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At least 37 records · Page 2Linked to original sources
PubMed2000

Expression of highly controllable genes in insect cells using a modified tetracycline-regulated gene expression system.

A modified tetracycline-responsive expression system (TRES) for use in insect cells was developed. The TRES contains two components: one encodes a tetracycline-controllable transactivator (tTA) and the other contains a tet operator DNA sequence to drive the luciferase gene. Our results show that the human cytomegalovirus (CMV) promoter, an essential part for strong tTA expression in mammalian system, was not functional in insect cells. Thus further modifications were required. Functional tTA was efficiently expressed in Sf9, Sf21, and TN368 cells by the p10 promoter of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) in plasmid form with virus co-infection. An increase of up to 258-fold of luciferase activity was detected in these cells when both components in modified TRES were co-transfected. In order to further simplify the experiment, tTA, which is driven by the p10 promoter, was inserted into AcMNPV. Luciferase activity was also strongly stimulated by the infection of this tTA expression-recombinant virus with the transfection of a plasmid containing the second TRES component expressing luciferase. The luciferase expressions in these systems, either in plasmids or the tTA gene in virus and luciferase in plasmid, were significantly suppressed by tetracycline. The time course kinetics of tetracycline action to the TRES were further studied. Within a time span of 50 h, the luciferase activities could be fully suppressed or activated, respectively, corresponding to the addition or removal of tetracycline. These experiments have established a well-regulated gene expression system for further broad applications of molecular biological studies in insect cells.

Animals↗
PubMed1999

Dissecting insect development: baculovirus-mediated gene silencing in insects.

A novel concept applying baculovirus-mediated gene silencing to study insect gene function and regulation is described in this paper. A recombinant baculovirus, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), was constructed with the juvenile hormone esterase (JHE) gene from the tobacco budworm Heliothis virescens in the antisense orientation, driven by the viral p10 promoter. Infection with this recombinant greatly reduced the haemolymph JHE level and resulted in aberrant morphogenesis of final-instar H. virescens larvae. The body organization remained larval, although the cuticle became hard and brown, similar to pupal cuticle. These results demonstrated that baculovirus-mediated gene silencing can be accomplished and utilized to dissect insect development and to design a new class of baculovirus insecticides.

Animals↗
PubMed2024

A PLA2 deletion mutant using CRISPR/Cas9 coupled to RNASeq reveals insect immune genes associated with eicosanoid signaling.

Eicosanoids mediate insect immune responses and synthesized by the catalytic activity of phospholipase A2 (PLA2). A uniquely encoded secretory PLA2 (sPLA2) is associated with immune responses of a lepidopteran insect, Spodoptera exigua. Its deletion mutant was generated using a CRISPR/Cas9 genome editing technology. Both wild and mutant lines were then immune-challenged, and the resulting transcripts were compared with their naïve transcripts by RNASeq using the Illumina-HiSeq platform. In total, 12,878 unigenes were further analyzed by differentially expressed gene tools. Over 69% of the expressed genes in S. exigua larvae are modulated in their expression levels by eicosanoids, recorded from CRISPR/Cas9 mutagenesis against an eicosanoid-synthetic gene, Se-sPLA2. Further, about 36% of the immune-associated genes are controlled by the eicosanoids in S. exigua. Indeed, the deletion mutant suffered significant immunosuppression in both cellular and humoral responses in response to bacterial challenge as well as severely reduced developmental and reproductive potentials.

Animals↗
PubMed1994

Structures associated with the expression of rabies virus structural genes in insect cells.

When rabies virus structural genes were expressed in insect cells, major observed alterations were a high level of cytoplasmic vacuolization caused by the matrix protein M2 and the glycoprotein G. Ring-like structures, 16 nm in diameter, were observed in cell-free extracts from insect cells that expressed the N protein alone. Hexagonally shaped structures, 16-20 nm in diameter, and regular lattice aggregates of the same structures appeared on co-expression of N and M1 proteins. Co-expression of the four structural proteins led to the formation of cell surface blebs containing the structures corresponding to N and M1 proteins.

Animals↗
PubMed2005

Diversification and independent evolution of troponin C genes in insects.

Troponin C (TpnC), the calcium-binding subunit of the troponin regulatory complex in the muscle thin filament, is encoded by multiple genes in insects. To understand how TpnC genes have evolved, we characterized the gene number and structure in a number of insect species. The TpnC gene complement is five genes in Drosophilidae as previously reported for D. melanogaster. Gene structures are almost identical in D. pseudoobscura, D. suboboscura, and D. virilis. Developmental patterns of expression are also conserved in Drosophila subobscura and D. virilis. Similar, but not completely equivalent, TpnC gene repertoires have been identified in the Anopheles gambiae and Apis mellifera genomes. Insect TpnC sequences can be divided into three groups, allowing a systematic classification of newly identified genes. The pattern of expression of the Apis mellifera genes essentially agrees with the pattern in Drosophilidae, providing further functional support to the classification. A model for the evolution of the TpnC genes is proposed including the most likely pathway of insect TpnC diversification. Our results suggest that the rapid increase in number and sequence specialization of the adult Type III isoforms can be correlated with the evolution of the holometabolous mode of development and the acquisition of asynchronous indirect flight muscle function in insects. This evolutionarily specialization has probably been achieved independently in different insect orders.

Amino Acid Sequence↗
PubMed1991

Expression of hepatitis B virus surface antigen gene in insect baculovirus vector systems.

The gene coding for the hepatitis B virus surface antigen (HBsAg) under the control of Autographa californicanuclear polyhedrosis virus polyhedrin promoter was successfully inserted into the genome of the Trichoplusia ni nuclear polyhdrosis virus. Infection of Spodoptera frugiperda cells with this recombinant virus produced a significant amount of HBsAg protein and secreted 22 nm particles containing the HBsAg. The expression of HBsAg gene was also obtained both in Trichoplusia ni larvae and in Philosamia cynthia ricini prepupae when infected with the recombinant virus. The HBsAg proteins expressed by baculovirus vector systems have morphological and antigenic properties identical to the 22 nm particles secreted by human cells.

Animals↗
PubMed2003

[Expression and purification of varicella-zoster virus glycoprotein I gene in insect cells].

OBJECTIVE: To express the cloned gene glycoprotein I (gpI) of varicella-zoster virus (VZV), Beijing VZV 84-7 strain in insect cells and to purify its expression product. METHODS: The gene coding for gpI of VZV was amplified from viral DNA by PCR and cloned into baculovirus transfer vector (pBacPAK9), and recombinant transfer vector plasmid pBacVZVgpI was obtained. The inserted gpI gene in the pBacVZVgpI was sequenced. Insect cells Sf 9 were co-transfected with the recombinant transfer vector plasmid pBacVZVgpI and wild type linear baculovirus BacPAK6 (digested with Bsu36I) DNA. The recombinant baculoviruses containing the VZV 84-7 gpI gene was isolated through several rounds of limited dilution. Recombinant protein gpI was expressed in insect cells Sf 9, postinfected with recombinant baculoviruses. The expressed recombinant gpI was purified by lectin affinity chromatography and its antigenicity and immunogenicity were investigated. RESULTS: The gene coding for gpI of VZV was obtained by PCR and the gpI gene of pBacPAK9 was confirmed by DNA sequencing. The recombinant gpI was expressed in insect cells Sf 9, post-infected with recombinant baculovirus and identified by SDS-PAGE and western blotting, with its product in cell culture reaching the peak in 72 hours and with a molecular mass of 58 kd and 70 kd, the same as theoretical values. Results of immunoassay with cell lysates infected by recombinant baculoviruses indicated that recombinant protein expressed in insect cells had ability of eliciting specific antibodies against native VZV in mice and complement-dependent neutralizing antibodies. The purified recombinant gpI gave a product with a purity of more than 80%. ELISA and Western-blot analysis demonstrated that purified protein had specific VZV antibody-binding activity. This suggested that the recombinant gpI expressed in insect cells had the same biological characteristics as its native counterpart. CONCLUSION: Baculovirus-insect cells could be used to express the gene of VZV gpI, which could provide a basis for quantitative analysis of VZV antigen, and preparation of its subunit vaccine.

Animals↗
PubMed1989

Alpha-amanitin resistant transcription of protein coding genes in insect and bloodstream form Trypanosoma brucei.

The variant cell surface glycoprotein (VSG) gene expression sites of the protozoan Trypanosoma brucei are transcribed by an unusual alpha-amanitin resistant RNA polymerase. All other protein coding genes of T.brucei examined to date are transcribed by an alpha-amanitin sensitive RNA polymerase, presumably RNA polymerase II. We now show that transcription of protein coding genes by alpha-amanitin resistant RNA polymerases is not unique to the bloodstream form expressed VSG gene expression sites, but also occurs in insect form trypanosomes, which do not express VSG genes. In insect form trypanosomes transcription of the procyclin or PARP genes is resistant to alpha-amanitin to a degree comparable with that of VSG and ribosomal RNA (rRNA) genes. Comparison of the alpha-amanitin resistantly transcribed PARP and VSG gene families shows that they both produce one of the most abundant mRNAs [1-3% of poly(A)+] and they both encode the major cell surface proteins of their respective life cycle stages. Transcription of a subset of functionally comparable protein coding genes is thus mediated by an RNA polymerase which differs from the regular RNA polymerase II.

Amanitins↗
PubMed1992

A synthetic early promoter from a baculovirus: roles of the TATA box and conserved start site CAGT sequence in basal levels of transcription.

Many baculovirus early genes and insect genes transcribed by RNA polymerase II have a conserved transcription start site sequence (CAGT) located downstream of a consensus TATA box. To examine the functions and interactions of these two motifs in initiating accurately positioned basal transcription, a 43-nt synthetic promoter was synthesized from the TATA box and start site sequences of the gp64 early promoter from the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV). The synthetic promoter initiated accurately and was also transactivated by the baculovirus transcriptional activator, IE1. To determine the roles of sequences within the 43-nt synthetic promoter, a series of linker-scanning and spacing mutations were analyzed for transcriptional activity, start site selection, and transactivation. Linker-scanning mutations were examined in vivo by transient expression and reporter gene assays. To examine transcription start site selection, promoter constructs were used for in vitro transcription in nuclear extracts from uninfected Spodoptera frugiperda (Sf9) cells. In vivo and in vitro analyses show that the TATA box, and not the start site CAGT, is the primary element controlling start site selection. Substitution of the conserved start site CAGT sequence resulted in a reduction of both reporter gene activity and in vitro transcripts, although transcripts initiated accurately. Data from linker-scanning and spacing mutations indicate that the conserved start site CAGT sequences are not required for accurate initiation but sequences at the start site play an important role in initiation efficiency.

Baculoviridae↗
PubMed2006

Evolutionary selective trends of insect/mosquito antimicrobial defensin peptides containing cysteine-stabilized alpha/beta motifs.

Insect defensins containing cysteine-stabilized alpha/beta motifs (Cs-alpha/beta defensin) are cationic, inducible antibacterial peptides involved in humoral defence against pathogens. To examine trends in molecular evolution of these antimicrobial peptides, sequences similar to the well-characterized Cs-alpha/beta defensin peptide of Anopheles gambiae, using six cysteine residues as landmarks, were retrieved from genomic and protein databases. These sequences were derived from different orders of insects. Genes of insect Cs-alpha/beta defensin appear to constitute a multigene family in which the copy number varies between insect species. Phylogenetic analysis of these sequences revealed two main lineages, one group comprising mainly lepidopteran insects and a second, comprising Hemiptera, Coleoptera, Diptera and Hymenoptera insects. Moreover, the topology of the phylogram indicated dipteran Cs-alpha/beta defensins are diverse, suggesting diversity in immune mechanisms in this order of insects. Overall evolutionary analysis indicated marked diversification and expansion of mature defensin isoforms within the species of mosquitoes relative to non-mosquito defensins, implying the presence of finely tuned immune responses to counter pathogens. The observed higher synonymous substitution rate relative to the nonsynonymous rate in almost all the regions of Cs-alpha/beta defensin of mosquitoes suggests that these peptides are predominately under purifying selection. The maximum-likelihood models of codon substitution indicated selective pressure at different amino acid sites in mosquito mature Cs-alpha/beta defensins is differ and are undergoing adaptive evolution in comparison to non-mosquito Cs-alpha/beta defensins, for which such selection was inconspicuous; this suggests the acquisition of selective advantage of the Cs-alpha/beta defensins in the former group. Finally, this study represents the most detailed report on the evolutionary strategies of Cs-alpha/beta defensins of mosquitoes in particular and insects in general, and indicates that insect Cs-alpha/beta defensins have evolved by duplication followed by divergence, to produce a diverse set of paralogues.

Amino Acid Motifs↗
PubMed2003

Molecular evolution of the insect chemoreceptor gene superfamily in Drosophila melanogaster.

The insect chemoreceptor superfamily in Drosophila melanogaster is predicted to consist of 62 odorant receptor (Or) and 68 gustatory receptor (Gr) proteins, encoded by families of 60 Or and 60 Gr genes through alternative splicing. We include two previously undescribed Or genes and two previously undescribed Gr genes; two previously predicted Or genes are shown to be alternative splice forms. Three polymorphic pseudogenes and one highly defective pseudogene are recognized. Phylogenetic analysis reveals deep branches connecting multiple highly divergent clades within the Gr family, and the Or family appears to be a single highly expanded lineage within the superfamily. The genes are spread throughout the Drosophila genome, with some relatively recently diverged genes still clustered in the genome. The Gr5a gene on the X chromosome, which encodes a receptor for the sugar trehalose, has transposed from one such tandem cluster of six genes at cytological location 64, as has Gr61a, and all eight of these receptors might bind sugars. Analysis of intron evolution suggests that the common ancestor consisted of a long N-terminal exon encoding transmembrane domains 1-5 followed by three exons encoding transmembrane domains 6-7. As many as 57 additional introns have been acquired idiosyncratically during the evolution of the superfamily, whereas the ancestral introns and some of the older idiosyncratic introns have been lost at least 48 times independently. Altogether, these patterns of molecular evolution suggest that this is an ancient superfamily of chemoreceptors, probably dating back at least to the origin of the arthropods.

Alternative Splicing↗
PubMed1996

Class 3 Hox genes in insects and the origin of zen.

We have cloned, from a beetle and a locust, genes that are homologous to the class 3 Hox genes of vertebrates. Outside the homeobox they share sequence motifs with the Drosophila zerknüllt (zen) and z2 genes, and like zen, are expressed only in extraembryonic membranes. We conclude that the zen genes of Drosophila derive from a Hox class 3 sequence that formed part of the common ancestral Hox cluster, but that in insects this (Hox) gene has lost its role in patterning the anterio-posterior axis of the embryo, and acquired a new function. In the lineage leading to Drosophila, the zen genes have diverged particularly rapidly.

Amino Acid Sequence↗
PubMed2002

[Transferring of multiple insect-resisting genes into two-line genetic male sterile rice Pei'ai 64S by particle bombardment].

Transformation of two-line genetic male sterile indica rice variety Pei'ai 64S was conducted by particle bombardment. Three insect-resisting genes ligased into plasmid vector pKC-3 was introduced into calli derived from mature embryo and a total of 33 transgenic plants had been obtained. The PCR and Southern blot analysis of each different gene in R0 plants and also PCR analysis of each different gene in R1 plants showed the integration of three insect-resisting genes into the genome of transgenic plants which could stably pass down.

Animals↗
PubMed2001

Insect segmentation: Genes, stripes and segments in "Hoppers".

Recent work has revealed that orthologues of several segmentation genes are expressed in the grasshopper embryo, in patterns resembling those shown in Drosophila. This suggests that, despite great differences between the embryos, a hierarchy of gap/pair-rule/segment polarity gene function may be a shared and ancestral feature of insect segmentation.

Animals↗
PubMed2003

[Inheritance and segregation of transformants in cotton with two types of insect-resistant genes].

A plant expression vector containing a chemeric Bt29K gene coding for the active Cry1Ac protein and the arrowhead proteinase inhibition gene API-B was introduced into an elite cotton cultivar Jihe 321 by Agrobactertium tumefaciens. Some insect-resistant cotton lines were developed. Segregation and stabilization of insect-resistant genes in six transformation lines were studied. Based on the results of kanamycin resistant test and insect bioassay using Heliethis armigera, PCR detection and Southern-blot, we found that the inheritance and segregation of Bt gene were complicated, some transformants were in accordance with Mendelian patterns of inheritance in the ratio of insect-resistant plants to non-resistant plants in Ti progeny, yet others were non-Mendelian patterns. But the inheritance and segregation of Bt gene in homozygous transformation lines were one or two pairs of major dominant genes through crossing of insect resistant homozygous lines with non-transformation cotton variety. That the insect resistance phenotype was conditioned by one or two pairs of dominant genes was ascertained in this study. There were two copies of Bt genes in two transformation lines DR248 and DR193, which was reported for the first time. The results were confirmed by Southern-blot. Through observation of segregation population of transgenic plants at different generations, we found that the exogenous Bt gene in cotton genome showed unstable in inheritance in early generations, but the gene could be stabilized through resistance screening generation by generation. The unstability of Bt gene may mean that it need time for the gene to compatibilize cotton genome.

Animals↗
PubMed1999

Construction of modular and versatile plasmid vectors for the high-level expression of single or multiple genes in insects and insect cell lines.

We have constructed a series of plasmid vectors for the expression of foreign genes in insects or insect cell lines. We incorporated the Drosophila hsp70 and actin 5C promoters, as well as the hr5 enhancer-driven baculovirus ie1 promoter, into plasmids that allow convenient cloning of heterologous genes into multiple cloning sites. We combined these promoters with either a short, double poly-adenylation site derived from the Heliothis virescens p63 chaperonin gene, or with a fusion of the small t intron with the early 3' untranslated region and poly-adenylation sites of SV40. Unique eight base cutter restriction sites flanking the promoters and poly-adenylation sequences make it possible to transfer the entire transcription units into other sequence contexts, for example, into transposable elements or into other plasmids bearing selectable marker genes. It is also convenient to combine two of our transcription units on the same plasmid in order to express multiple genes simultaneously. To test the ability of our vectors to drive expression of reporter genes, luciferase derivatives were made of the expression plasmids and introduced into Aedes albopictus C6/36 cells by electroporation or into Anopheles gambiae embryos by biolistic particle bombardment. All three promoters directed high levels of luciferase expression. However, there were differences in their relative activities in the two experimental systems. In C6/36 cells, the actin 5C and hr5-ie1 promoters were significantly more active than the hsp70 promoter. In Anopheles embryos, hsp70 and actin 5C had maximal activities, while hr5-ie1 was weaker. We also found that the constructs containing the SV40 small t intron and early 3' untranslated region sequences had higher expression levels than their counterparts containing the Heliothis poly-adenylation sequence. Our most active construct combines the actin 5C promoter with the SV40 intron and 3' untranslated region sequences. This vector was also used to drive expression of a visible marker, the enhanced green fluorescent protein gene, resulting in readily visible green fluorescent protein expression in C6/36 cells.

3' Untranslated Regions↗
PubMed1998

Examination of New Zealand's endemic Wiseana nucleopolyhedrovirus by analysis of the viral polyhedrin gene.

Insects of the genus Wiseana (Lepidoptera: Hepialidae) are major agricultural pests in New Zealand. Singly enveloped nucleopolyhedroviruses (SNPVs) isolated from three of the seven described Wiseana species have potential as biological control agents. As part of an effort to characterise the Wiseana SNPV genome the polyhedrin gene was cloned and the nucleotide sequence determined. The gene sequence was used, in conjunction with morphological and restriction endonuclease analysis, to compare isolates from different sites and species of Wiseana. Heterogeneity was detected within a single site, as well as between SNPV from separate Wiseana species. The extent of divergence between the nucleotide sequences was small enough, however, to consider three SNPVs from W. signata, W. cervinata and W. umbraculata as different strains of a single SNPV species. This improves the likely practicability of developing a single viral agent to control this pest complex. In addition, the virus polyhedrin gene sequence was used to estimate the phylogenetic relatedness of a W. signata SNPV to 16 other NPV from diverse insect genera. These comparisons suggest the Wiseana SNPV was unique within the Baculoviridae, but was more closely related to the group II NPVs.

Animals↗
PubMed1987

Expression of the human immunodeficiency virus gag gene in insect cells.

Regions of the gag-pol gene of human immunodeficiency virus (HIV), the causative agent of AIDS, have been cloned into the polyhedrin gene of the baculovirus Autographa californica nuclear polyhedrosis virus. When these recombinant viruses were used to infect insect cells, the cells produced gag-related proteins which could be immunoprecipitated with serum from AIDS patients. The major proteins produced by Acgag1, which contained the entire gag gene and a small portion of the pol gene, had molecular weights of 55,000 and 40,000 Da. Acgag2, which contained a larger portion of the pol gene in addition to the gag coding sequences, produced a major protein of 24,000 Da and only minor amounts of the 55,000- and 40,000-Da proteins. The implications of these results with respect to proteolytic processing of HIV gag proteins as well as the potential diagnostic use of this system are discussed.

Animals↗