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1.
BMC Biol ; 19(1): 41, 2021 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-33750380

RESUMO

BACKGROUND: The stable fly, Stomoxys calcitrans, is a major blood-feeding pest of livestock that has near worldwide distribution, causing an annual cost of over $2 billion for control and product loss in the USA alone. Control of these flies has been limited to increased sanitary management practices and insecticide application for suppressing larval stages. Few genetic and molecular resources are available to help in developing novel methods for controlling stable flies. RESULTS: This study examines stable fly biology by utilizing a combination of high-quality genome sequencing and RNA-Seq analyses targeting multiple developmental stages and tissues. In conjunction, 1600 genes were manually curated to characterize genetic features related to stable fly reproduction, vector host interactions, host-microbe dynamics, and putative targets for control. Most notable was characterization of genes associated with reproduction and identification of expanded gene families with functional associations to vision, chemosensation, immunity, and metabolic detoxification pathways. CONCLUSIONS: The combined sequencing, assembly, and curation of the male stable fly genome followed by RNA-Seq and downstream analyses provide insights necessary to understand the biology of this important pest. These resources and new data will provide the groundwork for expanding the tools available to control stable fly infestations. The close relationship of Stomoxys to other blood-feeding (horn flies and Glossina) and non-blood-feeding flies (house flies, medflies, Drosophila) will facilitate understanding of the evolutionary processes associated with development of blood feeding among the Cyclorrhapha.


Assuntos
Genoma de Inseto , Interações Hospedeiro-Parasita/genética , Controle de Insetos , Muscidae/genética , Animais , Reprodução/genética
2.
Mol Ecol ; 26(21): 5953-5960, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28902438

RESUMO

Among social insects, colony-level variation is likely to be widespread and has significant ecological consequences. Very few studies, however, have documented how genetic factors relate to behaviour at the colony level. Differences in expression of the foraging gene have been associated with differences in foraging and activity of a wide variety of organisms. We quantified expression of the red imported fire ant foraging gene (sifor) in workers from 21 colonies collected across the natural range of Texas fire ant populations, but maintained under standardized, environmentally controlled conditions. Colonies varied significantly in their behaviour. The most active colonies had up to 10 times more active foragers than the least active colony and more than 16 times as many workers outside the nest. Expression differences among colonies correlated with this colony-level behavioural variation. Colonies with higher sifor expression in foragers had, on average, significantly higher foraging activity, exploratory activity and recruitment to nectar than colonies with lower expression. Expression of sifor was also strongly correlated with worker task (foraging vs. working in the interior of the nest). These results provide insight into the genetic and physiological processes underlying collective differences in social behaviour. Quantifying variation in expression of the foraging gene may provide an important tool for understanding and predicting the ecological consequences of colony-level behavioural variation.


Assuntos
Formigas/genética , Formigas/fisiologia , Comportamento Apetitivo , Animais , Expressão Gênica , Genes de Insetos , Espécies Introduzidas , RNA Mensageiro/genética , Comportamento Social , Texas
4.
Nucleic Acids Res ; 40(14): 6978-91, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22492708

RESUMO

Integrating vectors such as viruses and transposons insert transgenes semi-randomly and can potentially disrupt or deregulate genes. For these techniques to be of therapeutic value, a method for controlling the precise location of insertion is required. The piggyBac (PB) transposase is an efficient gene transfer vector active in a variety of cell types and proven to be amenable to modification. Here we present the design and validation of chimeric PB proteins fused to the Gal4 DNA binding domain with the ability to target transgenes to pre-determined sites. Upstream activating sequence (UAS) Gal4 recognition sites harbored on recipient plasmids were preferentially targeted by the chimeric Gal4-PB transposase in human cells. To analyze the ability of these PB fusion proteins to target chromosomal locations, UAS sites were randomly integrated throughout the genome using the Sleeping Beauty transposon. Both N- and C-terminal Gal4-PB fusion proteins but not native PB were capable of targeting transposition nearby these introduced sites. A genome-wide integration analysis revealed the ability of our fusion constructs to bias 24% of integrations near endogenous Gal4 recognition sequences. This work provides a powerful approach to enhance the properties of the PB system for applications such as genetic engineering and gene therapy.


Assuntos
Marcação de Genes , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genoma , Células HEK293 , Humanos , Plasmídeos/genética , Proteínas Recombinantes de Fusão/metabolismo , Transposases/genética , Transposases/metabolismo
5.
Proc Natl Acad Sci U S A ; 107(18): 8117-22, 2010 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-20404201

RESUMO

Efficient integration of functional genes is an essential prerequisite for successful gene delivery such as cell transfection, animal transgenesis, and gene therapy. Gene delivery strategies based on viral vectors are currently the most efficient. However, limited cargo capacity, host immune response, and the risk of insertional mutagenesis are limiting factors and of concern. Recently, several groups have used transposon-based approaches to deliver genes to a variety of cells. The piggyBac (pB) transposase in particular has been shown to be well suited for cell transfection and gene therapy approaches because of its flexibility for molecular modification, large cargo capacity, and high transposition activity. However, safety considerations regarding transposase gene insertions into host genomes have rarely been addressed. Here we report our results on engineering helper-independent pB plasmids. The single-plasmid gene delivery system carries both the piggyBac transposase (pBt) expression cassette as well as the transposon cargo flanked by terminal repeat element sequences. Improvements to the helper-independent structure were achieved by developing new plasmids in which the pBt gene is rendered inactive after excision of the transposon from the plasmid. As a consequence, potentially negative effects that may develop by the persistence of an active pBt gene posttransposition are eliminated. The results presented herein demonstrate that our helper-independent plasmids represent an important step in the development of safe and efficient gene delivery methods that should prove valuable in gene therapy and transgenic approaches.


Assuntos
Técnicas de Transferência de Genes , Plasmídeos/genética , Transposases/genética , Animais , Sequência de Bases , Linhagem Celular , Dano ao DNA , Terapia Genética , Vetores Genéticos , Humanos , Camundongos
6.
Curr Microbiol ; 58(5): 478-82, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19159973

RESUMO

In our previous study we isolated 10 bacterial species from fourth-instar larval midguts of the red imported fire ant, Solenopsis invicta. Here we report the genetic transformation and reintroduction of three species (Kluyvera cryocrescens, Serratia marcescens, and isolate 38) into the fire ant host. All three species were transformed with the plasmid vector, pZeoDsRed. High expression levels of DsRed were observed and the plasmid is maintained in these bacteria at 37 degrees C in the absence of antibiotic selection for at least 9 days of subculturing. The transformed bacteria were successfully reintroduced into fire ant larvae and survived in the fire ant gut for at least 7 days. Upon pupal emergence, 7 days after reintroduction, transformed bacteria can still be isolated, however, most were passed out in the meconium. We further demonstrated that the engineered bacteria could be spread within the colony by feeding this meconium to naive larvae with the aid of worker fire ants.


Assuntos
Bactérias/crescimento & desenvolvimento , Bactérias/genética , Trato Gastrointestinal/microbiologia , Himenópteros/microbiologia , Transformação Genética , Animais , Bactérias/isolamento & purificação , Larva/microbiologia , Plasmídeos , Pupa/microbiologia
7.
Methods Mol Biol ; 435: 139-51, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18370073

RESUMO

Insertional mutagenesis can be achieved by a variety of approaches, including both random and targeted methods. In contrast to chemical mutagenesis, insertional mutagens provide a molecular tag, thereby allowing rapid identification of the mutated genomic region. Integration into defined genomic locations has great utility for both gene insertion and mutagenesis. Our laboratories have explored targeted integration through the use of transposases coupled to defined DNA-binding domains. This technology holds great promise for targeted insertional mutagenesis by biasing integration events to regions recognized by the chosen DNA-binding domain. Herein, we provide a brief background on targeted transposon integration and detailed protocols for testing chimeric transposases in both mammalian cell culture and insect embryos.


Assuntos
Elementos de DNA Transponíveis/genética , Mutagênese Insercional/métodos , Animais , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , DNA/genética , DNA/metabolismo , Humanos , Insetos/embriologia , Insetos/genética , Camundongos , Dados de Sequência Molecular , Plasmídeos/genética
8.
Mol Ther ; 15(1): 139-45, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17164785

RESUMO

Transposons are mobile genetic elements that can be used to integrate transgenes into host cell genomes. The piggyBac transposon system has been used for transgenesis of insects and for germline mutagenesis in mice. We compared transposition activity of piggyBac with Sleeping Beauty (SB), a widely used transposon system for preclinical gene therapy studies. An engineered piggyBac transposon with minimal length 5' and 3' terminal repeats exhibited greater transposition activity in transfected cultured human cells than a well-characterized hyperactive SB system. PiggyBac excision was very precise as evidenced by the typical absence of "footprint" mutations at the site of transposon excision. We mapped 575 piggyBac integration sites in human cells to determine site selectivity of genomic integration. PiggyBac demonstrated non-random integration site selectivity that differed from that previously reported for SB, including a higher preference for integrations in regions surrounding transcriptional start sites and within long terminal repeat elements. Importantly, overproduction inhibition was not observed with piggyBac, a major limitation of the SB system. This permitted the generation of combination "helper-independent" piggyBac transposase-transposon vectors that exhibited a 2-fold increase of transposition activity in human cells as compared with cells transfected with separate transposon and transposase plasmids. We conclude that piggyBac is a transposon system with certain properties, including high efficiency and lack of overproduction inhibition that are advantageous in preclinical development of transposon-based gene therapy.


Assuntos
Elementos de DNA Transponíveis/genética , Transgenes/genética , Sequência de Bases , Linhagem Celular , Vetores Genéticos/genética , Genoma Humano/genética , Humanos
9.
Nucleic Acids Res ; 31(22): e147, 2003 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-14602940

RESUMO

The excision of specific DNA sequences from integrated transgenes in insects permits the dissection in situ of structural elements that may be important in controlling gene expression. Furthermore, manipulation of potential control elements in the context of a single integration site mitigates against insertion site influences of the surrounding genome. The cre-loxP site-specific recombination system has been used successfully to remove a marker gene from transgenic yellow fever mosquitoes, Aedes aegypti. A total of 33.3% of all fertile families resulting from excision protocols showed evidence of cre-loxP-mediated site-specific excision. Excision frequencies were as high as 99.4% within individual families. The cre recombinase was shown to precisely recognize loxP sites in the mosquito genome and catalyze excision. Similar experiments with the FLP/FRT site-specific recombination system failed to demonstrate excision of the marker gene from the mosquito chromosomes.


Assuntos
Aedes/genética , Deleção de Genes , Integrases/metabolismo , Proteínas Virais/metabolismo , Animais , Animais Geneticamente Modificados , Sítios de Ligação Microbiológicos/genética , Sequência de Bases , Southern Blotting , DNA/genética , DNA Nucleotidiltransferases/genética , DNA Nucleotidiltransferases/metabolismo , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Feminino , Integrases/genética , Quinurenina 3-Mono-Oxigenase , Masculino , Oxigenases de Função Mista/genética , Dados de Sequência Molecular , Plasmídeos/genética , Recombinação Genética , Transformação Genética , Transgenes/genética , Proteínas Virais/genética
10.
Trends Biotechnol ; 23(8): 399-406, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15982766

RESUMO

A variety of technological advances in recent years have made permanent genetic manipulation of an organism a technical possibility. As the details of natural biological processes for genome modification are elucidated, the enzymes catalyzing these events (transposases, recombinases, integrases and DNA repair enzymes) are being harnessed or modified for the purpose of intentional gene modification. Targeted integration and gene repair can be mediated by the DNA-targeting specificity inherent to a particular enzyme, or rely on user-designed specificities. Integration sites can be defined by using DNA base-pairing or protein-DNA interaction as a means of targeting. This review will describe recent progress in the development of 'user-targetable' systems, particularly highlighting the application of custom DNA-binding proteins or nucleic acid homology to confer specificity.


Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/genética , Marcação de Genes/métodos , Melhoramento Genético/métodos , Terapia Genética/métodos , Genômica/métodos , Mutagênese Sítio-Dirigida/genética , Engenharia de Proteínas/métodos , Ácidos Nucleicos/genética
11.
Trends Biotechnol ; 23(8): 407-19, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15993503

RESUMO

The modification of mammalian genomes is an important goal in gene therapy and animal transgenesis. To generate stable genetic and biochemical changes, the therapeutic genes or transgenes need to be incorporated into the host genome. Ideally, the integration of the foreign gene should occur at sites that ensure their continual expression in the absence of any unwanted side effects on cellular metabolism. In this article, we discuss the opportunities provided by natural DNA-modifying enzymes, such as transposases, recombinases and integrases, to mediate the stable integration of foreign genes into host genomes. In addition, we discuss the approaches that have been taken to improve the efficiency and to modify the site-specificity of these enzymes.


Assuntos
Reparo do DNA , Exodesoxirribonucleases/genética , Marcação de Genes/métodos , Melhoramento Genético/métodos , Terapia Genética/métodos , Genômica/métodos , Mutagênese Sítio-Dirigida/genética , Engenharia de Proteínas/métodos , Animais , Humanos
12.
BMC Mol Biol ; 6: 16, 2005 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-15985163

RESUMO

BACKGROUND: One of the many ascribed functions of CCCTC-binding factor (CTCF) in vertebrates is insulation of genes via enhancer-blocking. Insulation allows genes to be shielded from "cross-talk" with neighboring regulatory elements. As such, endogenous insulator sequences would be valuable elements to enable stable transgene expression. Recently, CTCF joined Su(Hw), Zw5, BEAF32 and GAGA factor as a protein associated with insulator activity in the fruitfly, Drosophila melanogaster. To date, no known insulators have been described in mosquitoes. RESULTS: We have identified and characterized putative CTCF homologs in the medically-important mosquitoes, Aedes aegypti and Anopheles gambiae. These genes encode polypeptides with eleven C2H2 zinc fingers that show significant similarity to those of vertebrate CTCFs, despite at least 500 million years of divergence. The mosquito CTCFs are constitutively expressed and are upregulated in early embryos and in the ovaries of blood-fed females. We have uncovered significant bioinformatics evidence that CTCF is widespread, at least among Drosophila species. Finally, we show that the An. gambiae CTCF binds two known insulator sequences. CONCLUSION: Mosquito CTCFs are likely orthologous to the widely-characterized vertebrate CTCFs and potentially also serve an insulating function. As such, CTCF may provide a powerful tool for improving transgene expression in these mosquitoes through the identification of endogenous binding sites.


Assuntos
Aedes/genética , Anopheles/genética , DNA Complementar/genética , Proteínas de Ligação a DNA/genética , Proteínas Repressoras/genética , Aedes/crescimento & desenvolvimento , Sequência de Aminoácidos , Animais , Anopheles/crescimento & desenvolvimento , Fator de Ligação a CCCTC , Células Cultivadas/imunologia , Galinhas/genética , Clonagem Molecular/métodos , Drosophila/genética , Feminino , Perfilação da Expressão Gênica , Soros Imunes/metabolismo , Dados de Sequência Molecular , Ovário/metabolismo , Filogenia , Homologia de Sequência de Aminoácidos , Regulação para Cima/genética
13.
Insect Biochem Mol Biol ; 35(10): 1199-207, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16102425

RESUMO

The development of genetic strategies to control the spread of mosquito-borne diseases through the use of class II transposons has been hampered by suboptimal rates of transformation and the absence of post-integration mobility for all transposons evaluated to date. Two Mos1 mariner transposase mutants were produced by the site-directed mutagenesis of amino acids, E137 and E264, to K and R, respectively. The effects of these mutations on the transpositional activities of Mos1-derived transposon constructs were evaluated by interplasmid transposition assays in Escherichia coli and Aedes aegypti. The transpositional activities of two Mos1 transposons, one with imperfect wild type inverted terminal repeats (ITRs) and another that contained two perfectly matched 3' ITRs, were increased when the mutant transposases were supplied in trans in E. coli. The use of the perfect repeat transposon with wild type transposase did not result in an increase in transposition frequency in Ae. aegypti. However, an improvement in the integrity of the transposition process did occur, as evidenced by a lower rate of recombination events in which the transgene was transferred. An increase in the transpositional activity of the perfect repeat transposon was observed in the mosquito in the presence of either mutant transposase, and in the case of the E264R transposase, the observed increase in transposition frequency was also accompanied by a further improvement in the integrity of transposition. We discuss the possible contributions of these mutant residues to the transposition of the perfect repeat Mos1 transposon, the implications of these results with respect to the molecular evolution of Mos1, and the potential uses of the perfect repeat transposon and mutant transposases for the improvement of Mos1 mediated germ line transformation of Ae. aegypti.


Assuntos
Aedes/genética , Elementos de DNA Transponíveis/genética , Proteínas de Ligação a DNA/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Ligação a DNA/química , Dados de Sequência Molecular , Mutagênese , Fases de Leitura Aberta , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transposases
14.
PLoS One ; 10(12): e0143950, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26674802

RESUMO

Aedes aegypti, the principal vector of yellow fever and dengue fever, is responsible for more than 30,000 deaths annually. Compounds such as carbon dioxide, amino acids, fatty acids and other volatile organic compounds (VOCs) have been widely studied for their role in attracting Ae. aegypti to hosts. Many VOCs from humans are produced by associated skin microbiota. Staphyloccocus epidermidis, although not the most abundant bacteria according to surveys of relative 16S ribosomal RNA abundance, commonly occurs on human skin. Bacteria demonstrate population level decision-making through quorum sensing. Many quorum sensing molecules, such as indole, volatilize and become part of the host odor plum. To date, no one has directly demonstrated the link between quorum sensing (i.e., decision-making) by bacteria associated with a host as a factor regulating arthropod vector attraction. This study examined this specific question with regards to S. epidermidis and Ae. aegypti. Pairwise tests were conducted to examine the response of female Ae. aegypti to combinations of tryptic soy broth (TSB) and S. epidermidis wildtype and agr- strains. The agr gene expresses an accessory gene regulator for quorum sensing; therefore, removing this gene inhibits quorum sensing of the bacteria. Differential attractiveness of mosquitoes to the wildtype and agr- strains was observed. Both wildtype and the agr- strain of S. epidermidis with TSB were marginally more attractive to Ae. aegypti than the TSB alone. Most interestingly, the blood-feeder treated with wildtype S. epidermidis/TSB attracted 74% of Ae. aegypti compared to the agr- strain of S. epidermidis/TSB (P ≤ 0.0001). This study is the first to suggest a role for interkingdom communication between host symbiotic bacteria and mosquitoes. This may have implications for mosquito decision-making with regards to host detection, location and acceptance. We speculate that mosquitoes "eavesdrop" on the chemical discussions occurring between host-associated microbes to determine suitability for blood feeding. We believe these data suggest that manipulating quorum sensing by bacteria could serve as a novel approach for reducing mosquito attraction to hosts, or possibly enhancing the trapping of adults at favored oviposition sites.


Assuntos
Aedes/fisiologia , Insetos Vetores , Percepção de Quorum , Staphylococcus epidermidis/fisiologia , Animais , Humanos , Compostos Orgânicos Voláteis , Febre Amarela/transmissão
15.
BMC Mol Biol ; 5: 8, 2004 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-15251037

RESUMO

BACKGROUND: Aedes aegypti is the key vector of both the Yellow Fever and Dengue Fever viruses throughout many parts of the world. Low and variable transgene expression levels due to position effect and position effect variegation are problematic to efforts to create transgenic laboratory strains refractory to these viruses. Transformation efficiencies are also less than optimal, likely due to failure to detect expression from all integrated transgenes and potentially due to limited expression of the transposase required for transgene integration. RESULTS: Expression plasmids utilizing three heterologous promoters and three heterologous enhancers, in all possible combinations, were tested. The Hr3/IE1 enhancer-transactivator in combination with each of the constitutive heterologous promoters tested increased reporter gene expression significantly in transiently transfected Aedes albopictus C7-10 cells. CONCLUSIONS: The addition of the Hr3 enhancer to expression cassettes and concomitant expression of the IE1 transactivator gene product is a potential method for increasing the level of transgene expression in insect systems. This mechanism could also potentially be used to increase the level of transiently-expressed transposase in order to increase the number of integration events in transposon-mediated transformation experiments.


Assuntos
Aedes/genética , Baculoviridae/genética , Elementos Facilitadores Genéticos , Genes Precoces , Vetores Genéticos/genética , Transativadores , Actinas/genética , Animais , Células Cultivadas , Drosophila/genética , Expressão Gênica , Genes Reporter , Luciferases/genética , Nucleopoliedrovírus/genética , Plasmídeos , Poliubiquitina/genética , Regiões Promotoras Genéticas , Transfecção , Transgenes
16.
Gene ; 342(2): 293-301, 2004 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-15527988

RESUMO

Potato production in tropical and subtropical countries suffers from damage caused by the potato tuber moth (PTM), Phthorimaea operculella. The aim of this research was the development of the components required for a germline transformation system for the PTM. We tested three components that are critical to genetic transformation systems for insects: promoter activity, marker gene expression, and transposable element function. We compared the transcriptional activities of five different promoters, hsp70, hsp82, actin5C, polyubiquitin and immediate early 1 gene (ie1), within PTM embryos. The ie1 promoter, flanked by the hr5 enhancer element, showed a very high level of transcriptional activity compared to the other promoters. The fluorescence activity of EGFP was also determined and transient expression of EGFP was detected in 57% of injected embryos. The transpositional activity of the piggyBac transposable element was tested in an interplasmid transposition assay. The piggyBac element was shown to be mobile within the embryonic soma of the PTM with a transposition frequency of 4.2 x 10(-5) transpositions/donor plasmid. Incorporating a transactivator plasmid expressing the immediate early protein (IE1) from the Bombyx mori nuclear polyhedrosis virus enhanced the efficiency of piggyBac mobility.


Assuntos
Elementos de DNA Transponíveis/genética , Embrião não Mamífero/metabolismo , Mariposas/genética , Regiões Promotoras Genéticas/genética , Actinas/genética , Animais , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP90 , Proteínas de Choque Térmico/genética , Proteínas Imediatamente Precoces/genética , Luciferases/genética , Luciferases/metabolismo , Microinjeções , Mariposas/embriologia , Mutagênese Insercional , Plasmídeos/administração & dosagem , Plasmídeos/genética , Poliubiquitina/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae , Transativadores/genética
17.
Insect Biochem Mol Biol ; 34(9): 937-49, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15350613

RESUMO

The transition of fire ant queens from alates to dealates, following a mating flight, is associated with numerous important physiological changes. A molecular analysis of gene expression differences that occur between alates and dealates was performed using the suppression subtractive hybridization (SSH) method. 983 SSH clones were arrayed and screened by dot blot hybridization, followed by Northern blot analysis for selected clones. Gene expression profiles throughout fire ant development were determined using semi-quantitative reverse transcriptase polymerase chain reactions (RT-PCR). The cytochrome c oxidase subunit II and STARS (striated muscle activator of Rho signaling) transcripts were expressed at higher levels in dealates compared to alates and may be involved in the programmed cell death of the flight muscles. Three different vitellogenin genes and two unique yellow g-like genes were identified that may be closely associated with the reproductive system and/or nutrient transport. Two putative antibacterial peptides, abaecin and hymenoptaecin precursors, were highly expressed in dealate queens, suggesting that they are present as an immune system component during this important stage of fire ant development. The genes identified in this study may be utilized as novel targets for fire ant control and will also provide molecular markers for studies of other social insects.


Assuntos
Formigas/crescimento & desenvolvimento , Formigas/genética , Regulação da Expressão Gênica no Desenvolvimento , Sequência de Aminoácidos , Animais , Northern Blotting , Feminino , Voo Animal , Proteínas de Insetos/genética , Estágios do Ciclo de Vida/fisiologia , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase Via Transcriptase Reversa
18.
J Biomed Opt ; 9(6): 1347-57, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15568957

RESUMO

A rapid, simple, and reproducible assay is described that can be used to detect differences in the ability of oligonucleotides to influence the aggregation of colloidal gold nanoparticles. The aggregation reaction of the gold colloid was monitored through UV-visible absorption spectroscopy. Single isolated colloidal gold particles have a surface plasmon resonance manifested as a single absorbance peak at approximately 520 nm, and aggregated gold complexes develop new red-shifted peaks/shoulders depending on the nature and extent of the aggregated complex. A simple ratiometric study of the area under the single and aggregated plasmon resonance peaks thus gives information about the extent of the aggregation. It is postulated that differences in dynamic flexibility of the oligonucleotides affect their influence on the aggregation state of the gold nanoparticles. The results of this study provide new clues toward unraveling the causes behind the preferential affinity of the Hermes transposable element for certain insertion sites compared to other sequences that also contain recognizable target sites. The technique is robust and thus can potentially be used to study similar questions for numerous transposable elements and target sequences.


Assuntos
Análise Mutacional de DNA/métodos , Elementos de DNA Transponíveis/genética , Oligonucleotídeos/análise , Oligonucleotídeos/genética , Análise de Sequência de DNA/métodos , Espectrofotometria Ultravioleta/métodos , Coloide de Ouro/análise , Coloide de Ouro/química , Nanotubos/análise , Nanotubos/química , Oligonucleotídeos/química
19.
J Biomol Screen ; 18(6): 688-94, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23407365

RESUMO

Pesticides currently in widespread use often lack species specificity and also become less effective as resistance emerges. Consequently, there is a pressing need to develop novel agents that are narrowly targeted and safe to humans. A cell-based screening platform was designed to discover compounds that are lethal to mosquito (Anopheles and Aedes) cells but show little or no activity against other insect (Drosophila) or human cell lines. Mosquito-specific, aqueous-stable cytotoxins were recovered at rare frequencies. Three of these were profiled for structure-activity relationships and also assessed in whole-animal toxicity assays. In at least one test case, species-specific cytotoxicity seen in culture effectively translated to the whole-animal level, with potent toxicity against Anopheles yet none against Drosophila. Therefore, this initiative has the potential to advance novel mosquitocidal agents and, in a broader sense, could establish a versatile platform for developing customized pesticides that selectively target other disease vectors as well.


Assuntos
Aedes/efeitos dos fármacos , Anopheles/efeitos dos fármacos , Citotoxinas/farmacologia , Praguicidas/farmacologia , Aedes/metabolismo , Animais , Anopheles/metabolismo , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos/métodos , Insetos Vetores/efeitos dos fármacos , Insetos Vetores/metabolismo , Especificidade da Espécie , Relação Estrutura-Atividade
20.
Transgenic Res ; 16(3): 333-9, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17340207

RESUMO

This paper presents novel methods for producing transgenic animals, with a further emphasis on how these techniques may someday be applied in gene therapy. There are several passive methods for transgenesis, such as pronuclear microinjection (PNI) and Intracytoplasmic Sperm Injection-Mediated Transgenesis (ICSI-Tr), which rely on the repair mechanisms of the host for transgene (tg) insertion. ICSI-Tr has been shown to be an effective means of creating transgenic animals with a transfection efficiency of approximately 45% of animals born. Furthermore, because this involves the injection of the transgene into the cytoplasm of oocytes during fertilization, limited mosaicism has traditionally occurred using this technique. Current active transgenesis techniques involve the use of viruses, such as disarmed retroviruses which can insert genes into the host genome. However, these methods are limited by the size of the sequence that can be inserted, high embryo mortality, and randomness of insertion. A novel active method has been developed which combines ICSI-Tr with recombinases or transposases to increase transfection efficiency. This technique has been termed "Active Transgenesis" to imply that the tg is inserted into the host genome by enzymes supplied into the oocyte during tg introduction. DNA based methods alleviate many of the costs and time associated with purifying enzyme. Further studies have shown that RNA can be used for the transposase source. Using RNA may prevent problems with continued transposase activity that can occur if a DNA transposase is integrated into the host genome. At present piggyBac is the most effective transposon for stable integration in mammalian systems and as further studies are done to elucidate modifications which improve piggyBac's specificity and efficacy, efficiency in creating transgenic animals should improve further. Subsequently, these methods may someday be used for gene therapy in humans.


Assuntos
Animais Geneticamente Modificados/genética , Técnicas de Transferência de Genes , Animais , Vetores Genéticos/genética , Camundongos , Injeções de Esperma Intracitoplásmicas , Transfecção/métodos , Vírus/genética
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