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PubMed1995

DNA probes for two Microsporidia, Nosema bombycis and Nosema costelytrae.

Two DNA fragments which hybridize specifically with DNA of Nosema bombycis and Nosema costelytrae, respectively, were obtained from genomic DNA of each microsporidian species and sequenced. Neither fragment hybridized with genomic DNA from four other microsporidian isolates tested: Nosema apis, Vairimorpha sp. from cabbage white butterfly (Pieris rapae), and two isolates of Vavraia oncoperae, one from New Zealand grass grubs, Costelytrae zealandica, and another from porina caterpillars, Wiseana spp. The probe for N. bombycis did not hybridize with genomic DNA from N. costelytrae or with DNA from silkworms (Bombyx mori), the primary insect host of this microsporidium. Likewise, the probe for N. costelytrae did not hybridize with genomic DNA from N. bombycis or with DNA from grass grubs (C. zealandica). Both fragments were AT-rich (59 and 79% of total bases, respectively), had G+C/A+T ratios of 0.70 and 0.25, respectively, and represented repeated sequences dispersed throughout the genome.

Animals↗

PubMed2006

Unfolding the evolutionary story of polydnaviruses.

Polydnaviruses (PDVs) are fascinating viruses. Described in thousands of parasitoid wasp species they are unique viruses having both a segmented DNA genome in viral particles and an integrated form that persists as a provirus in the wasp genome. Parasitoid wasps inject their eggs in another insect host typically a lepidopteran. In these host-parasitoid interactions, the virus particles are co-injected along with the eggs and are essential to ensure wasp parasitism success. PDVs do not replicate in the lepidopteran host, but expression of viral gene products confers protection from the host immune defence response. Two genera of PDVs phylogenetically unrelated exist, the bracoviruses (BVs) and the ichnoviruses (IVs), associated with braconid and ichneumonid wasps, respectively. New data on the genomes of two bracoviruses (Microplitis demolitor BV and Cotesia congregata BV) and an ichnovirus associated with Campoletis sonorensis (CsIV) offers us new elements to discuss the central questions concerning the origin of these viral entities and how they have evolved. The results of sequencing approaches indicate that the tens of millions of years of mutualistic associations between PDVs and wasps have had a strong impact on PDV genomes that now ressemble eukaryotic regions both in organization and gene content.

Animals↗

PubMed2000

Evidence that a family of miniature inverted-repeat transposable elements (MITEs) from the Arabidopsis thaliana genome has arisen from a pogo-like DNA transposon.

Sequence similarities exist between terminal inverted repeats (TIRs) of some miniature inverted-repeat transposable element (MITE) families isolated from a wide range of organisms, including plants, insects, and humans, and TIRs of DNA transposons from the pogo family. We present here evidence that one of these MITE families, previously described for Arabidopsis thaliana, is derived from a larger element encoding a putative transposase. We have named this novel class II transposon Lemi1. We show that its putative product is related to transposases of the Tc1/mariner superfamily, being closer to the pogo family. A similar truncated element was found in a tomato DNA sequence, indicating an ancient origin and/or horizontal transfer for this family of elements. These results are reminiscent of those recently reported for the human genome, where other members of the pogo family, named Tiggers, are believed to be responsible for the generation of abundant MITE-like elements in an early primate ancestor. These results further suggest that some MITE families, which are highly reiterated in plant, insect, and human genomes, could have arisen from a similar mechanism, implicating pogo-like elements.

Amino Acid Sequence↗

PubMed2003

Recent horizontal transfer of mellifera subfamily mariner transposons into insect lineages representing four different orders shows that selection acts only during horizontal transfer.

We report the isolation and sequencing of genomic copies of mariner transposons involved in recent horizontal transfers into the genomes of the European earwig, Forficula auricularia; the European honey bee, Apis mellifera; the Mediterranean fruit fly, Ceratitis capitata; and a blister beetle, Epicauta funebris, insects from four different orders. These elements are in the mellifera subfamily and are the second documented example of full-length mariner elements involved in this kind of phenomenon. We applied maximum likelihood methods to the coding sequences and determined that the copies in each genome were evolving neutrally, whereas reconstructed ancestral coding sequences appeared to be under selection, which strengthens our previous hypothesis that the primary selective constraint on mariner sequence evolution is the act of horizontal transfer between genomes.

Amino Acid Sequence↗

PubMed1998

Expression of human interleukin 12 (hIL-12) in insect cells.

Human interleukin-12(hIL-12) is a herterodimer cytokine, which consists of two disulfide-linked subunits, p40 and p35. This paper reports the expression of hIL-12 using the Baculovirus Expression System in insect cells. First, we constructed two expression vectors pVL1392-hp40 and pVL1393-hp35, and then they were used to co-transfect the insect cells (Sf9) separately with linearized polyhedrosis virus genomic DNA. Two kinds of recombinant viruses AcNPV-hp40 and AcNPV-hp35 were visually screened out. Biological activity of the recombinant hIL-12 (rhIL-12) was detected in the conditioned medium using proliferation assay of PHA-activated human lymphocytes and the expression level was about 1.5 approximately 2 microg/10(6) cells. The results of real-time Biomolecular Interaction Analysis (BIA) and Northern blot demonstrated that the subunits of rhIL-12, hp35, and hp40 were expressed successfully in the insect cells. The apparent molecular weights of rhIL-12 and hp40 homodimer were 76 kDa and 92 kDa under non-reducing conditions of Western blot, respectively. The recombinant hp40 can significantly inhibit the biological activity of hIL-12.

Animals↗

PubMed2003

Functional genomic analysis of the 61D-61F region of the third chromosome of Drosophila melanogaster.

Assigning functional significance to completed genome sequences is one of the next challenges in biological science. Conventional genetic tools such as deficiency chromosomes help assign essential complementation groups to their corresponding genes. We describe an F2 genetic screen to identify lethal mutations within cytogenetic region 61D-61F of the third chromosome of Drosophila melanogaster. One hundred sixteen mutations were identified by their failure to complement both Df(3L)bab-PG and Df(3L)3C7. These alleles were assigned to 14 complementation groups and 9 deficiency intervals. Complementation groups were ordered using existing deficiencies, as well as new deficiencies generated in this study. With the aid of the genomic sequence, genetic and physical maps in the region were correlated by use of PCR to localize the breakpoints of deficiencies within a 268-kb genomic contig (GenBank accession No. AC005847). Six essential complementation groups were assigned to specific genes, including genes encoding a porphobilinogen deaminase and a Sac1-like protein.

Alleles↗

PubMed1994

Overexpression of human DNA topoisomerase I in insect cells using a baculovirus vector.

The 3645-bp human DNA topoisomerase I cDNA isolated by D'Arpa et al. (Proc. Natl. Acad. Sci. USA 85, 1988, 2543-2547) was integrated into the Autographa californica multiple nuclear polyhedrosis virus genome. The recombinant protein was expressed by infecting the SF9 insect cell line with this baculovirus and resulted in a 100-fold overexpression of human DNA topoisomerase I compared to the level found in human cell lines. This 100-kDa recombinant protein has the same electrophoretic mobility as the human DNA topoisomerase I from HeLa cells and is recognized by topoisomerase I-specific monoclonal antibody. The recombinant DNA topoisomerase I was isolated and purified to homogeneity with a two-step fractionation protocol and has a specific activity of 2 x 10(6) U/mg. Enzymatic properties such as stimulation by magnesium and inhibition by camptothecin resemble properties of the enzyme purified from human cell lines.

Animals↗

PubMed1995

Molecular cloning of mei-41, a gene that influences both somatic and germline chromosome metabolism of Drosophila melanogaster.

The mei-41 gene of Drosophila melanogaster plays an essential role in meiosis, in the maintenance of somatic chromosome stability, in postreplication repair and in DNA double-strand break repair. This gene has been cytogenetically localized to polytene chromosome bands 14C4-6 using available chromosomal aberrations. About 60 kb of DNA sequence has been isolated following a bidirectional chromosomal walk that extends over the cytogenetic interval 14C1-6. The breakpoints of chromosomal aberrations identified within that walk establish that the entire mei-41 gene has been cloned. Two independently derived mei-41 mutants have been shown to carry P insertions within a single 2.2 kb fragment of the walk. Since revertants of those mutants have lost the P element sequences, an essential region of the mei-41 gene is present in that fragment. A 10.5 kb genomic fragment that spans the P insertion sites has been found to restore methyl methanesulfonate resistance and female fertility of the mei-41D3 mutants. The results demonstrate that all the sequences required for the proper expression of the mei-41 gene are present on this genomic fragment. This study provides the foundation for molecular analysis of a function that is essential for chromosome stability in both the germline and somatic cells.

Animals↗

PubMed2002

Expression and functional characterization of a nucleotide sugar transporter from Drosophila melanogaster: relevance to protein glycosylation in insect cell expression systems.

Insect cells are used routinely to express recombinant mammalian glycoproteins. However, insect protein glycosylation pathways are not well understood and appear to differ from those of mammalian cells. One way to more clearly evaluate the protein glycosylation potential of insect cells is to use the Drosophila melanogaster genome to identify genes that might encode relevant functions. These genes can then be expressed and the functions of the gene products directly evaluated by biochemical assays. In this study, we used this approach to determine the function of a putative Drosophila nucleotide sugar transporter gene. The results showed that this gene encodes a protein that can transport UDP-galactose, but not CMP-sialic acid. Thus, Drosophila encodes at least some of the infrastructure needed to produce glycoproteins with complex glycans, but this particular gene product does not directly support glycoprotein sialylation. These findings are relevant to insect cell biology and to an informed consideration of insect cell expression systems as tools for recombinant glycoprotein production.

Amino Acid Sequence↗

PubMed1999

The nucleotide sequence of sacbrood virus of the honey bee: an insect picorna-like virus.

We have determined the nucleotide sequence of sacbrood virus (SBV), which causes a fatal infection of honey bee larvae. The genomic RNA of SBV is longer than that of typical mammalian picornaviruses (8832 nucleotides) and contains a single, large open reading frame (179-8752) encoding a polyprotein of 2858 amino acids. Sequence comparison with other virus polyproteins revealed regions of similarity to characterized helicase, protease and RNA-dependent RNA polymerase domains; structural genes were located at the 5' terminus with non-structural genes at the 3' end. Picornavirus-like agents of insects have two distinct genomic organizations; some resemble mammalian picornaviruses with structural genes at the 5' end and non-structural genes at the 3' end, and others resemble caliciviruses in which this order is reversed; SBV thus belongs to the former type. Sequence comparison suggested that SBV is distantly related to infectious flacherie virus (IFV) of the silk worm, which possesses an RNA of similar size and gene order.

Amino Acid Sequence↗

PubMed1985

Production of human c-myc protein in insect cells infected with a baculovirus expression vector.

A cDNA fragment coding for human c-myc was inserted into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedrin promoter. Insect cells infected with the recombinant virus produced significant amounts of c-myc protein, which constituted the major phosphoprotein component in these cells. By immunoprecipitation and immunoblot analysis, two proteins of 61 and 64 kilodaltons were detected with c-myc-specific antisera. The insect-derived proteins were compared with recombinant human c-myc-encoded proteins synthesized in Escherichia coli and Saccharomyces cerevisiae cells. The c-myc gene product was found predominantly in the nucleus by subcellular fractionation of infected insect cells.

Animals↗

PubMed1997

Assessment of the universality and utility of a set of conserved mitochondrial COI primers in insects.

A set of mitochondrial COI primers has been studied by genomic PCR and many primer combinations shown to work universally well across Insecta. They are able to amplify various amplicons with different variability which enables the selection of a particular amplicon as a suitable DNA marker for a project. The potential usefulness of different amplicons is examined, with analysis on published study cases employing these regions. With respect to their variability, amplicons UEA5/UEA6, UEA7/UEA8 and UEA5/UEA8 could be useful for low- to mid-level phylogenetic analysis, i.e. from species, genus to perhaps family level depending on taxa involved. UEA5/UEA6 will be too conserved for intraspecific studies. Amplicons UEA3/UEA4 and UEA9/UEA10 would be better suited to low-level phylogenetic investigations, such a analysis of relationships among closely related species and population genetic studies. However, these guidelines should not be over-generalized for the reasons given. Amplification conditions of various primer combinations, and general problems in the use of conserved PCR primers are discussed.

Conserved Sequence↗

PubMed2006

Sodium channel gene expression associated with pyrethroid resistant house flies and German cockroaches.

The voltage-gated sodium channels of the insect nervous system are the primary target of DDT and pyrethroid insecticides. The loss of target site sensitivity to insecticides resulting from a substitution of leucine to phenylalanine, termed the L-to-F kdr mutation, in the sodium channel of the insect nervous system is known to be important in insecticide resistance. Yet, little is known about the molecular basis underlying the genotype and kdr-mediated resistance phenotype relationship. Here we report a systematic study of resistance-associated kdr allelic expression within and among resistant and susceptible house fly and German cockroach populations. We compared genomic DNA and RNA sequences within the same individuals from different insect strains, finding no correlation for the kdr allele at the genomic DNA level with levels of susceptibility or resistance to insecticide. However, there was a strong correlation between kdr allele expression and the levels of insecticide resistance. This correlation is probably regulated through RNA variation and RNA editing. These results suggest a role for posttranscriptional regulation in the connection of the sodium channel genotype and its mutation-mediated resistance phenotype.

Alleles↗

PubMed2005

Site-specific antiphagocytic function of the Photorhabdus luminescens type III secretion system during insect colonization.

Photorhabdus is an entomopathogenic bacterium belonging to the Enterobacteriaceae. The genome of the TT01 strain of Photorhabdus luminescens was recently sequenced and a large number of toxin-encoding genes were found. Genomic analysis predicted the presence on the chromosome of genes encoding a type three secretion system (TTSS), the main role of which is the delivery of effector proteins directly into eukaryotic host cells. We report here the functional characterization of the TTSS. The locus identified encodes the secretion/translocation apparatus, gene expression regulators and an effector protein - LopT - homologous to the Yersinia cysteine protease cytotoxin YopT. Heterologous expression in Yersinia demonstrated that LopT was translocated into mammal cells in an active form, as shown by the appearance of a form of the RhoA GTPase with modified electrophoretic mobility. In vitro study showed that recombinant LopT was able to release RhoA and Rac from human and insect cell membrane. In vivo assays of infection of the cutworm Spodoptera littoralis and the locust Locusta migratoria with a TT01 strain carrying a translational fusion of the lopT gene with the gfp reporter gene revealed that the lopT gene was switched on only at sites of cellular defence reactions, such as nodulation, in insects. TTSS-mutant did not induce nodule formation and underwent phagocytosis by insect macrophage cells, suggesting that the LopT effector plays an essential role in preventing phagocytosis and indicating an unexpected link between TTSS expression and the nodule reaction in insects.

Amino Acid Sequence↗

PubMed2025

Tales of a Super Butterfly: Is Vanessa carye a Truly Migrant Species? Unraveling Migration Using Morphological and Genomics Approaches.

Among movement strategies, migratory behavior is particularly intriguing in insects. Home-breeding is often permanent, and return journeys can take several generations. Although migration is crucial to the ecological and evolutionary processes of the species involved, knowledge of insect migratory behavior needs to be better understood. Vanessa carye, a butterfly native to South America with a latitudinal range of ∼7,000 km, exemplifies this problem. This study analyzed samples collected across the species' range using single-nucleotide polymorphisms (SNPs) to assess population structure, genetic diversity, and geometric morphometrics to examine wing shape variation. Results indicate that V. carye forms a genetically homogeneous unit composed of only two potential populations spanning ∼5,000 km, geographically correlated with the Pacific Ocean and the Andes, maintaining constant gene flow, and with a mean heterozygosity of 5.74% (SE: ±0.048%). Geometric morphometrics detected no geographic differentiation in wing shapes and sizes across ∼7,000 km, suggesting an absence of local adaptation and indicating a conserved wing shape adapted to flight throughout the species' range. Our findings support V. carye as a migratory species with the longest migratory journey among American butterflies, revealing two migratory routes. With these approaches, we provide a consistent methodological framework for migratory studies in species with important gaps in knowledge of their natural history.

Animals↗

PubMed1992

Expression of the amino-terminal half of the NS1 region of the hepatitis C virus genome and detection of an antibody to the expressed protein in patients with liver diseases.

A cDNA fragment encompassing the 5'-terminal half of the NS1 region of the hepatitis C virus (HCV) genome was cloned. The cDNA was expressed in insect cells using a recombinant baculovirus, and a protein band of approximately 21K was identified by immunoblotting with a serum sample from a patient with chronic hepatitis C. Antibody to the protein was detected in sera from 13.4% of patients with chronic non-A, non-B hepatitis (NANBH), 20.8% of patients with liver cirrhosis and 16.8% of patients with hepatocellular carcinoma with no serum markers for hepatitis B virus infection. However, the antibody was not detected in sera from patients with acute NANBH. The prevalence of antibody to the protein encoded by the NS1 region was lower than that of antibody to the HCV core protein, but much higher than that of antibody to the envelope protein. Thus, the NS1 region of the HCV genome is suggested to encode a protein produced during the course of HCV replication.

Animals↗

PubMed2006

A recombinant truncated Cry1Ca protein is toxic to lepidopteran insects and forms large cuboidal crystals in insect cells.

A truncated version of the cry1Ca gene from Bacillus thuringiensis was introduced into the genome of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) under the control of two promoters. A recombinant virus (vSyncry1c) was isolated and used to infect insect cells in culture and insect larvae. Structural and ultrastructural analysis of insects infected with vSyncry1C showed the formation of large cuboidal crystals inside the cytoplasm of insect cells in culture and in insect cadavers late in infection. Infected insect cell extracts were analyzed by SDS-PAGE and Western blot and showed the presence of a 65-kDa polypeptide probably corresponding to the protease processed form of the toxin. Bioassays using purified recombinant toxin crystals showed a CL(50) of 19.49 ng/ml for 2(nd) instar A. gemmatalis larvae and 114.1 ng/ml for S. frugiperda.

Animals↗

PubMed2002

Isolation and molecular characterization of Musca domestica delta-9 desaturase sequences.

We have isolated fatty acyl-CoA desaturase cDNA (Mdomd9) and genomic sequences from the housefly, Musca domestica. Two approximately 1.66 kb cDNAs were recovered. They had identical coding regions and 3' untranslated regions (UTRs), but differed in their 5' UTRs. The open reading frame encodes a 380 amino acid (aa) protein with 82% identity to Drosophila melanogaster desat1, and significant (> 50%) identity with other insect delta-9 desaturases. Functional analyses in a yeast expression system confirmed the cDNA encodes a delta9 desaturase. Northern analysis indicated two transcripts of 1.7 and 2.9 kb that hybridized specifically to the open reading frame. PCR amplification of genomic templates revealed three intron sites that are conserved among other insect species. Southern analysis of genomic DNA indicated at least two desaturase gene copies per haploid genome. There is a high degree of polymorphism, most of which appears to be due to variable intron sequences; curiously, individual flies had varying morphs of intron II and intron III. Together, the data suggest that there are more delta9 desaturase alleles within the population studied than there are loci within the genome, and support other studies suggesting that insect fatty acyl-CoA desaturases are a dynamically evolving gene family.

Amino Acid Sequence↗