835).

The gypsy virus of Drosophila melanogaster is the first identified retrovirus of insects. Its infectious properties have been reported, and it is able to cause diverse mutations of the phenotype in the fruit fly. As a typical endogenous retrovirus, it is transmitted vertically as part of the host genome, but horizontal transmission has also been shown for the D. melanogaster gypsy virus. Using polymerase chain reaction with degenerate primers designed from the gypsy element of Drosophila subobscura, a gypsy-like sequence was amplified from the genome of the cat flea Ctenocephalides felis in a pool of laboratory and field strains. High identities with other gypsy-like sequences of Drosophila spp. can be detected. In contrast to some of these gypsy sequences, the 410-bp fragment of C. felis possesses two deletions, which are localised in the env gene region. Because of these deletions and the lack of virus particles in C. felis, the criteria for host infectivity are not fulfilled. Nevertheless, the general detection of a gypsy-like sequence in C. felis and the capability of the ectoparasite to act as transmitter of viral diseases can be of importance for the cat flea's position in the epidemiology of diseases, which are accompanied by a higher rate of mutation.

Animals↗

A human homoeo box gene specifically expressed in spinal cord during embryonic development.

Several genes involved in the determination of Drosophila body segments share a conserved DNA sequence of 183 nucleotides termed the homoeo box. Homologous homoeo box sequences have been detected in the genome of species ranging from insects and anellids to vertebrates, and a number of homoeo box-containing genomic DNA clones have been isolated from Xenopus, mouse and human. We have recently isolated human complementary DNA clones containing homoeo box sequences, representing transcripts from four different genes. We report here the nucleotide sequence of one of these clones (HHO.c10) and show that the corresponding gene is transcribed in human embryos and fetuses at 5-10 weeks post-conception. A major polyadenylated transcript of approximately 2.1 kilobases (kb), as well as RNA species of higher relative molecular mass (Mr), are specifically expressed at a constant level in spinal cord throughout this developmental period.

Base Sequence↗

Insect nicotinic acetylcholine receptor gene families: from genetic model organism to vector, pest and beneficial species.

Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic transmission in the insect nervous system and are targets of a major group of insecticides, the neonicotinoids. Analyses of genome sequences have shown that nAChR gene families remain compact in diverse insect species, when compared to their mammalian counterparts. Thus, Drosophila melanogaster and Anopheles gambiae each possess 10 nAChR genes while Apis mellifera has 11. Although these are among the smallest nAChR gene families known, receptor diversity can be considerably increased by alternative splicing and mRNA A-to-I editing, thereby generating species-specific subunit isoforms. In addition, each insect possesses at least one highly divergent nAChR subunit. Species-specific subunit diversification may offer promising targets for future rational design of insecticides that act on particular pests while sparing beneficial insects. Electrophysiological studies on cultured Drosophila cholinergic neurons show partial agonist actions of the neonicotinoid imidacloprid and super-agonist actions of another neonicotinoid, clothianidin, on native nAChRs. Recombinant hybrid heteromeric nAChRs comprising Drosophila Dalpha2 and a vertebrate beta2 subunit have been instructive in mimicking such actions of imidacloprid and clothianidin. Unitary conductance measurements on native nAChRs indicate that more frequent openings of the largest conductance state may offer an explanation for the superagonist actions of clothianidin.

Alternative Splicing↗

Complete genome sequence of the entomopathogenic and metabolically versatile soil bacterium Pseudomonas entomophila.

Pseudomonas entomophila is an entomopathogenic bacterium that, upon ingestion, kills Drosophila melanogaster as well as insects from different orders. The complete sequence of the 5.9-Mb genome was determined and compared to the sequenced genomes of four Pseudomonas species. P. entomophila possesses most of the catabolic genes of the closely related strain P. putida KT2440, revealing its metabolically versatile properties and its soil lifestyle. Several features that probably contribute to its entomopathogenic properties were disclosed. Unexpectedly for an animal pathogen, P. entomophila is devoid of a type III secretion system and associated toxins but rather relies on a number of potential virulence factors such as insecticidal toxins, proteases, putative hemolysins, hydrogen cyanide and novel secondary metabolites to infect and kill insects. Genome-wide random mutagenesis revealed the major role of the two-component system GacS/GacA that regulates most of the potential virulence factors identified.

Animals↗

Activation of antigen-presenting cells by immunostimulatory plant DNA: a natural resource for potential adjuvant.

Genomic DNA sequences (bacteria, insect, nematodes and molluscs) or synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs (CpG-DNA/ODN) are regarded as promising candidates for new medical adjuvants for their ability to stimulate the mammalian immune system and enhance immune responses to specific antigens. Here, we first report the immunostimulatory activity of total genomic DNA from two plants, Brassica chinensis L. and Zea may, the CpG methylation status of which is incomplete compared with E. coli DNA. These plant DNA can activate B cells to proliferate. Plant DNA promotes secretion of IL-12, and increases expression of MHC and costimulatory molecules by bone marrow-derived dendritic cells (BMDC). Plant DNA can also enhance antigen presentation capacity of BMDC and macrophages. When administrated in vivo, plant DNA can inhibit tumor growth in situ or metastasis in tumor-bearing mice. The immunostimulatory activity of plant DNA could be abolished by methylation. Our data showed that plant DNA can activate antigen-presenting cells (APC) including DC, macrophages and B cells, indicating that plant DNA is a new kind of potential adjuvant. Therefore, we conclude that plant DNA is another natural source of CpG-DNA, and that green plants may provide abundant resources for this potential medical adjuvant.

Adjuvants, Immunologic↗

Replication of the RNA segments of a bipartite viral genome is coordinated by a transactivating subgenomic RNA.

The insect nodavirus Flock house virus (FHV) has a small genome divided between two segments of positive-sense RNA, RNA1 and RNA2. RNA1 encodes the RNA-dependent RNA polymerase (RdRp) catalytic subunit and templates the synthesis of a subgenomic RNA (RNA3) that encodes two small nonstructural proteins. Replication of RNA2, which encodes a precursor to the viral capsid proteins, suppresses RNA3 synthesis. Here we report that RNA1 mutants deficient in RNA3 synthesis failed to support RNA2 replication. This effect was not caused by alterations in the RdRp catalytic subunit nor by a lack of the proteins encoded by RNA3. Furthermore, RNA3 supplied in trans from an exogenous source restored RNA2 replication. These data indicate that RNA3 transactivates the replication of RNA2, a novel property for a viral RNA. We propose that the RNA3 dependence of RNA2 replication serves to coordinate replication of the FHV genome segments.

Animals↗

Nuclear receptors of the honey bee: annotation and expression in the adult brain.

The Drosophila genome encodes 18 canonical nuclear receptors. All of the Drosophila nuclear receptors are here shown to be present in the genome of the honey bee (Apis mellifera). Given that the time since divergence of the Drosophila and Apis lineages is measured in hundreds of millions of years, the identification of matched orthologous nuclear receptors in the two genomes reveals the fundamental set of nuclear receptors required to 'make' an endopterygote insect. The single novelty is the presence in the A. mellifera genome of a third insect gene similar to vertebrate photoreceptor-specific nuclear receptor (PNR). Phylogenetic analysis indicates that this novel gene, which we have named AmPNR-like, is a new member of the NR2 subfamily not found in the Drosophila or human genomes. This gene is expressed in the developing compound eye of the honey bee. Like their vertebrate counterparts, arthropod nuclear receptors play key roles in embryonic and postembryonic development. Studies in Drosophila have focused primarily on the role of these transcription factors in embryogenesis and metamorphosis. Examination of an expressed sequence tag library developed from the adult bee brain and analysis of transcript expression in brain using in situ hybridization and quantitative RT-PCR revealed that several members of the nuclear receptor family (AmSVP, AmUSP, AmERR, AmHr46, AmFtz-F1, and AmHnf-4) are expressed in the brain of the adult bee. Further analysis of the expression of AmUSP and AmSVP in the mushroom bodies, the major insect brain centre for learning and memory, revealed changes in transcript abundance and, in the case of AmUSP, changes in transcript localization, during the development of foraging behaviour in the adult. Study of the honey bee therefore provides a model for understanding nuclear receptor function in the adult brain.

Amino Acid Sequence↗

Patterns in evolution: veins of the Drosophila wing.

The development of the Drosophila wing is a classical model for studying the genetic control of tissue size, shape and patterning. A detailed picture of how positional information is interpreted by cells in the imaginal disc and translated into the adult wing vein pattern has recently emerged. It highlights the central role of dose-dependent activation of distinct cell transcription programs in response to the Hedgehog (Hh) and Decapentaplegic (Dpp) morphogens, as well as an early role of Notch signalling, in connecting the positioning of vein primordia and vein differentiation proper. The biochemical basis of the cross-talk that operates between these different signalling pathways is less well understood. New strategies made possible by the genome sequencing of several insect models should provide an important complement to the knowledge obtained from >60 years of genetic studies.

Animals↗

Studies on overwintering of bluetongue viruses in insects.

Bluetongue viruses (BTVs) are economically important arboviruses that affect sheep and cattle. The overwintering mechanism of BTVs in temperate climates has eluded researchers for many years. Many arboviruses overwinter in their invertebrate vectors. To test the hypothesis that BTVs overwinter in their vertically infected insect vectors, Culicoides sonorensis larvae were collected from long-term study sites in northern Colorado, USA, and assayed for the presence of BTV RNA by nested RT-PCR. Sequences from BTV RNA segment 7 were detected in 30 % (17/56) of pools composed of larvae and pupae collected in 1998 and in 10 % (31/319) of pools composed of adults reared from larvae collected in 1996. BTV was not isolated from the insects. Additionally, Culicoides cell-culture lines derived from material collected at one of the sites, or derived from insect samples collected during a BTV outbreak, contained BTV RNA segment 7. In contrast, segment 2 RNA was detected at half the rate of segment 7 RNA in the field-collected larvae and was only detected in the Culicoides cell lines with one of two primer sets. These data suggest that BTVs could overwinter in the insect vector and that there is reduced expression of the outer capsid genes during persistent infection.

Animals↗

Providence virus: a new member of the Tetraviridae that infects cultured insect cells.

We identified a new member of the Tetraviridae, Providence virus (PrV), persistently infecting a midgut cell line derived from the corn earworm (Helicoverpa zea). Virus purified from these cells also productively infected a H. zea fat body cell line, and a cell line from whole embryos of the beet armyworm, Spodoptera exigua. PrV is thus the first tetravirus shown to replicate in cell culture. PrV virions are isometric particles composed of two structural proteins (60 and 7.4 kDa) that encapsidate both the genomic (6.4 kb) and the subgenomic (2.5 kb) RNAs. The monopartite organization of the PrV genome resembles that of Nudaurelia beta virus and Thosea asigna virus, members of the genus Betatetravirus. The predicted sequence of the PrV structural proteins demonstrates homology to tetraviruses in both genera. The infectivity of PrV for cultured cells uniquely permitted examination of tetravirus RNA and protein synthesis during synchronous infection. The discovery of PrV greatly facilitates studies of tetravirus molecular biology.

Animals↗

Sequence analysis of the mitochondrial genome of Sarcophyton glaucum: conserved gene order among octocorals.

The nucleotide sequence for an 11,715-bp segment of the mitochondrial genome of the octocoral Sarcophyton glaucum is presented, completing the analysis of the entire genome for this anthozoan member of the phylum Cnidaria. The genome contained the same 13 protein-coding and 2 ribosomal RNA genes as in other animals. However, it also included an unusual mismatch repair gene homologue reported previously and codes for only a single tRNA gene. Intermediate in length compared to two other cnidarians (17,443 and 18,911 bp), this organellar genome contained the smallest amount of noncoding DNA (428, compared to 1283 and 781 nt, respectively), making it the most compact one found for the phylum to date. The mitochondrial genes of S. glaucum exhibited an identical arrangement to that found in another octocoral, Renilla kolikeri, with five protein-coding genes in the same order as has been found in insect and vertebrate mitochondrial genomes. Although gene order appears to be highly conserved among octocorals, compared to the hexacoral, Metridium senile, few similarities were found. Like other metazoan mitochondrial genomes, the A + T composition was elevated and a general bias against codons ending in G or C was observed. However, an exception to this was the infrequent use of TGA compared to TGG to code for tryptophan. This divergent codon bias is unusual but appears to be a conserved feature among two rather distantly related anthozoans.

Amino Acid Sequence↗

Genome fragment of Wolbachia endosymbiont transferred to X chromosome of host insect.

The adzuki bean beetle, Callosobruchus chinensis, is triple-infected with distinct lineages of Wolbachia endosymbiont, wBruCon, wBruOri, and wBruAus, which were identified by their wsp (Wolbachia surface protein) gene sequences. Whereas wBruCon and wBruOri caused cytoplasmic incompatibility of the host insect, wBruAus did not. Although wBruCon and wBruOri were easily eliminated by antibiotic treatments, wBruAus persisted over five treated generations and could not be eliminated. The inheritance pattern of wBruAus was, surprisingly, explained by sex-linked inheritance in male-heterozygotic organisms, which agreed with the karyotype of C. chinensis (2n = 20, XY). Quantitative PCR analysis demonstrated that females contain around twice as much wsp titer as males, which is concordant with an X chromosome linkage. Specific PCR and Southern blot analyses indicated that the wBruAus-bearing strain of C. chinensis contains only a fraction of the Wolbachia gene repertoire. Several genome fragments of wBruAus were isolated using an inverse PCR technique. The fragments exhibited a bacterial genome structure containing a number of ORFs typical of the alpha-proteobacteria, although some of the ORFs contained disruptive mutations. In the flanking region of ftsZ gene, a non-long terminal repeat (non-LTR) retrotransposon sequence, which is typical of insects but not found from bacteria, was present. These results strongly suggest that wBruAus has no microbial entity but is a genome fragment of Wolbachia endosymbiont transferred to the X chromosome of the host insect.

Animals↗

Living with genome instability: the adaptation of phytoplasmas to diverse environments of their insect and plant hosts.

Phytoplasmas ("Candidatus Phytoplasma," class Mollicutes) cause disease in hundreds of economically important plants and are obligately transmitted by sap-feeding insects of the order Hemiptera, mainly leafhoppers and psyllids. The 706,569-bp chromosome and four plasmids of aster yellows phytoplasma strain witches' broom (AY-WB) were sequenced and compared to the onion yellows phytoplasma strain M (OY-M) genome. The phytoplasmas have small repeat-rich genomes. This comparative analysis revealed that the repeated DNAs are organized into large clusters of potential mobile units (PMUs), which contain tra5 insertion sequences (ISs) and genes for specialized sigma factors and membrane proteins. So far, these PMUs appear to be unique to phytoplasmas. Compared to mycoplasmas, phytoplasmas lack several recombination and DNA modification functions, and therefore, phytoplasmas may use different mechanisms of recombination, likely involving PMUs, for the creation of variability, allowing phytoplasmas to adjust to the diverse environments of plants and insects. The irregular GC skews and the presence of ISs and large repeated sequences in the AY-WB and OY-M genomes are indicative of high genomic plasticity. Nevertheless, segments of approximately 250 kb located between the lplA and glnQ genes are syntenic between the two phytoplasmas and contain the majority of the metabolic genes and no ISs. AY-WB appears to be further along in the reductive evolution process than OY-M. The AY-WB genome is approximately 154 kb smaller than the OY-M genome, primarily as a result of fewer multicopy sequences, including PMUs. Furthermore, AY-WB lacks genes that are truncated and are part of incomplete pathways in OY-M.

Animals↗

The densovirus of Junonia coenia (Jc DNV) as an insect cell expression vector.

An infectious genome of the Junonia coenia densovirus (Jc DNV) has been recently cloned and sequenced. We investigated the ability of this cloned genome to be used as expression vector by inserting the lacZ gene of Escherichia coli as fusion gene in the major open reading frame (ORF 1) of the viral sequence. The resulting recombinant plasmid designated pBRJlac Z was transfected into insect SPC-SL 52 cells and the expression of beta-galactosidase (beta-gal) was detected qualitatively or quantitatively by using Xgal or ONPG as chromogenic substrates. Western blot analysis revealed that beta-gal was expressed as chimeric capsid-beta-gal polypeptides. This provided evidence that ORF1 codes for structural polypeptides which share a common C-terminal sequence. Construction of plasmids with alterations or deletions in ORF2, 3 or 4, allowed us to implicate nonstructural (NS) functions in viral DNA replication. Deletions in inverted terminal repeats or in NS functions did not abolish expression of capsid polypeptides but reduced it dramatically. Encapsidation of Jlac Z recombinant genome was achieved by trans-complementation with plasmids bearing intact structural and nonstructural functions. Detection of a beta-gal activity in SPC-SL 52 cells following several subcultures post-transfection suggests that Jlac Z recombinant genome could be maintained in an integrative or episomal state.

Animals↗

Accurate processing and secretion in the baculovirus expression system of an erythroid-cell-stimulating factor consisting of a chimaera of insulin-like growth factor II and an insect insulin-like peptide.

A synthetic gene encoding the signal peptide and the N-terminal sequence of bombyxin, an insect insulin-like peptide, and the 58 amino acids of the C-terminal sequence of human insulin-like growth factor II (IGF II) has been expressed using the baculovirus system. This synthetic chimaera was obtained by amplification of four overlapping oligonucleotides using Taq polymerase and cloning into the transfer vector pBluebac. The construct was integrated by homologous recombination into the Autographa californica nuclear polyhedrosis genome. Spodoptera frugiperda Sf9 insect cells infected with the recombinant baculovirus secreted an accurately processed peptide consisting of the ten N-terminal amino acids of bombyxin and the 58 C-terminal amino acids of IGF II. The N-terminal glutamine of bombyxin was changed to asparagine to facilitate sequencing of the synthetic peptide. The chimaera was five times more potent than human recombinant IGF II in its capacity to stimulate thymidine incorporation into erythroid cells of fetal bovine liver in a serum-free medium. It stimulated erythroid colony formation in the presence of 2 microunits/ml erythropoietin in cells cultured over a monolayer of stromal cells of fetal liver. Artificial chimaeras as described here may prove useful for the production of insulin, IGF I and other peptides as secreted proteins in insect cells.

Amino Acid Sequence↗

Sex-specific organisation of middle repetitive DNA sequences in the mealybug Planococcus lilacinus.

Differential organisation of homologous chromosomes is related to both sex determination and genomic imprinting in coccid insects, the mealybugs. We report here the identification of two middle repetitive sequences that are differentially organised between the two sexes and also within the same diploid nucleus. These two sequences form a part of the male-specific nuclease-resistant chromatin (NRC) fraction of a mealybug Planococcus lilacinus. To understand the phenomenon of differential organisation we have analysed the components of NRC by cloning the DNA sequences present, deciphering their primary sequence, nucleosomal organisation, genomic distri-bution and cytological localisation. Our observations suggest that the middle repetitive sequences within NRC are functionally significant and we discuss their probable involvement in male-specific chromatin organisation.

Animals↗

Phytoplasmas: genetics, diagnosis and relationships with the plant and insect host.

Phytoplasmas cannot be cultivated in vitro, and remain the most poorly understood plant pathogens. Despite this limitation, the investigation of their nature with the aid of modern tools has produced noteworthy results during the last 20 years. Using biochemical and molecular approaches, the phylogeny of the phytoplasmas has been described, their chromosomal and extrachromosomal components are being studied, and information on the localization, movement, and metabolic interference occurring in their insect and plant hosts accumulated. At the same time, the application of the new findings in phylogeny and genetics has aided the development of powerful diagnostic tools that have improved the ability to manage diseases which are induced by phytoplasmas.

Animals↗