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At least 1,333 records · Page 74Linked to original sources
PubMed2004

Identification of two new peritrophic membrane proteins from larval Trichoplusia ni: structural characteristics and their functions in the protease rich insect gut.

Peritrophic membrane (PM) proteins are important determinants for the structural formation and function of the PM. We identified two new chitin binding proteins, named CBP1 and CBP2, from the PM of Trichoplusia ni larvae by cDNA cloning. The proteins contain 12 and 10 tandem chitin binding domains in CBP1 and CBP2, respectively. Chitin binding studies demonstrated the chitin binding activity of CBP1 and CBP2, and confirmed the chitin binding domain sequence predicted by sequence analysis. Both CBP1 and CBP2 were not mucin-like glycoproteins, however, they were highly resistant to proteolytic degradation by trypsin. We found that in CBP1 and CBP2, potential trypsin and chymotrypsin cleavage sites reside primarily within the chitin binding domain sequences, limiting exposure of the potential cleavage sites to the digestive proteinases. This finding suggests a proteinase-resistance mechanism for non-mucin PM proteins to function in the proteinase rich gut environment. Immunohistochemical analysis showed that CBP1 and CBP2 are specifically localized in the PM. However, intact CBP1 and CBP2 proteins were not present in the PM, indicating that their partially degraded fragments were assembled into the PM. This observation suggests that the presence of a large number of chitin binding domains in PM proteins allows the proteins to tolerate limited proteolytic degradation in the midgut without loss of their chitin binding activity with multiple chitin binding domains. Alignment of the chitin binding sequences suggested that CBP1 and CBP2 evolved by gene duplication and the tandem chitin binding domains in the proteins arose from domain duplications.

Amino Acid Sequence↗
PubMed2006

Structure and expressions of two circadian clock genes, period and timeless in the commercial silkmoth, Bombyx mori.

We cloned two circadian clock genes period (Bmper) and timeless (Bmtim) from the commercial silkmoth, Bombyx mori. Sequence analysis revealed a high degree of conservation among insects for both genes. BmPER predicted from the DNA sequence is a polypeptide of 1, 113 amino acids with functional domains such as PAS, PAC, nuclear localization signal (NLS) and cytoplasmic localization domain (CLD). Deduced BmTIM consists of 997 amino acids with PER interaction site (PIS) as well as NLS and CLD. Southern blot analyses revealed that Bmper and Bmtim are single copy genes. Northern blot analysis demonstrated that Bmper and Bmtim are expressed both in the head and peripheral tissues. We also examined temporal profiles of Bmper and Bmtim expressions in the head, flight muscle, testis and antenna of adult males under LD12:12 and LD16:8 by Real-Time PCR assays. Our data show that photoperiod differentially affects the temporal expression patterns of Bmper and Bmtim. The mRNA expression of Bmper and Bmtim in the head had a phase lead under LD12:12 compared to that under LD16:8, whereas photoperiod did not affect expression patterns in peripheral tissues relative to light-on. Photoperiod affected not only the phase relationship but also the expression level. In the testis and antenna, the level of transcription of Bmtim was low in LD12:12 but high in LD16:8. The daily differences in amplitudes of the Bmper and Bmtim expression rhythms were 2-fold in the head and 1.5-2.5 folds in the peripheral tissues examined.

Amino Acid Sequence↗
PubMed2002

Isolation, characterization, and functional expression of kynurenine aminotransferase cDNA from the yellow fever mosquito, Aedes aegypti(1).

This study describes the molecular and biochemical characterization of kynurenine aminotransferase (KAT) from Aedes aegypti mosquitoes. Through screening an A. aegypti larval cDNA library, a 1695-bp full-length cDNA clone with a 1434-bp open reading frame (ORF) was isolated. Its deduced amino acid sequence consists of 477 amino acid residues with a predicted molecular mass of 53,490 and the amino acid sequence shares 47 and 42% identity with KATs from Homo sapiens and Rattus norvegicus, respectively. This putative A. aegypti KAT (AeKAT) has four potential N-glycosylation sites (Asn-Xxx-Trp/Ser) and a typical mitochondrial leader sequence consisting of 49 amino acids at its NH(2)-terminus with a putative cleavage site between Met-49 and Ser-50. A consensus pyridoxal 5'-phosphate (PLP) binding domain (Gly-Ser-Ala-Gly-Lys-Thr-Phe-Ser) is present in the central region of the ORF. Using a baculovirus/insect cell expression system, a full-length AeKAT and a truncated AeKAT, lacking the mitochondrial leader sequence, were expressed. The full-length AeKAT expressed in Sf9 insect cells is insoluble and has no detectable KAT activity, but the truncated AeKAT is soluble and capable of catalyzing the transamination of kynurenine to kynurenic acid in the presence of pyruvate as an amino group acceptor. However, the expressed truncated protein is not active to 3-hydroxykynurenine. Northern analysis indicates that transcription of the AeKAT occurs at all stages during mosquito development, but higher levels of mRNA are observed during the pupal and adult stages. Results indicate that a specific KAT is present in mosquitoes and the enzyme may play an important physiological role in A. aegypti.

Aedes↗
PubMed2001

Structural, functional, and evolutionary characterization of novel members of the allatostatin receptor family from insects.

By using degenerate primers based on known mammalian somatostatin receptors and the recently identified Drosophila allatostatin receptors (AlstR), we have cloned a novel receptor for the neuropeptide, allatostatin, from the cockroach Periplaneta americana. The receptor exhibits about 60% amino acid identity in the transmembrane regions when compared to the two known AlstRs from Drosophila melanogaster. In addition, two cDNA fragments were obtained from the stick insect Carausius morosus, one of which is similar to Drosophila AlstR, whereas the other is more similar to mammalian somatostatin receptors. Functional expression in Xenopus oocytes shows that the Periplaneta-AlstR exhibits high affinity to endogenous cockroach allatostatin peptides. Studies with synthetic peptides demonstrate that agonistic activity is mediated by the conserved C-terminal pentapeptide YXFGL-amide; in this sequence, amidation of the C-terminus is obligatory to maintain affinity. Thus, our studies provide a molecular basis for understanding the widespread biological activities of the allatostatin peptides.

Amino Acid Sequence↗
PubMed2006

Mosquito transgenesis: what is the fitness cost?

The generation of transgenic mosquitoes with a minimal fitness load is a prerequisite for the success of strategies for controlling mosquito-borne diseases using transgenic insects. It is important to assemble as much information as possible on this subject because realistic estimates of transgene fitness costs are essential for modeling and planning release strategies. Transgenic mosquitoes must have minimal fitness costs, because such costs would reduce the effectiveness of the genetic drive mechanisms that are used to introduce the transgenes into field mosquito populations. Several factors affect fitness of transgenic mosquitoes, including the potential negative effect of transgene products and insertional mutagenesis. Studies to assess fitness of transgenic mosquitoes in the field (as opposed to the laboratory) are still needed.

Animals↗
PubMed2002

Polymerase chain reaction-restriction fragment length polymorphism of ribosomal internal transcribed spacer region analysis on polyacrylamide gel electrophoresis reveals two haplotypes coexisting in Myzus persicae.

Aphids are geographically and economically the most important group of phytophagous insects. They are responsible for various damage on cultivated plants. In our laboratory, we are interested in their ability to transmit phytoviruses such as Rhabdovirus, Cucumovirus, Potyvirus, and Caulimavirus which cause many crop diseases. The present work deals with the study of vectors as analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of internal transcribed spacer (ITS) regions. Electrophoresis of PCR-RFLP products using EcoRV, AluI and DraI restriction enzymes on 10% polyacrylamid gel reveals the coexistence of two different haplotypes in the same individual genome. This result seems to explain some features of aphid evolution.

Animals↗
PubMed1995

Expression and characterization of recombinant Manduca sexta serpin-1B and site-directed mutants that change its inhibitory selectivity.

Hemolymph of Manduca sexta contains a number of serine proteinase inhibitors from the serpin superfamily. During formation of a stable complex between a serpin and a serine proteinase, the enzyme cleaves a specific peptide bond in an exposed loop (the reactive-site region) at the surface of the serpin. The amino acid residue on the amino-terminal side of this scissile bond, the P1 residue, is important in defining the selectivity of a serpin for inhibiting different types of serine proteinases. M. sexta serpin-1B, with alanine at the position predicted from sequence alignments to be the P1 residue, was previously named alaserpin. This alanyl residue was changed by site-directed mutagenesis to lysine (A343K) and phenylalanine (A343F). The serpin-1B cDNA and its mutants were inserted into an expression vector, H6pQE-60, and the serpin proteins were expressed in Escherichia coli. Affinity-purified recombinant serpins selectively inhibited mammalian serine proteinases: serpin-1B inhibited elastase; serpin-1B(A343K) inhibited trypsin, plasmin, and thrombin; serpin-1B(A343F) inhibited chymotrypsin as well as trypsin. All three serpins inhibited human cathepsin G. This insect serpin and its site-directed mutants associated with mammalian serine proteinases at rates similar to those reported for mammalian serpins. Serpin-1B and its mutants formed SDS-stable complexes with the enzymes they inhibited. The scissile bond was determined to be between residues 343 and 344 in wild-type serpin-1B and in serpin-1B with mutations at residue 343. These results demonstrate that the P1 alanine residue defines the primary selectivity of serpin-1B for elastase-like enzymes, and that this selectivity can be altered by mutations at this position.

Amino Acid Sequence↗
PubMed1998

Seven-up, the Drosophila homolog of the COUP-TF orphan receptors, controls cell proliferation in the insect kidney.

Morphogenesis of the insect kidney, the Malpighian tubules, is controlled in Drosophila by a single large cell, the tip cell. It has been postulated that this cell sends out a mitogenic signal that induces the division of neighboring cells. The signal and the molecules that receive it have remained elusive. We show that the COUP-TF-related nuclear orphan receptor Seven-up is a key component that becomes induced in response to mitogenic EGF receptor signaling activity emanating from the tip cell. Seven-up in turn is capable of regulating the transcription of cell cycle regulators.

Animals↗
PubMed2000

cDNA cloning, biochemical characterization and inhibition by plant inhibitors of the alpha-amylases of the Western corn rootworm, Diabrotica virgifera virgifera.

We report the characterization and cDNA cloning of two alpha-amylase isozymes from larvae of the Western corn rootworm (Diabrotica virgifera virgifera LeConte). Larvae raised on artificial media have very low levels of amylase activity, and much higher levels are found in larvae raised on maize seedlings. At pH 5.7, the optimum pH for enzyme activity, the alpha-amylases are substantially but not completely inhibited by amylase inhibitors from the common bean (Phaseolus vulgaris) and from wheat (Triticum aestivum). Using the reverse transcriptase polymerase chain reaction (RT-PCR), we cloned two cDNAs with 83% amino acid identity that encode alpha-amylase-like polypeptides. Expression of one of the two cDNAs in insect cells with a baculovirus vector shows that this cDNA encodes an active amylase with a mobility that corresponds to that of one of the two isozymes present in larval extracts. The expressed enzyme is substantially inhibited by the same two inhibitors. We also show that expression in Arabidopsis of the cDNA that encodes the amylase inhibitor AI-1 of the common bean results in the accumulation of active inhibitor in the roots, and the results are discussed with reference to the possibility of using amylase inhibitors as a strategy to genetically engineer maize plants that are resistant to Western corn rootworm larvae.

Amino Acid Sequence↗
PubMed2000

DNA structures common for chironomid telomeres terminating with complex repeats.

Tandem repeats, 340 bp long, have been shown to terminate the chromosomes in Chironomus pallidivittatus and similar DNA may be used for this purpose by related insects. In view of the importance of Chironomus in telomere studies, representing in principle a third system after short repeats and Drosophila telomeric retrotransposons, we have investigated the related Chironomus dilutus, to learn what DNA structures are conserved at the chromosome ends. Interspersed subrepeats in the telomeric repeats, which contain a long palindrome, and a zone of about 100 bp of relatively constant subtelomeric DNA towards the junction to the telomeric DNA, are characteristic for C. dilutus as for previously investigated species. C. dilutus has similar subtelomeric DNA at all chromosome ends, but typical telomeric repeats in only seven of the pairs since the eighth telocentric pair contains centromere-specific repeats.

Animals↗
PubMed2005

Expression and localization of three G protein alpha subunits, Go, Gq, and Gs, in adult antennae of the silkmoth (Bombyx mori).

In insect olfactory receptor neurons, rapid and transient increases in inositol triphosphate (IP3) and Ca2+ are detected upon stimulation with pheromone or nonpheromonal odorants. This suggests that heterotrimeric guanine nucleotide binding proteins (G proteins) may transduce some odorant responses in insects. We obtained cDNA clones encoding three classes of G protein alpha subunits, Bm Go, Bm Gq, and Bm Gs, from the antennae of the adult male silkmoth (Bombyx mori). RT-PCR experiments showed that the mRNA of these G protein alpha subunits was also present in the various tissues of adult and larval insects. We used immunocytochemistry to localize these G protein alpha subunits in adult male and female antennae. In the adult male antennae, anti-Go antiserum stained the nerve bundles. In contrast, anti-Gq and anti-Gs antisera stained the inner and outer dendritic segments of the putative olfactory receptor neuron. The localizations of Bm Go, Bm Gq, and Bm Gs in the female antennae were the same as in the male antennae. The localizations of Bm Gq and Bm Gs suggest that each subunit mediates a subset of the odorant response.

Amino Acid Sequence↗
PubMed1995

Comparison of the allatostatin neuropeptide precursors in the distantly related cockroaches Periplaneta americana and Diploptera punctata.

Allatostatins are a family of insect neuropeptides that inhibit juvenile hormone biosynthesis by the corpora allata. We have characterized cDNA and genomic DNA sequences that specify a preproallatostatin precursor in the oviparous cockroach Periplaneta americana. Comparison of this precursor with that previously described [Donly, B. C., Ding, Q., Tobe, S. S. & Bendena, W. G. (1993) Proc. Natl Acad. Sci. USA 90, 8807-8811] for the viviparous cockroach Diploptera punctata revealed several common features. First, the precursors are remarkably similar in size and the organization of the peptides within the precursor is conserved. The separation of the peptides into groups by acidic domains within the precursor has been maintained. The P. americana precursor contains 14 allatostatin-like peptides that contain the core C-terminal sequence (Tyr/Phe)-Xaa-Phe-Gly-(Leu/Ile)-NH2, as compared to the D. punctata precursor, which contains 13. Five of the peptides are perfectly conserved between the two species. The remainder, with one exception, contain amino acid substitutions in the N-terminal address portion of the peptide. Several features of expression are also similar between these two species. In both, a single copy gene specifies a large allatostatin transcript of 5.0 kb in P. americana and 9.2 kb in D. punctata. In P. americana, allatostatin transcripts appear to be produced by numerous cells in different regions of the brain.

Amino Acid Sequence↗
PubMed2003

Amplifying whole insect genomes with multiple displacement amplification.

Many applications in insect genetics require repeated analyses on individual organisms. These include population genetic and genomics, linkage mapping, molecular systematics and map-based positional cloning. However, using the polymerase chain reaction, a limited number of analyses are possible with DNA isolated from whole bodies or parts of small or preserved specimens. We describe the optimization of a new technique, Multiple Displacement Amplification, for 100-400-fold amplification of whole mosquito genomes. We demonstrate that MDA amplifies genomic DNA directly from an adult leg or from organisms as small as a first instar mosquito larva. Genetic polymorphisms revealed at individual loci are the same whether using the original genomic DNA or MDA DNA as a template.

Aedes↗
PubMed1997

A Hedgehog activity gradient contributes to AP axial patterning of the Drosophila wing.

The secreted protein Hedgehog (Hh) transmits a signal from posterior to anterior cells that is essential for limb development in insects and vertebrates. In Drosophila, Hh has been thought to act primarily to induce localized expression of Decapentaplegic and Wingless which in turn relay patterning cues at long range. We report here that Hh plays an additional role in patterning the wing. By replacing endogenous Hh activity with that of a membrane-tethered form of Hh, we show that Hh acts directly to pattern the central region of the wing, in addition to its role as an inducer of Dpp. Comparing the biological activities of secreted and membrane-tethered Hh provides evidence that Hh forms a local concentration gradient and functions as a concentration-dependent morphogen in the fly wing.

Animals↗
PubMed1996

Expression of polydnavirus genes under polydnavirus promoter regulation in insect larvae infected with baculovirus recombinants.

We have evaluated the use of baculoviruses to deliver Campoletis sonorensis polydnavirus (CsPDV) genomic DNA into lepidopteran larvae to facilitate the identification of functional CsPDV genes. Genomic fragments consisting of regulatory (promoter) and coding sequences for two CsPDV genes (VHv1.1 and WHv1.6) were used to generate CsPDV-baculovirus recombinants and evaluate the expression of genes under the regulation of the CsPDV promoters. Northern blot and primer extension studies established that CsPDV genes were expressed under the control of their own promoters in these CsPDV-baculovirus recombinants. Transcripts were detected as early as 4 h post-infection indicating that temporal activity of CsPDV promoters was retained. The VHv1.1 gene product as expressed from CsPDV-baculovirus recombinants was identical in size and in functional properties to that produced in CsPDV-infected insects. CsPDV-baculovirus recombinants may be useful for the screening and characterization of polydnavirus genes with functional activities that can only be evaluated in insect larvae.

Animals↗
PubMed2001

Recombinant baculoviruses for insect control.

Baculoviruses are double-stranded DNA viruses which are highly selective for several insect groups. They are valuable natural control agents, but their utility in many agricultural applications has been limited by their slow speed of kill and narrow host specificity. Baculoviruses have been genetically modified to express foreign genes under powerful promoters in order to accelerate their speed of kill. In our and other laboratories, the expression of genes coding for insect juvenile hormone esterases and various peptide neurotoxins has resulted in recombinant baculoviruses with promise as biological insecticides. These viruses are efficacious in the laboratory, greenhouse and field and dramatically reduce damage caused by insect feeding. The recombinant viruses synergize and are synergized by classical pesticides such as pyrethroids. Since they are highly selective for pest insects, they can be used without disrupting biological control. Because the recombinant virus produces fewer progeny in infected larvae than the wild-type virus, they are rapidly out-competed in the ecosystem. The viruses can be used effectively with crops expressing endotoxins of Bacillus thuringiensis. They can be produced industrially but also by village industries, indicating that they have the potential to deliver sustainable pest control in developing countries. It remains to be seen, however, whether the current generation of recombinant baculoviruses will be competitive with the new generation of synthetic chemical pesticides. Current research clearly indicates, though, that the use of biological vectors of genes for insect control will find a place in agriculture. Baculoviruses will also prove valuable in testing the potential utility of proteins and peptides for insect control.

Animals↗
PubMedDate not supplied

Development and partial characterization of heliothine cell lines from embryonic and differentiated tissues.

The goal of this study was to generate cell lines from a variety of insect tissues that could be useful for developing in vitro assays with tissue-specific properties. In this article, we describe the establishment of new cell cultures from differentiated (primarily neural) and undifferentiated tissues (primarily embryonic) and their initial characterization. Cell lines were established from the following tissues of the budworm, Heliothis virescens, and the bollworm, Helicoverpa zea: larval ventral nerve cords (4 lines), larval midguts (1 line), adult ovaries (1 line), and embryonic tissues (11 lines). Cell lines were primarily characterized by morphological examination and polymerase chain reaction (PCR) (both deoxyribonucleic acid amplification fingerprinting and inter-simple sequence repeats PCR).

Animals↗
PubMed1993

Molecular cloning and sequence of a novel ommochrome-binding protein cDNA from an insect, Manduca sexta.

An ommochrome-binding protein (OBP) from the hemolymph of Manduca sexta has recently been purified and characterized. A cDNA clone was isolated from a fifth instar larval cDNA expression library utilizing antiserum against OBP. Northern blot analysis of total fat body RNA detected a transcript of approximately 1.2 kilobases in fifth instar wandering larvae RNA. The complete nucleotide sequence of the 905-base pair cDNA insert was determined by the dideoxy chain termination method. The OBP cDNA encodes a polypeptide of 274 residues with a predicted molecular weight of 30,580 and with one consensus N-linked glycosylation site. Comparison of the NH2-terminal sequence of the mature protein and the cDNA sequence revealed a typical signal peptide of 18 amino acids. In wandering stage larvae, the OBP transcript appeared to be at least 250-fold less abundant than ribosomal RNA.

Amino Acid Sequence↗