RESUMO
Temperature is one of the critical factors affecting gene expression in bacteria. Despite the general interest in the link between bacterial phenotypes and environmental temperature, little is known about temperature-dependent gene expression in plant pathogenic Pectobacterium atrosepticum, a causative agent of potato blackleg and tuber soft rot worldwide. In this study, twenty-nine P. atrosepticum SCRI1043 thermoregulated genes were identified using Tn5-based transposon mutagenesis coupled with an inducible promotorless gusA gene as a reporter. From the pool of 29 genes, 14 were up-regulated at 18 °C, whereas 15 other genes were up-regulated at 28 °C. Among the thermoregulated loci, genes involved in primary bacterial metabolism, membrane-related proteins, fitness-corresponding factors, and several hypothetical proteins were found. The Tn5 mutants were tested for their pathogenicity in planta and for features that are likely to remain important for the pathogen to succeed in the (plant) environment. Five Tn5 mutants expressed visible phenotypes differentiating these mutants from the phenotype of the SCRI1043 wild-type strain. The gene disruptions in the Tn5 transposon mutants caused alterations in bacterial generation time, ability to form a biofilm, production of lipopolysaccharides, and virulence on potato tuber slices. The consequences of environmental temperature on the ability of P. atrosepticum to cause disease symptoms in potato are discussed.
Assuntos
Elementos de DNA Transponíveis/genética , Pectobacterium/genética , Doenças das Plantas/genética , Solanum tuberosum/genética , Resistência à Doença/genética , Regulação Bacteriana da Expressão Gênica/genética , Estudo de Associação Genômica Ampla , Pectinas/química , Pectinas/genética , Pectobacterium/patogenicidade , Doenças das Plantas/microbiologia , Solanum tuberosum/microbiologia , Temperatura , Transposases/genéticaRESUMO
The objective of this study was to optimise the electroporation conditions for efficient integration of Venus construct in buffalo fetal fibroblasts using Sleeping Beauty (SB) based transposition and to produce Venus expressing transgenic cloned embryos through handmade cloning (HMC) approach. Primary culture of buffalo fetal fibroblast cells was established and subsequently cultured cells were co-transfected with Venus and helper plasmid at different combinations of electroporation condition. In different combinations of voltage, time and plasmid dose, we observed that 300â¯V, single pulse for 10â¯ms in 2â¯mm cuvette and 1.5-2.0⯵g transposons with 200-300â¯ng transposase dose was optimum for expressing Venus fluorescence in cells via electroporation. After electroporation, the cells were cultured for 2-3â¯days and then Venus expressing cells were picked with the help of a Pasteur pipette under the fluorescence microscope to enrich them through single cell culture method before using as donor cells for HMC. In vitro matured oocytes were reconstructed with either transfected or non-transfected buffalo somatic cells by electric fusion followed by activation. The reconstructed, activated embryos were cultured in 400⯵L of Research Vitro Cleave medium supplemented with 1% fatty acid-free BSA in 4-well dish, covered with mineral oil and incubated in an incubator (5% CO2 in air) at 38.5⯰C for 8â¯days and the developmental competence was observed. The percentage of cleaved, 4-8 and 8-16 cells stage embryos generated through Venus expressing cells were comparable with control, whereas, the morula (21.0 vs 53.0%) and blastocysts (10.5 vs 30.6%) produced through Venus expressing cells was found low as compared to control. These results indicate that fetal fibroblasts transfected with Venus could be used as donor cells for buffalo cloning and that Venus gene can be safely used as a marker of foreign gene in buffalo transgenesis.
Assuntos
Animais Geneticamente Modificados/genética , Elementos de DNA Transponíveis/genética , Engenharia Genética/métodos , Transposases/genética , Animais , Búfalos , Células Cultivadas , Clonagem de Organismos/métodos , Eletroporação/métodos , Embrião de Mamíferos , Fibroblastos , Corantes Fluorescentes , Técnicas de Transferência NuclearRESUMO
The integrase and transposase enzymes of retrovirus and transposons, respectively, share the catalytic DDE domain. In vitro assays showed that inhibitors of HIV-1 integrase generally inhibit the mariner Mos1 transposase. Using a Drosophila strain in which the mobilisation of the mariner element can be quantified by mosaic eyes, we showed that flies maintained in medium containing 210 µM to 4 mM of raltegravir, or 1 or 2 mM of dolutegravir, which are HIV-1 integrase inhibitor used in AIDS treatment, have 23-33% less somatic mobilisation in mosaic eyes when treated with raltegravir and 28-32% when treated with dolutegravir. The gene expression of the mariner transposase gene, estimated by qPCR, is similar among treated and control flies. The results suggest that in vivo assays using Drosophila can be used as a primary screening of inhibitory drugs for transposase and retroviral integrase. The advantages of this assay are that it is easy, quick, cheap and is an in vivo test, meaning that the tested substance has to have been taken in by cells and has arrived at the target site, which is not the case when in vitro assays are applied.
Assuntos
Proteínas de Ligação a DNA/genética , Inibidores de Integrase de HIV/farmacologia , HIV-1 , Transposases/genética , Animais , Elementos de DNA Transponíveis , Proteínas de Ligação a DNA/metabolismo , Drosophila/anatomia & histologia , Drosophila/genética , Drosophila/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Compostos Heterocíclicos com 3 Anéis/farmacologia , Oxazinas , Fenótipo , Piperazinas , Piridonas , Raltegravir Potássico/farmacologia , Transposases/metabolismoRESUMO
Intact retrotransposon and DNA transposons inserted in a single gene were characterized in onions (Allium cepa) and their transcription and copy numbers were estimated in this study. While analyzing diverse onion germplasm, large insertions in the DFR-A gene encoding dihydroflavonol 4-reductase (DFR) involved in the anthocyanin biosynthesis pathway were found in two accessions. A 5,070-bp long terminal repeat (LTR) retrotransposon inserted in the active DFR-A (R4) allele was identified from one of the large insertions and designated AcCOPIA1. An intact ORF encoded typical domains of copia-like LTR retrotransposons. However, AcCOPIA1 contained atypical 'TG' and 'TA' dinucleotides at the ends of the LTRs. A 4,615-bp DNA transposon was identified in the other large insertion. This DNA transposon, designated AcCACTA1, contained an ORF coding for a transposase showing homology with the CACTA superfamily transposable elements (TEs). Another 5,073-bp DNA transposon was identified from the DFR-A (TRN) allele. This DNA transposon, designated AchAT1, belonged to the hAT superfamily with short 4-bp terminal inverted repeats (TIRs). Finally, a 6,258-bp non-autonomous DNA transposon, designated AcPINK, was identified in the ANS-p allele encoding anthocyanidin synthase, the next downstream enzyme to DFR in the anthocyanin biosynthesis pathway. AcPINK also possessed very short 3-bp TIRs. Active transcription of AcCOPIA1, AcCACTA1, and AchAT1 was observed through RNA-Seq analysis and RT-PCR. The copy numbers of AcPINK estimated by mapping the genomic DNA reads produced by NextSeq 500 were predominantly high compared with the other TEs. A series of evidence indicated that these TEs might have transposed in these onion genes very recently, providing a stepping stone for elucidation of enormously large-sized onion genome structure.
Assuntos
Oxirredutases do Álcool/genética , Elementos de DNA Transponíveis/genética , Cebolas/genética , Oxigenases/genética , Retroelementos/genética , Alelos , Sequência de Aminoácidos , Sequência de Bases , DNA Complementar/genética , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Dados de Sequência Molecular , Cebolas/enzimologia , Filogenia , Pigmentos Biológicos/genética , Proteínas de Plantas/genética , Alinhamento de Sequência , Análise de Sequência de RNA , Sequências Repetidas Terminais/genética , Transposases/genéticaRESUMO
Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial pathogen which establishes chronic pulmonary infections in patients with cystic fibrosis (CF). The overproduction of a capsular polysaccharide called alginate, also known as mucoidy, promotes the formation of mucoid biofilms which are more resistant than planktonic cells to antibiotic chemotherapy and host defenses. Additionally, the conversion from the nonmucoid to mucoid phenotype is a clinical marker for the onset of chronic infection in CF. Alginate overproduction by P. aeruginosa is an endergonic process which heavily taxes cellular energy. Therefore, alginate production is highly regulated in P. aeruginosa. To better understand alginate regulation, we describe a protocol using the mini-himar1 transposon mutagenesis for the identification of novel alginate regulators in a prototypic strain PAO1. The procedure consists of two basic steps. First, we transferred the mini-himar1 transposon (pFAC) from host E. coli SM10/λpir into recipient P. aeruginosa PAO1 via biparental conjugation to create a high-density insertion mutant library, which were selected on Pseudomonas isolation agar plates supplemented with gentamycin. Secondly, we screened and isolated the mucoid colonies to map the insertion site through inverse PCR using DNA primers pointing outward from the gentamycin cassette and DNA sequencing. Using this protocol, we have identified two novel alginate regulators, mucE (PA4033) and kinB (PA5484), in strain PAO1 with a wild-type mucA encoding the anti-sigma factor MucA for the master alginate regulator AlgU (AlgT, σ(22)). This high-throughput mutagenesis protocol can be modified for the identification of other virulence-related genes causing change in colony morphology.
Assuntos
Elementos de DNA Transponíveis , Mutagênese Insercional/métodos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Alginatos , Proteínas de Ligação a DNA/genética , Ácido Glucurônico/biossíntese , Ácidos Hexurônicos , Transposases/genéticaRESUMO
Coxiella burnetii is the agent of Q fever , an emergent worldwide zoonosis of wide clinical spectrum. Although C. burnetii infection is typically associated with acute infection, atypical pneumonia and flu-like symptoms, endocarditis, osteoarticular manifestations and severe disease are possible, especially when the patient has a suppressed immune system; however, these severe complications are typically neglected. This study reports the sequencing of the repetitive element IS1111 of the transposase gene of C. burnetii from blood and bronchoalveolar lavage (BAL) samples from a patient with severe pneumonia following methotrexate therapy, resulting in the molecular diagnosis of Q fever in a patient who had been diagnosed with active seronegative polyarthritis two years earlier. To the best of our knowledge, this represents the first documented case of the isolation of C. burnetii DNA from a BAL sample.
Assuntos
Artrite/microbiologia , Coxiella burnetii/genética , DNA Bacteriano/genética , Febre Q/diagnóstico , Sequências Repetitivas de Ácido Nucleico/genética , Transposases/genética , Doença Aguda , Adulto , Lavagem Broncoalveolar , Coxiella burnetii/isolamento & purificação , Humanos , MasculinoRESUMO
Coxiella burnetii is the agent of Q fever , an emergent worldwide zoonosis of wide clinical spectrum. Although C. burnetii infection is typically associated with acute infection, atypical pneumonia and flu-like symptoms, endocarditis, osteoarticular manifestations and severe disease are possible, especially when the patient has a suppressed immune system; however, these severe complications are typically neglected. This study reports the sequencing of the repetitive element IS1111 of the transposase gene of C. burnetii from blood and bronchoalveolar lavage (BAL) samples from a patient with severe pneumonia following methotrexate therapy, resulting in the molecular diagnosis of Q fever in a patient who had been diagnosed with active seronegative polyarthritis two years earlier. To the best of our knowledge, this represents the first documented case of the isolation of C. burnetii DNA from a BAL sample.
Assuntos
Adulto , Humanos , Masculino , Artrite/microbiologia , Coxiella burnetii/genética , DNA Bacteriano/genética , Febre Q/diagnóstico , Sequências Repetitivas de Ácido Nucleico/genética , Transposases/genética , Doença Aguda , Lavagem Broncoalveolar , Coxiella burnetii/isolamento & purificaçãoRESUMO
XopN is a type III effector protein from Xanthomonas campestris pathovar vesicatoria that suppresses PAMP-triggered immunity (PTI) in tomato. Previous work reported that XopN interacts with the tomato 14-3-3 isoform TFT1; however, TFT1's role in PTI and/or XopN virulence was not determined. Here we show that TFT1 functions in PTI and is a XopN virulence target. Virus-induced gene silencing of TFT1 mRNA in tomato leaves resulted in increased growth of Xcv ΔxopN and Xcv ΔhrpF demonstrating that TFT1 is required to inhibit Xcv multiplication. TFT1 expression was required for Xcv-induced accumulation of PTI5, GRAS4, WRKY28, and LRR22 mRNAs, four PTI marker genes in tomato. Deletion analysis revealed that the XopN C-terminal domain (amino acids 344-733) is sufficient to bind TFT1. Removal of amino acids 605-733 disrupts XopN binding to TFT1 in plant extracts and inhibits XopN-dependent virulence in tomato, demonstrating that these residues are necessary for the XopN/TFT1 interaction. Phos-tag gel analysis and mass spectrometry showed that XopN is phosphorylated in plant extracts at serine 688 in a putative 14-3-3 recognition motif. Mutation of S688 reduced XopN's phosphorylation state but was not sufficient to inhibit binding to TFT1 or reduce XopN virulence. Mutation of S688 and two leucines (L64,L65) in XopN, however, eliminated XopN binding to TFT1 in plant extracts and XopN virulence. L64 and L65 are required for XopN to bind TARK1, a tomato atypical receptor kinase required for PTI. This suggested that TFT1 binding to XopN's C-terminal domain might be stabilized via TARK1/XopN interaction. Pull-down and BiFC analyses show that XopN promotes TARK1/TFT1 complex formation in vitro and in planta by functioning as a molecular scaffold. This is the first report showing that a type III effector targets a host 14-3-3 involved in PTI to promote bacterial pathogenesis.
Assuntos
Proteínas 14-3-3/metabolismo , Doenças das Plantas/imunologia , Proteínas de Plantas/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Solanum lycopersicum/microbiologia , Transposases/metabolismo , Xanthomonas campestris/patogenicidade , Proteínas 14-3-3/genética , Proteínas 14-3-3/imunologia , Sistemas de Secreção Bacterianos/genética , Sistemas de Secreção Bacterianos/imunologia , Inativação Gênica , Solanum lycopersicum/genética , Solanum lycopersicum/imunologia , Solanum lycopersicum/metabolismo , Mutação , Doenças das Plantas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , RNA Mensageiro/análise , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Reconhecimento de Padrão/genética , Receptores de Reconhecimento de Padrão/imunologia , Transposases/genética , Transposases/imunologia , Virulência/genética , Xanthomonas campestris/enzimologia , Xanthomonas campestris/genéticaRESUMO
Genome-wide analyses of repetitive DNA suggest a significant impact particularly of transposable elements on genome size and evolution of virtually all eukaryotic organisms. In this study, we analyzed the abundance and diversity of the hAT transposon superfamily of the sugar beet (B. vulgaris) genome, using molecular, bioinformatic and cytogenetic approaches. We identified 81 transposase-coding sequences, three of which are part of structurally intact but nonfunctional hAT transposons (BvhAT), in a B. vulgaris BAC library as well as in whole genome sequencing-derived data sets. Additionally, 116 complete and 497 truncated non-autonomous BvhAT derivatives lacking the transposase gene were in silico-detected. The 116 complete derivatives were subdivided into four BvhATpin groups each characterized by a distinct terminal inverted repeat motif. Both BvhAT and BvhATpin transposons are specific for species of the genus Beta and closely related species, showing a localization on B. vulgaris chromosomes predominantely in euchromatic regions. The lack of any BvhAT transposase function together with the high degree of degeneration observed for the BvhAT and the BvhATpin genomic fraction contrasts with the abundance and activity of autonomous and non-autonomous hAT transposons revealed in other plant species. This indicates a possible genus-specific structural and functional repression of the hAT transposon superfamily during Beta diversification and evolution.
Assuntos
Beta vulgaris/genética , Elementos de DNA Transponíveis , Sequência de Aminoácidos , Sequência de Bases , Cromossomos Artificiais Bacterianos , Cromossomos de Plantas , Biblioteca Gênica , Genoma de Planta , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Filogenia , Mapeamento Físico do Cromossomo , Transposases/genéticaRESUMO
BACKGROUND: Analogues of vitamin D3 are extensively used in the treatment of various illnesses, such as osteoporosis, inflammatory skin diseases, and cancer. Functional testing of new vitamin D3 analogues and formulations for improved systemic and topical administration is supported by sensitive screening methods that allow a comparative evaluation of drug properties. As a new tool in functional screening of vitamin D3 analogues, we describe a genomically integratable sensor for sensitive drug detection. This system facilitates assessment of the pharmacokinetic and pharmadynamic properties of vitamin D3 analogues. The tri-cistronic genetic sensor encodes a drug-sensoring protein, a reporter protein expressed from an activated sensor-responsive promoter, and a resistance marker. RESULTS: The three expression cassettes, inserted in a head-to-tail orientation in a Sleeping Beauty DNA transposon vector, are efficiently inserted as a single genetic entity into the genome of cells of interest in a reaction catalyzed by the hyperactive SB100X transposase. The applicability of the sensor for screening purposes is demonstrated by the functional comparison of potent synthetic analogues of vitamin D3 designed for the treatment of psoriasis and cancer. In clones of human keratinocytes carrying from a single to numerous insertions of the vitamin D3 sensor, a sensitive sensor read-out is detected upon exposure to even low concentrations of vitamin D3 analogues. In comparative studies, the sensor unveils superior potency of new candidate drugs in comparison with analogues that are currently in clinical use. CONCLUSIONS: Our findings demonstrate the use of the genetic sensor as a tool in first-line evaluation of new vitamin D3 analogues and pave the way for new types of drug delivery studies in sensor-transgenic animals.
Assuntos
Colecalciferol/farmacologia , Elementos de DNA Transponíveis/genética , Queratinócitos/efeitos dos fármacos , Elementos de Resposta/genética , Animais , Calcitriol/análogos & derivados , Calcitriol/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colecalciferol/análogos & derivados , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Hidroxicolecalciferóis/farmacologia , Queratinócitos/citologia , Queratinócitos/metabolismo , Microscopia de Fluorescência , Mutagênese Insercional , Receptores X de Retinoides/genética , Transfecção , Transposases/genética , Transposases/metabolismo , Proteína de Ligação a Vitamina D/genéticaRESUMO
The green blowfly species Lucilia cuprina and Lucilia sericata are economically important pests for the sheep industries of Australia and New Zealand. L. cuprina has long been considered a good target for a genetic pest management program. In addition, L. sericata maggots are used in the cleaning of wounds and necrotic tissue of patients suffering from ulcers that are difficult to treat by other methods. Development of efficient transgenesis methods would greatly facilitate the development of strains ideal for genetic control programs or could potentially improve "maggot therapy". We have previously reported the germ-line transformation of L. cuprina and the design of a "female killing system" that could potentially be applied to this species. However, the efficiency of transformation obtained was low and transformed lines were difficult to detect due to the low expression of the EGFP marker used. Here we describe an efficient and reliable method for germ-line transformation of L. cuprina using new piggyBac vector and helper plasmids containing the strong promoter from the L. cuprina hsp83 gene to drive expression of the transposase and fluorescent protein marker gene. We also report, for the first time, the germ-line transformation of L. sericata using the new piggyBac vector/helper combination.
Assuntos
Animais Geneticamente Modificados/metabolismo , Dípteros/genética , Transformação Genética , Animais , Austrália , Feminino , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Genes de Insetos , Células Germinativas/metabolismo , Proteínas de Fluorescência Verde/análise , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Larva/genética , Larva/metabolismo , Nova Zelândia , Controle Biológico de Vetores/métodos , Plasmídeos/genética , Plasmídeos/metabolismo , Ovinos , Transposases/genética , Transposases/metabolismo , Ferimentos e Lesões/terapiaRESUMO
The maize Activator/Dissociation (Ac/Ds) transposable element system was introduced into sugar beet. The autonomous Ac and non-autonomous Ds element excise from the T-DNA vector and integrate at novel positions in the sugar beet genome. Ac and Ds excisions generate footprints in the donor T-DNA that support the hairpin model for transposon excision. Two complete integration events into genomic sugar beet DNA were obtained by IPCR. Integration of Ac leads to an eight bp duplication, while integration of Ds in a homologue of a sugar beet flowering locus gene did not induce a duplication. The molecular structure of the target site indicates Ds integration into a double strand break. Analyses of transposase transcription using RT-PCR revealed low amounts of alternatively spliced mRNAs. The fourth intron of the transposase was found to be partially misspliced. Four different splice products were identified. In addition, the second and third exon were found to harbour two and three novel introns, respectively. These utilize each the same splice donor but several alternative splice acceptor sites. Using the SplicePredictor online tool, one of the two introns within exon two is predicted to be efficiently spliced in maize. Most interestingly, splicing of this intron together with the four major introns of Ac would generate a transposase that lacks the DNA binding domain and two of its three nuclear localization signals, but still harbours the dimerization domain.
Assuntos
Beta vulgaris/enzimologia , Beta vulgaris/genética , Transposases/genética , Zea mays/enzimologia , Zea mays/genética , Processamento Alternativo , Sequência de Bases , Primers do DNA/genética , Elementos de DNA Transponíveis , DNA Bacteriano/genética , DNA de Plantas/genética , Vetores Genéticos , Plantas Geneticamente Modificadas , Sítios de Splice de RNA , Nicotiana/enzimologia , Nicotiana/genética , Ativação TranscricionalRESUMO
The smallest known DNA transposases are those from the IS200/IS605 family. Here we show how the interplay of protein and DNA activates TnpA, the Helicobacter pylori IS608 transposase, for catalysis. First, transposon end binding causes a conformational change that aligns catalytically important protein residues within the active site. Subsequent precise cleavage at the left and right ends, the steps that liberate the transposon from its donor site, does not involve a site-specific DNA-binding domain. Rather, cleavage site recognition occurs by complementary base pairing with a TnpA-bound subterminal transposon DNA segment. Thus, the enzyme active site is constructed from elements of both protein and DNA, reminiscent of the interdependence of protein and RNA in the ribosome. Our structural results explain why the transposon ends are asymmetric and how the transposon selects a target site for integration, and they allow us to propose a molecular model for the entire transposition reaction.
Assuntos
Elementos de DNA Transponíveis/genética , Transposases/metabolismo , Alanina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Catálise , Cristalização , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/metabolismo , Dimerização , Ativação Enzimática , Helicobacter pylori/enzimologia , Ligação de Hidrogênio , Modelos Genéticos , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Transposases/química , Transposases/genética , Tirosina/genética , Tirosina/metabolismoRESUMO
We report the first cDNA-sequencing project of the entomopathogenic nematode, Heterorhabditis bacteriophora. A total of 1246 expressed sequence tags (ESTs) were generated by random sequencing of clones from a cDNA library of the infective juvenile stage. The ESTs were annotated resulting in 1072 useful ESTs that were categorized into functional categories according to Kyoto Encyclopedia of Genes and Genomes. Approximately 459 of 1072 ESTs (43%) had significant similarities to annotated sequences in GenBank. Of these, 417 had significant similarities to the free-living nematode Caenorhanditis elegans proteins. Most ESTs (18%) belonged to the genetic information processing category followed by metabolism (15% ESTs) and environmental information processing (15%) pathways. Several interesting ESTs were found that may have roles in the infectivity and survival of infective juveniles. These included proteases, dauer pathway genes (akt-1, pdk-1 & daf-7) and aging and stress resistance genes such as superoxide dismutase (sod-4), heat shock genes (hsp-4 & hsp-6), and eat genes, and signaling proteins like G-protein coupled receptors, regulators of G-protein signaling (rgs), and serine/threonine kinases. Other interesting ESTs include systemic RNAi defective protein (sid-1), ribonuclease III family members (rnh-2 &rnc) and transposase gene (Tc3A). About 67% of the ESTs did not find matches in any of the searched databases suggesting potentially novel genes in this enomopathogenic nematode. Note: Sequences described in this paper have been deposited in Genbank under the accessions DN 152655-DN 152999, and DN 153000-DN 153726.
Assuntos
Etiquetas de Sequências Expressas , Genoma Helmíntico , Rabditídios/genética , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Envelhecimento/genética , Animais , Caenorhabditis elegans/genética , DNA Complementar , DNA de Helmintos/química , DNA de Helmintos/genética , Proteínas de Choque Térmico/genética , Proteínas de Membrana/genética , Dados de Sequência Molecular , Peptídeo Hidrolases/genética , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas RGS/genética , Receptores Acoplados a Proteínas G/genética , Ribonuclease III/genética , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Superóxido Dismutase/genética , Fator de Crescimento Transformador beta/genética , Transposases/genéticaRESUMO
A 3,165-bp chromosomally integrated transposon, designatedTn3692, of the gram-positive strain Lactobacillus crispatus CHCC3692 contains an erm(B) gene conferring resistance to erythromycin at concentrations of up to 250 micrograms/ml. Loss of this resistance can occur spontaneously, but the rate is substantially increased by heat shock treatment. Heat shock treatment at 60 degrees C resulted in an almost 40-fold increase in the frequency of erythromycin-sensitive cells (erythromycin MIC, 0.047 micrograms/ml). The phenotypic change was followed by a dramatic increase in transcription of the transposase gene and the concomitant loss of an approximately 2-kb DNA fragment carrying the erm(B) gene from the 3,165-bp erm transposon. In cells that were not subjected to heat shock, transcription of the transposase gene was not detectable. The upstream sequence of the transposase gene did not show any homology to known heat shock promoters in the gene data bank. Significant homology (>99%) was observed between the erythromycin resistance-encoding gene from L. crispatus CHCC3692 and the erm(B) genes from other gram-positive bacteria, such as Streptococcus agalactiae, Streptococcus pyogenes, Enterococcus faecium, and Lactobacillus reuteri, which strongly indicates a common origin of the erm(B) gene for these species. The transposed DNA element was not translocated to other parts of the genome of CHCC3692, as determining by Southern blotting, PCR analysis, and DNA sequencing. No other major aberrations were observed, as judged by colony morphology, growth performance of the strain, and pulsed-field gel electrophoresis. These observations suggest that heat shock treatment could be used as a tool for the removal of unwanted antibiotic resistance genes harbored in transposons flanked by insertion sequence elements or transposases in lactic acid bacteria used for animal and human food production.
Assuntos
Antibacterianos/farmacologia , Elementos de DNA Transponíveis/genética , Farmacorresistência Bacteriana/genética , Eritromicina/farmacologia , Regulação Bacteriana da Expressão Gênica , Temperatura Alta , Lactobacillus/efeitos dos fármacos , Animais , Cromossomos Bacterianos , Resposta ao Choque Térmico , Lactobacillus/genética , Lactobacillus/fisiologia , Metiltransferases/genética , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Análise de Sequência de DNA , Suínos , Transposases/genéticaRESUMO
The Arabidopsis transposon Tag1 undergoes late excision during vegetative and germinal development in plants containing 35S-Tag1-GUS constructs. To determine if transcriptional regulation can account for the developmental control of Tag1 excision, the transcriptional activity of Tag1 promoter-GUS fusion constructs of various lengths was examined in transgenic plants. All constructs showed expression in the reproductive organs of developing flowers but no expression in leaves. Expression was restricted to developing gametophytes in both male and female lineages. Quantitative RT-PCR analysis confirmed that Tag1 expression predominates in the reproductive organs of flower buds. These results are consistent with late germinal excision of Tag1, but they cannot explain the vegetative excision activity of Tag1 observed with 35S-Tag1-GUS constructs. To resolve this issue, Tag1 excision was reexamined using elements with no adjacent 35S promoter sequences. Tag1 excision in this context is restricted to germinal events with no detectable vegetative excision. If a 35S enhancer sequence is placed next to Tag1, vegetative excision is restored. These results indicate that the intrinsic activity of Tag1 is restricted to germinal excision due to targeted expression of the Tag1 transposase to developing gametophytes and that this activity is altered by the presence of adjacent enhancers or promoters.
Assuntos
Arabidopsis/genética , Elementos de DNA Transponíveis/genética , DNA de Plantas/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica de Plantas/genética , Sequências Reguladoras de Ácido Nucleico , Transposases/genética , Arabidopsis/citologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Flores/crescimento & desenvolvimento , Marcação de Genes , Células Germinativas/citologia , Glucuronidase/genética , Plasmídeos , Pólen/crescimento & desenvolvimento , Regiões Promotoras Genéticas , Reprodução/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Deleção de Sequência , Transcrição Gênica , TransgenesRESUMO
MuDR controls transposition of the Mu transposable element family in Zea mays L. It produces two major transcripts: mudrA and mudrB, mudrA encodes the MURA transposase, but no specific function has been ascribed to mudrB, which lacks strong homology to known genes. Using transient expression assays in onion epidermal cells, we defined three monopartite nuclear localization signals (NLSs) of MURA; each was functionally sufficient for nuclear targeting of MURA:GUS fusion proteins. Interestingly, one NLS (NLS-A3) is produced by the splicing of the third intron. In contrast, there were no clear NLS in MURB, and the major form of MURB aggregated in the cytoplasm. Self-interaction of MURA and of MURB was also shown in a yeast two-hybrid assay. To test whether interactions of MURA and MURB can occur at the level of protein translocation into the nucleus, a cytoplasmically localized MURB:GFP was co-expressed with MURA or with the GUS fusion proteins. Co-expression did not change the localization pattern of either MURA or MURB; MURA and MURB do not detectably interact in a yeast two-hybrid assay. These results suggest that MURA and MURB do not mutually affect their localization, at least in the forms examined here.
Assuntos
Elementos de DNA Transponíveis/genética , Proteínas de Plantas/metabolismo , Transposases/metabolismo , Zea mays/genética , Sequência de Aminoácidos , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Glucuronidase/genética , Glucuronidase/metabolismo , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Sinais de Localização Nuclear/genética , Cebolas/citologia , Cebolas/metabolismo , Proteínas de Plantas/genética , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Transposases/genética , Técnicas do Sistema de Duplo-Híbrido , Zea mays/enzimologiaRESUMO
Transposon mediated insertional mutagenesis is one of the approaches for the unique gene cloning. A wild species of Cruciferae family Orychophragmus violaceus (L.) O.E. Schulz, which is of interest for practical breeding as a donor of improved plant oil, was an object of the investigation. Plasmid construction used in the experiments included selective NPT II gene, reported GUS gene serving as an excision marker, structural BAR gene located within the dSpm element and Spm transposase. The GUS gene of this plasmid had not his own promoter and became functional only after Spm-transposition. Transformed Orychophragmus violaceus (L.) O.E. Schulz. plants were obtained by direct mesophyll protoplast transformation as well as Agrobacterium tumefaciens-mediated root explant transformation. Gene transfer and the transposition event were confirmed by the GUS activity and the PCR analysis. Relative transformation efficiency using protoplasts was 5.8%.
Assuntos
Agrobacterium tumefaciens/genética , Brassicaceae/genética , Elementos de DNA Transponíveis/genética , Plantas Geneticamente Modificadas/genética , Transformação Genética , Brassicaceae/enzimologia , Genes de Plantas/genética , Genes Reporter/genética , Vetores Genéticos , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Plantas Geneticamente Modificadas/enzimologia , Reação em Cadeia da Polimerase , Protoplastos/metabolismo , Seleção Genética , Transposases/genéticaRESUMO
Inversions of short genomic sequences may play a central role in the generation of protein complexity. We report here the existence of an heterogeneous group of proteins (the trefoil precursors MUC-1 and MUA-1, six preproendothelins, and five classes of zinc finger knot proteins) having both cysteine signatures (Cs) and their inverse complementary sequences (Cs) in the same polypeptide chain. We have also found cases in which the (Cs) of a given signature is not present in the same protein, but elsewhere. TGEKPYK, a cysteine-free motif of the human transcription factor, Krab, coexists with its inverse complementary sequence in 31 proteins; the inverse complementary alone is present in a great number of proteins. Our findings suggest that short DNA inversions are a widespread feature of the genome.