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At least 811 records · Page 45Linked to original sources
PubMed1996

Synthesis and expression of genes encoding putative insect neuropeptide precursors in tobacco.

Neuropeptides are the key molecules in a multiplicity of physiological processes and their use in pest control has recently been suggested. Most neuropeptides are produced in the form of a precursor that is cleaved by proteolysis to yield various biologically active peptides. To mimic this structure, a method has been developed for synthesizing genes that code for putative polyneuropeptide precursors. As a model neuropeptide, the 5-amino-acid proctolin, one of the best studied invertebrate neuropeptides, functioning both as a visceral and a skeletal neuromuscular transmitter, was chosen. The synthetic gene was introduced into bacteria and tobacco plants, where it was efficiently transcribed. We present our results as a possible approach for the expression, in a variety of organisms, of synthetic genes coding for a wide repertoire of insect neuropeptides.

Amino Acid Sequence↗
PubMed1991

Retrotransposable elements R1 and R2 interrupt the rRNA genes of most insects.

A large number of insect species have been screened for the presence of the retrotransposable elements R1 and R2. These elements integrate independently at specific sites in the 28S rRNA genes. Genomic blots indicated that 43 of 47 insect species from nine orders contained insertions, ranging in frequency from a few percent to greater than 50% of the 28S genes. Sequence analysis of these insertions from 8 species revealed 22 elements, 21 of which corresponded to R1 or R2 elements. Surprisingly, many species appeared to contain highly divergent copies of R1 and R2 elements. For example, a parasitic wasp contained at least four families of R1 elements; the Japanese beetle contained at least five families of R2 elements. The presence of these retrotransposable elements throughout Insecta and the observation that single species can harbor divergent families within its rRNA-encoding DNA loci present interesting questions concerning the age of these elements and the possibility of cross-species transfer.

Amino Acid Sequence↗
PubMed2002

Intron-dependent stimulation of marker gene expression in cultured insect cells.

We tested in a systematic fashion the effect of an intron on the level of luciferase expression in cultured C6/36 Aedes albopictus cells. The intron was inserted in both orientations, upstream and downstream of the luciferase coding region in two different luciferase expression vectors. The two parental luciferase expression vectors differed only in their promoters, one containing the Drosophila melanogaster actin5C promoter and the other the Autographa californica nuclear polyhedrosis virus hr5/ie1 enhancer/promoter. All resulting intron-containing constructs were tested for their ability to express luciferase in transient assays following electroporation into C6/36 cells. We found that the introns stimulate luciferase expression between twelve and sixtyfold, depending on the promoter. Enhanced expression was only seen when the intron was present in the correct orientation upstream of the luciferase ORF. When the 3' splice sites of the enhanced intron-containing constructs were mutated, the expression level dropped back to below the level of the intronless parental constructs, suggesting that the intron-dependent stimulation of luciferase expression is depending on splicing and is not due to other effects the intron may have on transcription or translation. The luciferase transcripts of all constructs were analysed by reverse transcription, PCR amplification and sequencing, and the results show a perfect correlation between efficient splicing of the intron and elevated levels of luciferase expression. Our findings have the potential to be very useful for boosting expression of foreign proteins in the widely used baculoviral or non-viral systems in insect cells.

Aedes↗
PubMedDate not supplied

[Improved efficient gene transfer into insect and mammalian cells by baculovirus vectors].

The fragments of genomics DNA of the nuclear polyhedrosis virus (NPV) containing genes of late viral proteins p10, p35, p39, were cloned, the promoter regions of this genes were used to design baculovirus transfer vectors. A double-promoter and triple-promoter baculovirus transfer vectors were obtained. Recombinant baculovirus vectors containing mammalian expression cassette with cytomegalovirus (CMV) promoter, the gene for green or red fluorescent protein, SV40pA and polylinker MCS were constructed for the delivery of foreign genes into mammalian cells.

Animals↗
PubMed1989

Insect baculoviruses: powerful gene expression vectors.

Baculovirus vectors have proven useful in producing high levels of biologically active eukaryotic proteins and providing cellular fractions which are enriched in the protein of interest. Expression occurs in infected insect cells which also provide a suitable environment for post-translational modification and folding of the protein product. Stable baculovirus vectors can be constructed rapidly with a minimum of viral manipulation.

DNA Viruses↗
PubMed1996

Detection of baculovirus gene expression in insect cells and larvae by low light video image analysis.

Recombinant baculovirus isolates BmNPVluc and AcNPVluc (Kopylova-Sviridova et al., 1990) expressing the luc gene in Bombyx mori N-4, and in Sf 9 and Trichoplusia ni 368 cells, respectively, were studied. Luc gene expression driven by baculovirus regulatory elements was detected by enzyme and photometric assays. The expression of recombinant AcNPVluc and BmNPVluc genes in infected larvae of the cabbage looper, T. ni and the tomato hornworm Manduca sexta was analyzed by low-light video image methods. Expression of the luc gene was detected at high levels in both the lepidopteran cells and in third to fifth instar T. ni larvae. However, no light emission was detected in M. sexta caterpillars. High levels of light emission were detected in T. ni larvae when occlusion bodies containing both wild type and recombinant virus were fed to larvae. The results of these experiments demonstrate that video image analysis can be used to monitor the progression of baculovirus infection in susceptible insect cells and larvae. Bioluminescence in recombinant virus infected larvae can be used to determine virus host range, to monitor latent virus infection in insect cells and to assess the spread of recombinant viruses in the environment. Video image analysis was found to be a sensitive method for rapid detection and semiquantitative measurement of luc gene expression in baculovirus infected cells and for monitoring virus infection in larval tissues.

Animals↗
PubMed2006

Insect small nuclear RNA gene promoters evolve rapidly yet retain conserved features involved in determining promoter activity and RNA polymerase specificity.

In animals, most small nuclear RNAs (snRNAs) are synthesized by RNA polymerase II (Pol II), but U6 snRNA is synthesized by RNA polymerase III (Pol III). In Drosophila melanogaster, the promoters for the Pol II-transcribed snRNA genes consist of approximately 21 bp PSEA and approximately 8 bp PSEB. U6 genes utilize a PSEA but have a TATA box instead of the PSEB. The PSEAs of the two classes of genes bind the same protein complex, DmSNAPc. However, the PSEAs that recruit Pol II and Pol III differ in sequence at a few nucleotide positions that play an important role in determining RNA polymerase specificity. We have now performed a bioinformatic analysis to examine the conservation and divergence of the snRNA gene promoter elements in other species of insects. The 5' half of the PSEA is well-conserved, but the 3' half is divergent. Moreover, within each species positions exist where the PSEAs of the Pol III-transcribed genes differ from those of the Pol II-transcribed genes. Interestingly, the specific positions vary among species. Nevertheless, we speculate that these nucleotide differences within the 3' half of the PSEA act similarly to induce conformational alterations in DNA-bound SNAPc that result in RNA polymerase specificity.

Animals↗
PubMed2006

Phylogeny of the sex-determining gene Sex-lethal in insects.

The Sex-lethal (SXL) protein belongs to the family of RNA-binding proteins and is involved in the regulation of pre-mRNA splicing. SXL has undergone an obvious change of function during the evolution of the insect clade. The gene has acquired a pivotal role in the sex-determining pathway of Drosophila, although it does not act as a sex determiner in non-drosophilids. We collected SXL sequences of insect species ranging from the pea aphid (Acyrtho siphom pisum) to Drosophila melanogaster by searching published articles, sequencing cDNAs, and exploiting homology searches in public EST and whole-genome databases. The SXL protein has moderately conserved N- and C-terminal regions and a well-conserved central region including 2 RNA recognition motifs. Our phylogenetic analysis shows that a single orthologue of the Drosophila Sex-lethal (Sxl) gene is present in the genomes of the malaria mosquito Anopheles gambiae, the honeybee Apis mellifera, the silkworm Bombyx mori, and the red flour beetle Tribolium castaneum. The D. melanogaster, D. erecta, and D. pseudoobscura genomes, however, contain 2 paralogous genes, Sxl and CG3056, which are orthologous to the Anopheles, Apis, Bombyx, and Tribolium Sxl. Hence, a duplication in the fly clade generated Sxl and CG3056. Our hypothesis maintains that one of the genes, Sxl, adopted the new function of sex determiner in Drosophila, whereas the other, CG3056, continued to serve some or all of the yet-unknown ancestral functions.

Amino Acid Sequence↗
PubMed1994

Characterization and transcriptional profiles of a Drosophila gene encoding an insect defensin. A study in insect immunity.

Insect defensins are a family of 4-kDa, cationic, inducible antibacterial peptides which bear six cysteine residues engaged in three intramolecular disulfide bridges. They owe their name to certain sequence similarities with defensins from mammalian neutrophiles and macrophages. We report the characterization of a novel defensin isoform from Drosophila and the cloning of the gene encoding a preprodefensin. The gene, which is intronless and present in a single copy/haploid genome, maps at position 46CD on the right arm of the second chromosome. The analysis of the upstream region of the gene reveals the presence of multiple putative cis-regulatory sequences similar to mammalian regulatory motifs of acute-phase-response genes. Transcriptional profiles indicate that the Drosophila defensin gene is induced by bacterial challenge with acute-phase kinetics. It is also expressed in the absence of immune challenge during metamorphosis. These and other data on the Drosophila defensin gene lead us to suggest that insect and mammalian defensins have evolved independently.

Amino Acid Sequence↗
PubMed2003

Human peptidoglycan recognition protein-L is an N-acetylmuramoyl-L-alanine amidase.

Peptidoglycan recognition proteins (PGRPs) are pattern recognition molecules coded by up to 13 genes in insects and 4 genes in mammals. In insects PGRPs activate antimicrobial pathways in the hemolymph and cells, or are peptidoglycan (PGN)-lytic amidases. In mammals one PGRP is an antibacterial neutrophil protein. We report that human PGRP-L is a Zn2+-dependent N-acetylmuramoyl-l-alanine amidase (EC 3.5.1.28), an enzyme that hydrolyzes the amide bond between MurNAc and l-Ala of bacterial PGN. The minimum PGN fragment hydrolyzed by PGRP-L is MurNAc-tripeptide. PGRP-L has no direct bacteriolytic activity. The other members of the human PGRP family, PGRP-Ialpha, PGRP-Ibeta, and PGRP-S, do not have the amidase activity. The C-terminal region of PGRP-L, homologous to bacteriophage and bacterial amidases, is required and sufficient for the amidase activity of PGRP-L, although its activity (in the N-terminal delta1-343 deletion mutant) is reduced. The Zn2+ binding amino acids (conserved in PGRP-L and T7 amidase) and Cys-419 (not conserved in T7 amidase) are required for the amidase activity of PGRP-L, whereas three other amino acids, needed for the activity of T7 amidase, are not required for the activity of PGRP-L. These amino acids, although required, are not sufficient for the amidase activity, because changing them to the "active" configuration does not convert PGRP-S into an active amidase. In conclusion, human PGRP-L is an N-acetylmuramoyl-l-alanine amidase and this function is conserved in prokaryotes, insects, and mammals.

Amino Acid Sequence↗
PubMed1998

Of mites and zen: expression studies in a chelicerate arthropod confirm zen is a divergent Hox gene.

We have cloned, from an oribatid mite, a gene homologous to the zerknült (zen) genes of insects and the Hox 3 genes of vertebrates. Hox genes specify cell fates in specific regions of the body in all metazoans studied and are expressed in antero-posteriorly restricted regions of the embryo. This is true of the vertebrate Hox 3 but not of the zen genes, the insect homologs, and it has been proposed that the zen genes have lost their Hox-like function in the ancestor of the insects. We studied expression of a mite Hox 3/zen homolog and found that it is expressed in a discrete antero-posterior region of the body with an anterior boundary coinciding with that of the chelicerate homolog of the Drosophila Hox gene, proboscipedia, and propose that its loss of Hox function in insects is due to functional redundancy due to this overlap with another Hox gene.

Amino Acid Sequence↗
PubMed1996

Studies of Choristoneura fumiferana nuclear polyhedrosis virus gene expression in insect cells.

To investigate the mechanisms regulating baculovirus virulence and host range we have begun to study Choristoneura fumiferana nuclear polyhedrosis virus (CfMNPV) and its gene expression in permissive and nonpermissive cells. We have identified and mapped three genes on the CfMNPV genome. The polyhedrin gene is located from 0.0 to 2.0 m.u. and two other genes, dnapol and p143, both of which are essential for baculovirus DNA replication, are located from 35.3 to 40.9 m.u. and 55.5 to 63.4 m.u., respectively. To gain insight into the expression of CfMNPV genes in permissive C. fumiferana and nonpermissive Spodoptera frugiperda cells, we constructed three expression plasmids in which the promoter region of the dnapol, the p143, and polyhedrin genes were placed in front of a chloramphenicol acetyltransferase reporter gene. All three CfMNPV promoters were active in nonpermissive cells in the presence of Autographa californica nuclear polyhedrosis virus (AcMNPV) DNA, but no activity was detected in permissive cells either in the presence of CfMNPV DNA or AcMNPV DNA. This lack of promoter activity was not due to failure of viral or plasmid DNA to enter the cell nucleus. It was possible that the reporter plasmids were inefficient templates for transcriptional transactivation so we developed a CfMNPV transfer vector and generated a recombinant virus in which the polyhedrin promoter driving CAT gene cassette was integrated into the CfMNPV genome. In this case, the CfMNPV polyhedrin promoter was highly active in the permissive cells.

Animals↗
PubMed1995

Homeotic genes and diversification of the insect body plan.

The homeotic genes regulate segmental identity by repressing the formation of or influencing the morphology of segmental structures during development. The specific roles these genes have played in the generation of insect diversity are discussed relative to abdominal limb formation and to the evolution of wings as novel repeated structures. Other recent findings discussed suggest possible 'homeotic' origins for the non-homeotic genes found within the homeotic complexes of higher insects.

Animals↗
PubMed2001

Social insects and selfish genes.

Sometimes science advances because of a new idea. Sometimes, it's because of a new technique. When both occur together, exciting times result. In the study of social insects, DNA-based methods for measuring relatedness now allow increasingly detailed tests of Hamilton's theory of kin selection.

Altruism↗
PubMed1997

Construction of a gene encoding the insect bactericidal protein attacin. Studies on its expression in Escherichia coli.

Attacin, a bactericidal small protein is produced by the giant silk moth Hyalophora cecropia. This paper deals with our efforts to clone the attacin cDNA in a bacterial vector to express it in Escherichia coli and produce the protein in sufficient amount, for further studies. We chose two inducible expression vector/bacterial cell systems: pPL-lambda/N99cI+ cells which is able to be induced by nalidixic acid, and pET3d/BL21(DE3) cells carrying a T7 RNA polymerase gene which is IPTG-inducible. After cloning in the pPL-lambda system and under no addition of the inducer, isolated transformants carried this plasmid with at least 2 concurrent deletions that drastically affected attacin expression, even though attacin gene seems to be intact as deduced by its PCR amplification. It was concluded that basal attacin expression occurred in this system and bacterial growth was limited. Plasmid deletions may have emerged by selection pressure as a way to avoid bactericidal expression and allow bacteria survival. The second cloning attempt was done in pET3d vector/BL21 cells, that should not express the cloned sequence (they lack T7 RNA polymerase gene). Transformed BL21 cells gave 3 recombinant plasmids, 2 of them presented a C deletion that generated an early stop signal in the attacin coding region. The third clone, pET-ATT18, carrying an intact gene, was transferred to BL21(DE3)-IPTG inducible cells in order to be expressed. Attacin was undetectable in stained gels or by Western blot analysis. However, expression was visualized in grown cells after 30 min of IPTG induction and 5 min of [35S]-methionine labeling, as a 22.5 kDa protein band by using gel electrophoresis and fluorography. This low level of expression drastically affected bacterial growth. Considering that attacin has no lytic activity, these results suggest that this molecule should block bacterial growth directly at the cytoplasm by an unknown mechanism, since no signal peptide coding sequence was incorporated in this gene construction, precluding periplasmic or external destination of this protein.

Anti-Bacterial Agents↗
PubMed2003

Annotation pattern of ESTs from Spodoptera frugiperda Sf9 cells and analysis of the ribosomal protein genes reveal insect-specific features and unexpectedly low codon usage bias.

MOTIVATION: A whole set of Expressed Sequence Tags (ESTs) from the Sf9 cell line of Spodoptera frugiperda is presented here for the first time. By this way we want to identify both conserved and specific genes of this pest species. We also expect from this analysis to find a class of protein sequences providing a tool to explore genomic features and phylogeny of Lepidoptera. RESULTS: The ESTs display both housekeeping as well as developmentally regulated genes, and a high percentage of sequences with unknown function. Among the identified ORFs, almost all ribosomal proteins (RPs) were found with high EST redundancy and hence sequence accuracy. The codon usage found among RP genes is in average surprisingly much less biased in Lepidoptera than in other organisms. Other Spodoptera genes also displayed a low bias, suggesting a general genome expression feature in this Lepidoptera. We also found that the L35A and L36 RP sequences, respectively, display 40 and 10 amino-acid insertions, both being present only in insects. Sequence analysis suggests that they are probably not subjected to a strong selective pressure and may be good phylogenetic markers for Lepidoptera. Most interestingly, the Lepidoptera sequences of 9 RP genes displayed a specific signature different from the canonical one. We conclude that the RP family allows valuable comparative genomics and phylogeny of Lepidoptera. AVAILABILITY: All EST sequence data are available from the private 'Spodo-Base' upon request.

Abstracting and Indexing↗