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PubMed1998

Identification of a broad complex-regulated enhancer in the developing visual system of Drosophila.

During metamorphosis, the central nervous system (CNS) is reconstructed through the concerted action of cell birth, death, and remodeling, so that it can serve the novel and complex behavioral needs of the adult insect. In Drosophila, Broad Complex (BRC) zinc-finger transcription factors are essential for many aspects of metamorphosis, including reorganization of the CNS. In particular, we showed previously that some mutant alleles disrupt the assembly of visual system synaptic neuropils. Using an enhancer-detector screen, we have now identified a candidate BRC target gene, H217, that is normally expressed in visual system neural precursor cells of the inner proliferative center. Moreover, the P-element insertion in the H217 line has caused a hypomorphic mutation in an essential gene, with an optic lobe disorganization phenotype very similar to that seen in BRC mutants. In BRC mutants of the br complementation group (but not in rbp or 2Bc mutants), the H217 enhancer is ectopically expressed in lamina precursor cells (LPCs) whose proliferation is regulated by signals from photoreceptor axons. As predicted by the current model of BRC structure-function relationships, we demonstrated that BRC-Z2 isoforms, when induced during the third larval instar, can repress H217 enhancer activity in the LPCs, whereas BRC-Z3 cannot. Taken together, the data suggest that the H217 P element has tagged an essential gene repressed by BRC-Z2 in LPCs and required for the normal architecture of the retinotopically connected visual system neuropils.

Animals↗
PubMed1997

Studies on the Expression of the 28 kD Glutathione S-transferase Gene from Schistosoma japonicum in the Silkworm (Bombyx mori) Larvae and Insect Cells.

The gene of the 28 kD glutathione S-transferase (GST) from the Chinese strain of Schistosoma japonicum had been expressed in the silkworm (Bombyx mori) cells and larvae by Bombyx mori nuclear polyhedrosis virus (BmNPV) vector which had been modified. The GST gene was inserted into the right position of the BmNPV genome as identified by Southern hybridization. The product was a 28 kD protein, had GST activity and antigenicity. The yield in BmN cells (1x10(6) cells/ml) was 0.77 mg and more than 5 mg in a silkworm larvae. All the results showed that it is possible to develop the GST expressed in the silkworm larvae to a new kind of genetic engineered schistosomiasis vaccine.

Journal Article↗
PubMed2006

Evolution of insect P450.

The first fully sequenced insect genomes were those of the fruitfly and the mosquito, both from the order Diptera. Now, with an increasing number and diversity of insect genomes becoming available, the diversity of insect P450 genes can be better appreciated and tentative ideas about the evolution of the CYP (cytochrome P450) superfamily in insects can be proposed. There are four large clades of insect P450 genes that existed before the divergence of the class Insecta and that are also represented by CYP families in vertebrates: the CYP2 clade, the CYP3 clade, the CYP4 clade and the mitochondrial P450 clade. P450s with known or suspected physiological functions are present in each of these clades and only a dozen genes appear to have orthologues or very close paralogues in each insect genome. P450 enzymes from each of these clades have been linked to insecticide resistance or to the metabolism of natural products and xenobiotics. In particular, insects appear to maintain a repertoire of mitochondrial P450 paralogues devoted to the response to environmental challenges.

Amino Acid Sequence↗
PubMed2004

Assembly of Marek's disease virus (MDV) capsids using recombinant baculoviruses expressing MDV capsid proteins.

The genes UL18, UL19, UL26, UL26.5, UL35 and UL38 of Marek's disease virus 1 (MDV-1) strain RB1B, encoding the homologues of herpes simplex virus type 1 (HSV-1) capsid proteins VP23, VP5, VP21-VP24, preVP22a, VP26 and VP19C, were identified and sequenced. Recombinant baculoviruses were used to express the six capsid genes in insect cells. Coexpression of the six genes or of UL18, UL19, UL26.5 and UL38 in insect cells resulted in the formation of capsids with a large core. In addition, electron microscopy of thin sections clearly revealed the presence of large numbers of small spherical particles. Experimental coinfection demonstrated that these small particles were associated with production of the preVP22a protein.

Animals↗
PubMed2006

Differential gene expression in abdomens of the malaria vector mosquito, Anopheles gambiae, after sugar feeding, blood feeding and Plasmodium berghei infection.

BACKGROUND: Large scale sequencing of cDNA libraries can provide profiles of genes expressed in an organism under defined biological and environmental circumstances. We have analyzed sequences of 4541 Expressed Sequence Tags (ESTs) from 3 different cDNA libraries created from abdomens from Plasmodium infection-susceptible adult female Anopheles gambiae. These libraries were made from sugar fed (S), rat blood fed (RB), and P. berghei-infected (IRB) mosquitoes at 30 hours after the blood meal, when most parasites would be transforming ookinetes or very early oocysts. RESULTS: The S, RB and IRB libraries contained 1727, 1145 and 1669 high quality ESTs, respectively, averaging 455 nucleotides (nt) in length. They assembled into 1975 consensus sequences--567 contigs and 1408 singletons. Functional annotation was performed to annotate probable molecular functions of the gene products and the biological processes in which they function. Genes represented at high frequency in one or more of the libraries were subjected to digital Northern analysis and results on expression of 5 verified by qRT-PCR. CONCLUSION: 13% of the 1965 ESTs showing identity to the A. gambiae genome sequence represent novel genes. These, together with untranslated regions (UTR) present on many of the ESTs, will inform further genome annotation. We have identified 23 genes encoding products likely to be involved in regulating the cellular oxidative environment and 25 insect immunity genes. We also identified 25 genes as being up or down regulated following blood feeding and/or feeding with P. berghei infected blood relative to their expression levels in sugar fed females.

Abdomen↗
PubMed1997

Characterisation of the ultraviolet-sensitive opsin gene in the honey bee, Apis mellifera.

The cDNA sequence of the ultraviolet-sensitive opsin in the honey-bee, Apis mellifera, with associated 5' and 3' untranslated regions, is presented. The analysis of genomic structure reveals seven introns in the coding region of the gene, with six at novel positions for an insect opsin gene. The equivalent site to the counterion in vertebrate opsins is occupied by a Tyr residue. This contrasts with the presence of Phe at this site in the ultraviolet-sensitive opsins of Drosophila sps. A comparison of the amino acid sequence within the seven alpha-helical transmembrane regions of insect ultraviolet/blue-sensitive opsins identifies substitution at five sites that involve either replacement of a polar with a non-polar residue, or a change in charge. Such changes are known to result in spectral shifts in vertebrate pigments. Phylogenetic analysis indicates that the ultraviolet-sensitive pigments represent an ancient class of insect opsins.

Amino Acid Sequence↗
PubMed2005

[Sequence analysis of alpha-amylase inhibitors genes with resistance to insects in wheat and Aegilops].

The alpha-amylase inhibitors have been proposed as possibly important weapons against pests. Thus, it is of importance to identify the specificity of them. Based on the EST data of alpha-amylase inhibitor genes that were retrieved from NCBI, BBSRC and GrainGenes, two PCR primers were designed. The coding sequences of 24 kD dimeric alpha-amylase inhibitors with resistance to insects in 17 wheat and Aegilops accessions were investigated and 17 new genes were obtained. Only one 24 kD alpha-amylase inhibitor gene was found in each diploid wheat and Aegilops accession, whereas 8 genes were characterized from one hexaploid wheat variety, indicating that the 24 kD alpha-amylase inhibitors in hexaploid wheat were encoded by multi-gene. The deduced amino acid sequences of 2 genes from common wheat and 1 gene from Ae. tauschii were the same as the sequence of the inhibitor 0.19, and the deduced amino acid sequence of another gene from common wheat was similar to the inhibitor 0.53 with only one amino acid difference. The amino acid sequences of 24 kD dimeric alpha-amylase inhibitors shared very high coherence (91.2%). These results suggest that the alpha-amylase inhibitors in 24 kD family were derived from common ancestral genes by phylogenesis.

Amino Acid Sequence↗
PubMed2000

Insect chitin synthase cDNA sequence, gene organization and expression.

Chitin is a major component of the cuticle of arthropods. However, the synthesis of chitin is poorly understood. Feeding larvae of the insect Lucilia cuprina on the fungal chitin synthase competitive inhibitor, nikkomycin Z resulted in strong concentration-dependent mortality of the larvae (LD50 = 280 nM). This result demonstrates that chitin is an essential component of this insect. The complete cDNA and deduced amino-acid sequences of the first arthropod chitin synthase-like protein, LcCS-1, from the larvae of the insect L. cuprina have been determined. The cDNA sequence is 5757 bp in length and codes for a large complex protein containing 1592 amino acids (Mr = 180 717). Analysis of the whole protein sequence reveals low, but significant, similarity to yeast chitin synthases with stronger areas of conservation centred on local regions implicated in the active sites of the yeast enzymes. Strikingly, LcCS-1 contains 15-18 potential transmembrane segments, indicating that the protein is an integral membrane protein. Two alternative topographical models of LcCS-1 are described, which involve its association with either the plasma membrane or the membrane of intracellular vesicles. LcCS-1 mRNA is produced in all life stages of the insect with expression in the larval stage limited to the integument and trachea. In a third instar larva the mRNA was localized to a single layer of epidermal cells immediately underlying the procuticle region of the integument. cDNA or genomic sequences that are highly related to fragments of LcCS-1 were demonstrated in three insect orders, one arachnid and Caenorhabditis elegans, thereby attesting to the importance of this enzyme in these chitin-producing organisms. Bioinformatics has been used to deduce the gene sequence and organization of the highly homologous Drosophila melanogaster orthologue of LcCS-1, DmCS-1.

Amino Acid Sequence↗
PubMed2006

Patterns of conservation and change in honey bee developmental genes.

The current insect genome sequencing projects provide an opportunity to extend studies of the evolution of developmental genes and pathways in insects. In this paper we examine the conservation and divergence of genes and developmental processes between Drosophila and the honey bee; two holometabolous insects whose lineages separated approximately 300 million years ago, by comparing the presence or absence of 308 Drosophila developmental genes in the honey bee. Through examination of the presence or absence of genes involved in conserved pathways (cell signaling, axis formation, segmentation and homeobox transcription factors), we find that the vast majority of genes are conserved. Some genes involved in these processes are, however, missing in the honey bee. We have also examined the orthology of Drosophila genes involved in processes that differ between the honey bee and Drosophila. Many of these genes are preserved in the honey bee despite the process in which they act in Drosophila being different or absent in the honey bee. Many of the missing genes in both situations appear to have arisen recently in the Drosophila lineage, have single known functions in Drosophila, and act early in developmental pathways, while those that are preserved have pleiotropic functions. An evolutionary interpretation of these data is that either genes with multiple functions in a common ancestor are more likely to be preserved in both insect lineages, or genes that are preserved throughout evolution are more likely to co-opt additional functions.

Amino Acid Sequence↗
PubMed1998

Molecular evolution of the globin gene cluster E in two distantly related midges, Chironomus pallidivittatus and C. thummi thummi.

We have studied the evolutionary dynamics of a cluster of insect globin genes by comparing the organization and sequence of the gene group in two distantly related species, Chironomus pallidivittatus and C. t. thummi. Although the general architecture of the globin gene cluster has been conserved, we have found an additional, previously undescribed gene (named Cpa F) in C. pallidivittatus which shows signs of accelerated sequence evolution at nonsynonymous codon positions. This new gene is clearly functional, as demonstrated by Northern analysis. Comparison of paralogous and orthologous genes reveals patterns of intraspecific sequence homogenization. The head-to-head-oriented globin 3 and 4 gene pairs in C. t. thummi and the gb 4 gene pair in C. pallidivittatus have been efficiently homogenized, probably by gene conversion, in their promoter and coding regions. Inverted transcriptional orientation seems to favor efficient conversion. The orthologous genes from C. t. thummi and C. pallidivittatus reveal different levels of sequence conservation, ranging from 85.3 to 94.7% amino acid identity. Surprisingly, globin gene E, for which up to now no corresponding protein has been detected in the larval hemolymph of C. t. thummi, shows the highest degree of interspecies sequence conservation. This points to an essential, as yet unknown function of this globin. The usefulness of globin gene comparisons for dating speciation events in Chironomus is discussed.

Amino Acid Sequence↗
PubMed1997

Adjacent GATA and kappa B-like motifs regulate the expression of a Drosophila immune gene.

The GATA motif is a well known positive cis -regulatory element in vertebrates. In this work we report experimental evidence for the direct participation of a GATA motif in the expression of the Drosophila antibacterial peptide gene Cecropin A1 . Previously we have shown that a kappaB-like site is necessary for Cecropin A1 gene expression. Here we present evidence that the Drosophila Rel protein which binds to the kappaB-like site requires an intact GATA site for maximal Dif-mediated transactivation of the Cecropin A1 gene. We show that a Drosophila blood cell line contains factors binding specifically to the GATA motif of the Cecropin A1 gene. The GATA binding activity is likely to include member(s) of the GATA family of transcriptional regulators. We show that the promoters of several inducible insect immune genes possess GATA sites 0-12 base pairs away from kappaB-like sites in functionally important promoter regions. Clusters of GATA and kappaB sites are also observed in the promoters of two important mammalian immune genes, namely IL6 and IL3. The consistent proximity of GATA and kappaB sites appears to be a common theme in the immune gene expression of insects and mammals.

Animals↗
PubMed2005

A dominant lethal genetic system for autocidal control of the Mediterranean fruitfly.

The Sterile Insect Technique (SIT) used to control insect pests relies on the release of large numbers of radiation-sterilized insects. Irradiation can have a negative impact on the subsequent performance of the released insects and therefore on the cost and effectiveness of a control program. This and other problems associated with current SIT programs could be overcome by the use of recombinant DNA methods and molecular genetics. Here we describe the construction of strains of the Mediterranean fruit fly (medfly) harboring a tetracycline-repressible transactivator (tTA) that causes lethality in early developmental stages of the heterozygous progeny but has little effect on the survival of the parental transgenic tTA insects. We show that these properties should prove advantageous for the implementation of insect pest control programs.

Animals↗
PubMed2002

A transgene-based, embryo-specific lethality system for insect pest management.

Biological approaches to insect pest management offer alternatives to pesticidal control. In area-wide control programs that cover entire regions, the sterile insect technique (SIT) can be used to successfully suppress economically important pest species by the mass release of sterilized pest organisms. However, conventional sterilization by ionizing radiation reduces insect fitness, which can result in reduced competitiveness of the sterilized insects. Here we report a transgene-based, dominant embryonic lethality system that allows for generation of large quantities of competitive but sterile insects without the need of irradiation. The system involves the ectopic expression of a hyperactive pro-apoptotic gene that causes embryo-specific lethality when driven by the tetracycline-controlled transactivator (tTA) under the regulation of a cellularization gene enhancer-promoter. We have successfully tested this system in Drosophila melanogaster. The embryonic lethality can be suppressed maternally, which will allow it to be combined with transgenic female-specific lethality systems to raise only vigorous but sterile males.

Animals↗
PubMed1998

Plasmid-mediated gene transfer between insect-resident bacteria, Enterobacter cloacae, and plant-epiphytic bacteria, Erwinia herbicola, in guts of silkworm larvae.

Five strains of Enterobacter cloacae isolated from several species of plants and insects were able to grow in the guts of silkworm larvae. A much larger population of Ent. cloacae strains was detected in the insect guts and feces collected 3 and 6 days than in samples collected 1 day after feeding artificial diets contaminating these bacteria. Furthermore, insect-origin strains of Ent. cloacae were mated with a donor strain, epiphytic Erwinia herbicola, harboring RSF1010 and pBPW1::Tn7 plasmids in the insect guts by introducing these bacteria through separate artificial diets administered at different times. A number of transconjugants, Ent. cloacae strains which had acquired RSF1010 plasmid, were detected from guts and fecal samples at transfer frequencies of 10(-2) to 10(-3) per recipient. Thus, gene transfer between epiphytic Er. herbicola and insect-resident Ent. cloacae strains in the insect guts was confirmed. These findings may provide significant information about the role of "in insecta mating" in the evolution of these bacteria.

Animals↗
PubMed2002

Critical evaluation of the role of the Toll-like receptor 18-Wheeler in the host defense of Drosophila.

Essential aspects of innate immune responses to microbial infections appear to be conserved between insects and mammals. In particular, in both groups, transmembrane receptors of the Toll superfamily play a crucial role in activating immune defenses. The Drosophila Toll family member 18-Wheeler had been proposed to sense Gram-negative infection and direct selective expression of peptides active against Gram-negative bacteria. Here we re-examine the role of 18-Wheeler and show that in adults it is dispensable for immune responses. In larvae, 18wheeler is required for normal fat body development, and in mutant larvae induction of all antimicrobial peptide genes, and not only of those directed against Gram-negative bacteria, is compromised. 18-Wheeler does not qualify as a pattern recognition receptor of Gram-negative bacteria.

Animals↗
PubMed1993

An amplified mosquito dihydrofolate reductase gene: amplicon size and chromosomal distribution.

The dihydrofolate reductase amplicon in methotrexate-resistant mosquito cells provides an amplified gene in insects that can be compared directly to the corresponding amplified locus in mammalian cells. A cloned Aedes albopictus dihydrofolate reductase gene was used as a probe to examine the structure of dihydrofolate reductase alleles in sensitive and resistant cells. In wild type cells, two distinct alleles could be distinguished by restriction fragment length polymorphisms, one of which was amplified in methotrexate-resistant cells. Subsequent to amplification, an additional polymorphism at a ten base-pair XmnI recognition site indicated that the amplified mosquito gene is subject to genetic changes similar to those that have been described in amplified genes from mammalian cells. Pulsed-field gel electrophoresis was used to determine that the minimum size of the mosquito dihydrofolate reductase amplicon was 140 kilobases; ethidium bromide staining patterns suggested a size of at least 233 kilobases. Dihydrofolate reductase probes hybridized to distinct locations in five of the thirteen chromosomes in Mtx-5011-128 cells, indicating that the amplified DNA probably occurs as tandem direct or inverted repeats.

Aedes↗
PubMed2002

Dominant lethality and insect population control.

Recent advances in insect genetic engineering have opened up new possibilities in the genetic control of insect vectors of human diseases. We outline the current state of the sterile insect technique and show how the use of engineered dominant lethals can greatly increase the effectiveness of this approach. We consider alternative strategies based on the use of conditional dominant lethals that are not always active in the environment.

Animals↗