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At least 829 records · Page 46Linked to original sources
PubMed1990

Expression of cauliflower mosaic virus gene I in insect cells using a novel polyhedrin-based baculovirus expression vector.

An improved polyhedrin-based baculovirus expression vector was constructed to expedite distinguishing infections by putative baculovirus recombinants from infections by wild-type (wt) baculovirus. The vector utilizes the Escherichia coli beta-galactosidase gene (lacZ) as a genetic marker for positive recombination between wt Autographa californica nuclear polyhedrosis virus and the baculovirus transfer vector. The marker gene/expression cassette was constructed so that lacZ and the deleted polyhedrin gene were transcribed in opposite orientations, both terminating in a simian virus 40 DNA fragment which acts as a bidirectional terminator. In the constructed vector, lacZ is transcribed from the Drosophila melanogaster heat-shock promoter (hsp 70), which is constitutively expressed in baculovirus-infected Spodoptera frugiperda (Sf) cells, thereby making the site of the deleted polyhedrin gene available for the insertion and expression of foreign genes under the control of the polyhedrin promoter. Recombinant baculoviruses are readily selected in plaque assays by the development of a blue colour upon the addition of X-Gal. The colour selection renders the retrieval of recombinants less dependent on a high frequency of recombination between the transfer vector and wt baculovirus DNA. The usefulness of this new vector was illustrated by expressing gene I of cauliflower mosaic virus, which encodes a protein of Mr 46,000. Expression of gene I was at the same level as in cells infected with a conventional polyhedrin-based expression vector. Gene I protein formed large hollow fibre-like structures in the cytoplasm of infected Sf cells. This is the first plant virus protein to be expressed in insect cells by a recombinant baculovirus.

Animals↗
PubMed1999

Homeotic genes and the arthropod head: expression patterns of the labial, proboscipedia, and Deformed genes in crustaceans and insects.

cDNA fragments of the homologues of the Drosophila head homeotic genes labial (lab), proboscipedia (pb), and Deformed (Dfd) have been isolated from the crustacean Porcellio scaber. Because the accumulation domains of the head homeotic complex (Hox) genes had not been previously reported for crustaceans, we studied the expression patterns of these genes in P. scaber embryos by using in situ hybridization. The P. scaber lab homologue is expressed in the developing second antennal segment and its appendages. This expression domain in crustaceans and in the homologous intercalary segment of insects suggests that the lab gene specified this metamere in the last common ancestor of these two groups. The expression domain of the P. scaber pb gene is in the posterior part of the second antennal segment. This domain, in contrast to that in insects, is colinear with the domains of other head genes in P. scaber, and it differs from the insect pb gene expression domain in the posterior mouthparts, suggesting that the insect and crustacean patterns evolved independently from a broader ancestral domain similar to that found in modern chelicerates. P. scaber Dfd is expressed in the mandibular segment and paragnaths (a pair of ventral mouthpart structures associated with the stomodeum) and differs from insects, where expression is in the mandibular and maxillary segments. Thus, like pb, Dfd shows a divergent Hox gene deployment. We conclude that homologous structures of the mandibulate head display striking differences in their underlying developmental programs related to Hox gene expression.

Amino Acid Sequence↗
PubMed1988

Synthesis of a gene coding for an insect-specific scorpion neurotoxin and attempts to express it using baculovirus vectors.

We have explored the possibility of improving baculovirus pesticides by incorporating an insect-specific neurotoxin gene into a baculovirus genome. A 112-bp gene (BeIt) encoding insectotoxin-1 of the scorpion Buthus eupeus was synthesized and cloned in Escherichia coli. For expression, BeIt was transferred to the DNA genome of Autographa californica nuclear polyhedrosis virus (AcMNPV). Three different recombinant AcMNPVs, carrying BeIt under the control of the strong AcMNPV polyhedrin promoter, were constructed and expression of BeIt was monitored upon infection of Spodoptera frugiperda (Sf) cells. Toxin expression was low using a recombinant virus in which BeIt was inserted 6 nucleotides (nt) downstream from the intact polyhedrin mRNA leader. More expression was observed when a signal-peptide was attached in-frame to the N terminus of BeIt. The highest level of expression was observed with a fusion gene comprised of the 58 N-terminal codons of polyhedrin fused to BeIt; however, the level of expression was ten- to twenty-fold below that for polyhedrin. Polyhedrin promoter-directed transcripts of all three recombinants accumulated to levels similar to those of wild-type polyhedrin transcripts, indicating that the limitation to expression of unfused BeIt was not at the level of transcription but rather at the posttranscriptional level including translation or protein stability. Paralytic activity of the toxin products was not detected.

Amino Acid Sequence↗
PubMed1999

RNA editing-like phenomenon in paramyxovirus V gene mRNA observed in insect cells infected with a recombinant baculovirus.

The V gene of the paramyxovirus human parainfluenza virus type 2 (hPIV2) is transcribed into both V and P mRNA. The V mRNA is a faithful transcript of the V gene; however, the P mRNA is transcribed by an RNA-editing mechanism in hPIV2-infected mammalian cells. Recombinant baculoviruses (rBV) were constructed containing the wild-type V gene, which has seven G residues at its editing site, and a manipulated V gene with ten G residues at its editing site. A small amount of the P protein was synthesized, in addition to the V protein, when the wild-type V gene was expressed in rBV-infected insect cells. Furthermore, synthesis of the P protein increased when rBV containing the manipulated V gene was used to infect insect cells. Both the P and V proteins were detected after in vitro translation of mRNA from rBV-infected cells. Moreover, G-residue insertions and a deletion were detected in mRNA. Since the P protein was not detected after in vitro translation of V RNA that had been transcribed in vitro by T7 RNA polymerase, these results suggest that the non-encoded G residues were inserted and deleted during transcription in insect cells. This RNA editing-like phenomenon and the implications of the length of the G cluster are discussed.

Animals↗
PubMed1990

Characterization of the transcript for a depressant insect selective neurotoxin gene with an isolated cDNA clone from the scorpion Buthotus judaicus.

The poly(A)+ mRNA isolated from the venomous terminal segments of the scorpion Buthotus judaicus was reversed transcribed into cDNA. PCR amplification of the cDNA in presence of oligonucleotide primers prepared on basis of the known amino acid sequence of the depressant insect toxin II yielded a 125 bp long product. This fragment was cloned and its sequence determined. The deduced amino acid sequence has revealed a complete homology with the amino acid sequence of the toxin. This clone was used to probe a Northern blot resolving the poly(A)+ and poly(A)- fractions derived from the scorpion. An organ specific 360 nucleotide transcript which might be the processed product of a approximately 4.0 kb precursor was elucidated. This cDNA clone may pave the way for a molecular genetic approach to study the structure-function relationship of scorpion selective insect toxins.

Amino Acid Sequence↗
PubMed2004

Gene-for-gene disease resistance: bridging insect pest and pathogen defense.

Active plant defense, also known as gene-for-gene resistance, is triggered when a plant resistance (R) gene recognizes the intrusion of a specific insect pest or pathogen. Activation of plant defense includes an array of physiological and transcriptional reprogramming. During the past decade, a large number of plant R genes that confer resistance to diverse group of pathogens have been cloned from a number of plant species. Based on predicted protein structures, these genes are classified into a small number of groups, indicating that structurally related R genes recognize phylogenetically distinct pathogens. An extreme example is the tomato Mi-1 gene, which confers resistance to potato aphid (Macrosiphum euphorbiae), whitefly (Bemisia tabaci), and root-knot nematodes (Meloidogyne spp.). While Mi-1 remains the only cloned insect R gene, there is evidence that gene-for-gene type of plant defense against piercing-sucking insects exists in a number of plant species.

Animals↗
PubMed2000

A maize sesquiterpene cyclase gene induced by insect herbivory and volicitin: characterization of wild-type and mutant alleles.

Plants can defend themselves from herbivorous insects by emitting volatile chemical signals that attract natural enemies of the herbivore. For example, maize seedlings attacked by beet armyworm larvae (Spodoptera exigua) produce a mixture of terpenoid and indole volatiles that serve to attract parasitic wasps. A key step in terpenoid biosynthesis is the conversion of acyclic prenyl diphosphates to terpenoid compounds by specific terpenoid synthases (cyclases). We have cloned a maize sesquiterpene cyclase gene, stc1, by transposon tagging and have identified two deletion mutations of the gene. The stc1 gene is located on chromosome 9S and does not seem to have a closely related ortholog in the maize genome. It is induced 15- to 30-fold in maize leaves by beet armyworm larvae feeding or by application of purified volicitin, the insect-derived elicitor, at a mechanically wounded site. stc1 induction is systemic, because undamaged leaves of the same plant show a similar increase in stc1 transcription. Analysis of volatiles from volicitin-treated seedlings revealed that a major naphthalene-based sesquiterpene was present in wild-type seedlings but absent in the Ac-insertion and x-ray-deletion mutants. Therefore, we have identified a maize gene that responds to caterpillar herbivory by producing a chemical defense signal that most likely serves to attract natural enemies of the herbivore.

Alleles↗
PubMed2005

Synergistic action of E74B and ecdysteroid receptor in activating a 20-hydroxyecdysone effector gene.

A number of insect effector genes activated by the steroid hormone 20-hydroxyecdysone (20E) are dually controlled by the ecdysteroid receptor (EcR/USP) and products of ecdysteroid early responsive genes (E74, E75, and Broad). However, the molecular mechanism of this dual action is poorly understood. Here we examined transcriptional activation of the vitellogenin (Vg) gene in the yellow fever mosquito, Aedes aegypti, by EcR/USP and E74 in response to an elevation of 20E titers. There are two isoforms of the Aedes E74 gene, AaE74A and AaE74B, which have a common C-terminal Ets DNA-binding domain and isoform-specific N termini in the female mosquito. Inhibiting expression of AaE74B but not AaE74A by RNA interference led to substantial reduction in the Vg gene expression. AaE74B and the ecdysteroid receptor synergistically enhanced 20E-induced transcription of the Vg promoter. This action required the E74-binding sites and the ecdysone response elements in the Vg 5' regulatory region. Two-hybrid assays and coimmunoprecipitation analyses demonstrated direct interaction between AaE74B and AaEcR/AaUSP. Moreover, disruption of this interaction by a dominant negative E74 mutant abolished the enhanced activation of Vg. Therefore, the cooperative interaction between AaE74B and the ecdysteroid receptor is required for high-level expression of the Vg gene in vivo. The synergistic activation is accomplished through their 20E-dependent protein-protein interaction on the gene promoter. This study reveals how the 20E direct-indirect regulation of an effector gene is achieved at the molecular level.

Aedes↗
PubMed1990

Geminivirus coat protein gene replacement alters insect specificity.

Chimeric clones have been constructed in which the coat protein encoded by DNA A of the bipartite genome of the geminivirus African cassava mosaic virus (ACMV) has been replaced by that of beet curly top virus (BCTV). Constructs containing the coding region inserted in either orientation were infectious when co-inoculated with ACMV DNA B onto Nicotiana benthamiana, producing symptoms typical of ACMV infection. The onset of symptom production was delayed relative to plants inoculated with parental ACMV clones and remission of symptoms was observed. When inserted in the correct orientation for expression from the ACMV coat protein promoter, the BCTV gene was expressed in plants and the coat protein synthesized encapsidated ssDNA of both ACMV genomic components. The BCTV leafhopper vector, Circulifer tenellus (Baker), transmitted both BCTV and the chimeric virus but not ACMV when injected with virus preparations and transferred to N. benthamiana seedlings. The results show that the specificity of leafhopper transmission from insect to plant resides with the coat protein.

Animals↗
PubMed1993

Identification of two expressed flagellin genes in the insect pathogen Bacillus thuringiensis subsp. alesti.

Flagellin from Bacillus thuringiensis subspecies alesti strain Bt75 was isolated both from the culture medium and from flagella. Two protein forms with molecular masses close to 32 kDa were obtained from flagella; one form was identical to the flagellin purified from the culture medium. The N-terminal amino acid sequences were identical for both forms. Two genes coding for flagellin have been identified in B. thuringiensis subsp. alesti. The flaB gene was cloned and sequenced in its entire length. The clone containing the flaA gene was incomplete. Both genes were expressed in the mid-exponential growth phase. The flaB gene was flanked by long (355 bp) direct repeats protruding into the coding region in both the N- and C-terminal parts of the gene. DNA sequences related to the flaB gene were found in most other B. thuringiensis subspecies, and in two of them, subsp. kurstaki and subsp. entomocidus, such sequences were present in multiple copies.

Amino Acid Sequence↗
PubMed2006

Alternative starts of transcription, several paralogues, and almost-fixed interspecific differences of the gene fruitless in a hemimetabolous insect.

In fruit flies, the gene fruitless (fru) governs the establishment of the potential for male sexual behavior. We partially cloned fru from a hemimetabolous insect for the first time and we compared fru among three closely related and acoustically communicating grasshopper species: Chorthippus biguttulus, C. brunneus, and C. mollis. The fru of grasshoppers is organized similarly to fru of holometabolous insects, with a BTB and Zn-finger domains separated by a nonconserved repetitive linker. As in Drosophila, several transcripts of fru are found in grasshoppers. We also present evidence for the coexistence of several copies of fru in the grasshopper genome. Within species these copies are almost identical and carry almost-fixed species-specific differences. This suggests that the paralogous copies of fru in grasshoppers do not evolve independently from each other.

Amino Acid Sequence↗
PubMed2000

Tagging bloodmeals with phagemids allows feeding of multiple-sample arrays to single cages of mosquitoes (Diptera: Culicidae) and the recovery of single recombinant antibody fragment genes from individual insects.

A recombinant single-chain variable-region human antibody fragment (scFv) was expressed in Escherichia coli, extracted in hypertonic sucrose, mixed directly with blood and fed to Anopheles gambiae Giles mosquitoes. When E. coli containing the phagemids that encode these scFv were included in bloodmeals, phagemids could be recovered from the mosquito midgut for up to 3 d after feeding. Furthermore, large arrays of such gene-tagged scFv-containing bloodmeals could be fed to cages of mosquitoes using microtiter plates. Arrays of phagemids with and without an antibody insert were fed to single cages of mosquitoes to test whether individual mosquitoes fed from single wells of such arrays. Phagemids were recovered from 95% of blood-fed females and > 80% of these phagemids were monoclonal. Therefore, it is possible to feed multiple sample arrays of recombinant proteins to single cages of mosquitoes and to recover the genetic material that encodes for only one of the array elements from individual mosquitoes. This demonstration indicates that multiple-sample feeding and recovery strategies are feasible and may represent a viable strategy for future rapid screening of biologically active genes, gene products or microorganisms in live arthropods.

Animals↗
PubMed2001

Pax group III genes and the evolution of insect pair-rule patterning.

Pair-rule genes were identified and named for their role in segmentation in embryos of the long germ insect Drosophila. Among short germ insects these genes exhibit variable expression patterns during segmentation and thus are likely to play divergent roles in this process. Understanding the details of this variation should shed light on the evolution of the genetic hierarchy responsible for segmentation in Drosophila and other insects. We have investigated the expression of homologs of the Drosophila Pax group III genes paired, gooseberry and gooseberry-neuro in short germ flour beetles and grasshoppers. During Drosophila embryogenesis, paired acts as one of several pair-rule genes that define the boundaries of future parasegments and segments, via the regulation of segment polarity genes such as gooseberry, which in turn regulates gooseberry-neuro, a gene expressed later in the developing nervous system. Using a crossreactive antibody, we show that the embryonic expression of Pax group III genes in both the flour beetle Tribolium and the grasshopper Schistocerca is remarkably similar to the pattern in Drosophila. We also show that two Pax group III genes, pairberry1 and pairberry2, are responsible for the observed protein pattern in grasshopper embryos. Both pairberry1 and pairberry2 are expressed in coincident stripes of a one-segment periodicity, in a manner reminiscent of Drosophila gooseberry and gooseberry-neuro. pairberry1, however, is also expressed in stripes of a two-segment periodicity before maturing into its segmental pattern. This early expression of pairberry1 is reminiscent of Drosophila paired and represents the first evidence for pair-rule patterning in short germ grasshoppers or any hemimetabolous insect.

Amino Acid Sequence↗
PubMed1999

Directional substitution and evolution of nucleotide content in the cytochrome oxidase II gene in earwigs (dermapteran insects).

The cytochrome oxidase subunit II (COII) gene was sequenced for six dermapteran species. The nucleotide composition of this gene is biased in most animals. While the CG content of other insect orders is low (mean, 27.6%; range, 19.5%-33.1%), species from the Forficula genus showed unusually high values (mean, 42.4%; range, 37.3%-44.1%), mostly due to high CG frequencies at third codon positions: the mean CG content at these positions was around 45% (range, 43.9%-46.9%) for Forficula, compared with only 13.3% for other insects. This effect was so strong that in one species, Forficula lesnei, there was no significant difference between the frequencies of the four bases. During evolution, this loss of bias has involved a significant increase in the synonymous substitution rate and an increase of transitions over transversions compared with other insects. A strong directionality of substitutions has favored T-->C and A-->G changes. This phenomenon was also observed between two conspecific populations of Forficula auricularia. A species from a closely related genus, Anechura bipunctata, was intermediate between Forficula and other insects for these parameters, while two remotely related dermapteran species, Labidura riparia and Euborellia moesta, were similar to other insects. These results suggest that the evolution of Forficula DNA content has been both rapid and recent.

Animals↗
PubMed1997

Treatment of l(2)mbn Drosophila tumorous blood cells with the steroid hormone ecdysone amplifies the inducibility of antimicrobial peptide gene expression.

Insects rely on both humoral and cellular mechanisms to defend themselves against microbial infections. The humoral response involves synthesis of a battery of potent antimicrobial peptides by the fat body and, to a lesser extent, by blood cells. The cellular response on the other hand consists of phagocytosis of small microorganisms and melanization and encapsulation of larger parasites. The l(2)mbn cell line, established from tumorous larval hemocytes, represents a system of choice to dissect the molecular events controlling cellular immunity. We report here that l(2)mbn cells can be efficiently induced to differentiate in adherent, macrophage-like cells by treatment with 20-hydroxyecdysone. Ecdysone treatment increases both the phagocytic capacity of l(2)mbn cells and their competence to express antimicrobial genes in response to immune challenge. We also report that expression of several regulatory molecules thought to be involved in the immune response is up-regulated by ecdysone in l(2)mbn cells.

Animals↗
PubMed1998

Expression, processing and secretion of a proteolytically-sensitive insect diuretic hormone by Saccharomyces cerevisiae requires the use of a yeast strain lacking genes encoding the Yap3 and Mkc7 endoproteases found in the secretory pathway.

A system is described for the heterologous expression of peptides in Saccharomyces cerevisiae. A synthetic gene encoding a precursor of the 41 amino acid Manduca sexta diuretic hormone (Mas-DH) was expressed at 0.8 mg/l purified peptide. A precursor of a mutant peptide of Mas-DH, Mas-DH[K22Q] was also expressed. The peptides were purified, then treated with peptidylglycine alpha-amidating enzyme to generate the alpha-amidated, mature, form of Mas-DH or Mas-DH[K22Q], which were biologically active. Successful expression of full-length Mas-DH+Gly depended upon the use of a protease-deficient yeast strain. In wild-type strains, Mas-DH+Gly was recovered only as proteolytic fragments, even in the presence of various protease inhibitors. Expression of Mas-DH+Gly in strains deficient in either the Mkc7 or the Yap3 protease reduced proteolysis, while no proteolysis of Mas-DH+Gly was detectable in a strain lacking both proteases. This protease-deficient strain may prove of general utility for expression of peptides. Analysis of recovered proteolytic fragments revealed a complex pattern of cleavage sites. Both the Yap3 and Mkc7 proteases preferred to cleave at a single Glu-Lys downward arrow-Glu-Arg site. Analysis of secondary cleavage sites showed that Yap3 preferred to cleave after either Lys or Arg and Mkc7 after Lys. This paper is the first report on the in vivo activity and specificity of Yap3 and Mkc7 expressed at physiological levels.

Amino Acid Sequence↗