Molecular analysis of a mutant Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) shows an interruption of an inhibitor of apoptosis gene (iap-3) by a new class-II piggyBac-related insect transposon

A new piggyBac-related transposable element (TE) was found in the genome of a mutant Anticarsia gemmatalis multiple nucleopolyhedrovirus interrupting an inhibitor of apoptosis gene. This mutant virus induces apoptosis upon infection of an Anticarsia gemmatalis cell line, but not in a Trichoplusia ni cell line. The sequence of the new TE (which was named IDT for iap disruptor transposon) has 2531 bp with two DNA sequences flanking a putative Transposase (Tpase) ORF of 1719 bp coding for a protein with 572 amino acids. These structural features are similar to the piggyBac TE, also reported for the first time in the genome of a baculovirus. We have also isolated variants of this new TE from different lepidopteran insect cells and compared their Tpase sequences.

msechBari, a new MITE-like element in Drosophila sechellia related to the Bari transposon

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Transposon Tn1721 distribution among strains of Xylella fastidiosa

Transposons are mobile genetic elements found within the genomes of various organisms including bacteria, fungi, plants and animals. Fragments of the transposon Tn1721 were found included in the genome of Xylella fastidiosa strain 9a5c. Regions from such fragments were PCR-amplified using specially designed primers (TNP1 and TNP2). In order to detect insertions of the Tn1721 element, both primers were used and one of them included a region of the transposon (TNP1) and the other one had the right repeat and part of the bacterial chromosome (TNP2). The PCR products obtained from strain 9a5c were used as a pattern for fragment size comparisons when DNA samples from other X. fastidiosa strains were used as template for the PCR assays. Differences were observed concerning the PCR products of such amplifications when some X. fastidiosa strains isolated from grapevine and plum were used. For the citrus-derived strains only the strains U187d and GP920b produced fragments with different sizes or weak band intensity. Such variations in the X. fastidiosa genome related to disrupted Tn1721 copies are probably due to the possibility of such a transposon element being still able to duplicate even after deletion events might have taken place and also because the bacterial strains in which the main differences were detected are derived from different host plants cultivated under different climate conditions from the one used as reference. © 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Transposon display supports transpositional activity of P elements in species of the saltans group of Drosophila

Mobilization of two P element subfamilies (canonical and O-type) from Drosophila sturtevanti and D. saltans was evaluated for copy number and transposition activity using the transposon display (TD) technique. Pairwise distances between strains regarding the insertion polymorphism profile were estimated. Amplification of the P element based on copy number estimates was highly variable among the strains (D. sturtevanti, canonical 20.11, O-type 9.00; D. saltans, canonical 16.4, O-type 12.60 insertions, on average). The larger values obtained by TD compared to our previous data by Southern blotting support the higher sensitivity of TD over Southern analysis for estimating transposable element copy numbers. The higher numbers of the canonical P element and the greater divergence in its distribution within the genome of D. sturtevanti (24.8%) compared to the O-type (16.7%), as well as the greater divergence in the distribution of the canonical P element, between the D. sturtevanti (24.8%) and the D. saltans (18.3%) strains, suggest that the canonical element occupies more sites within the D. sturtevanti genome, most probably due to recent transposition activity. These data corroborate the hypothesis that the O-type is the oldest subfamily of P elements in the saltans group and suggest that the canonical P element is or has been transpositionally active until more recently in D. sturtevanti. © Indian Academy of Sciences.

Few substitutions affect the bioluminescence spectra of Phrixotrix (Coleoptera: Phengodidae) luciferases: a site-directed mutagenesis survey

Phrixotrix (railroad worm) luciferases produce bioluminescence in the green and red regions of the spectrum, depending on the location of the lanterns, and are the only luciferases naturally producing red bioluminescence. Comparison of the luciferase sequences showed a set of substitutions that could be involved in bioluminescence colour determination: (a) unique substitutions in the red luciferase replacing otherwise invariant residues; (b) conserved basic residues in the green-yellow emitting luciferases; and (c) an additional R353 residue in red-emitting luciferase (Viviani et al., 1999). To investigate whether these sites have a functional role in bioluminescence colour determination, we performed a site-directed mutagenesis. Natural substitutions in the region 220-344 and residues in the putative luciferin-binding site were also investigated. With the exception of the previously identified substitution of R215 and T226 (Viviani et al., 2002), which display dramatic red-shift effects on the spectrum of green-yellow-emitting luciferases, only a few substitutions had a moderate effect on the spectrum of the green-emitting luciferase. In contrast, no single substitution affected the spectrum of the red-emitting luciferase. The results suggest that the identity of the active site residues is not so critical for determining red bioluminescence in PxRE luciferase. Rather, the conformation assumed during the emitting step could be critical to set up proper interactions with excited oxyluciferin. Copyright ©2007 John Wiley & Sons, Ltd.

Bacillus Thuringiensis mutant increase activity against Apodoptera Frugiperda larvae

Bacillus thuringiensis is a Gram-positive bacterium which main characteristic is the production of Cry proteins, that is toxic to some insects. These proteins, when ingested by susceptible insects, become active causing their death. In nature, it is possible to found B. thuringiensis strains which produce these proteins, but they differ in productivity (some of these isolates are more productive then others), and as to the toxicity levels of the produced proteins. Two B. thuringiensis strains that were highly effective against Spodoptera frugiperda larvae were chosen to verifying genetic mutation implication on Cry proteins productivity. One strain with a prolific spores production, while the other one only produced small amounts of spores. A genomic mutant library of these two isolates was, separately, constructed by genome Tn-5 transposon random insertion. Data analysis showed that mutation had a direct effect on the spores production, inducing an increase as well as a decrease in the production, according to the different strain observed. These results indicate, for the first time, that it is possible to use the described technique with B. thuringiensis, as well as the possibility to genetically breeding this bacteria. Another possibility introduced here is the possibility to do functional genetic studies mediated by mutagenesis in this bacterium.

Lentinula edodes (Shiitake) modulates chemically induced mutagenesis by enhancing pitting

This study was undertaken to understand how Lentinula edodes modulates in vivo mutagenesis induced by alkylating agents in bone marrow and peripheral blood as described in our previous article. Male Swiss mice were pretreated for 15 consecutive days with aqueous extracts prepared from L. edodes, after which, the number of circulating blood cells, normal erythroid bone marrow cell cycling, and phagocytosis of micronucleated reticulocyte (MNRET) and activation of spleen macrophages were assessed. The results indicate that the antimutagenicity seen in bone marrow and peripheral blood is exerted by distinct compounds with different actions. The antimutagenic effect in bone marrow is exerted by compounds subject to degradation at deep-freeze storage temperature of -20 C. On the other hand, compounds responsible for antimutagenicity in peripheral blood are not subject to degradation at -20 C. The results also indicate that the antimutagenic action in peripheral blood leading to the reduction of circulating MNRET occurs in the spleen primarily through a phagocytic activity due to higher macrophage numbers and probably not due to the enhanced activation state of individual cells. © Mary Ann Liebert, Inc.

Tumor venéreo transmissível espontâneo canino: a inserção do transposon line-1 no gene C-MYC e os critérios de malignidade

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Análise da expressão gênica global de mutantes de Xanthomonas citri subsp. citri

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Análise de uma biblioteca de mutantes de Xanthomonas citri subsp. citri quanto à patogenicidade

Pós-graduação em Microbiologia Agropecuária - FCAV

Identification and characterization of ISXax2, a TN3-like transposable element potentially related with the virulence and pathogenicity of Xanthomonas citri subsp. citri

Pós-graduação em Agronomia (Genética e Melhoramento de Plantas) - FCAV

The Mycobacterium leprae hsp65 displays proteolytic activity. Mutagenesis studies indicate that the M. leprae hsp65 Proteolytic activity is catalytically related to the HslVU protease?

The present study reports, for the first time, that the recombinant hsp65 from Mycobacterium leprae (chaperonin 2) displays a proteolytic activity toward oligopeptides. The M. leprae hsp65 proteolytic activity revealed a trypsin-like specificity toward quenched fluorescence peptides derived from dynorphins. When other peptide substrates were used (β-endorphin, neurotensin, and angiotensin I), the predominant peptide bond cleavages also involved basic amino acids in P 1, although, to a minor extent, the hydrolysis involving hydrophobic and neutral amino acids (G and F) was also observed. The amino acid sequence alignment of the M. leprae hsp65 with Escherichia coli Hs1VU protease suggested two putative threonine catalytic groups, one in the N-domain (T 136, K 168, and Y 264) and the other in the C-domain (T 375, K 409, and S 502). Mutagenesis studies showed that the replacement of K 409 by A caused a complete loss of the proteolytic activity, whereas the mutation of K 168 to A resulted in a 25% loss. These results strongly suggest that the amino acid residues T 375, K 409, and S 502 at the C-domain form the catalytic group that carries out the main proteolytic activity of the M. leprae hsp65. The possible pathophysiological implications of the proteolytic activity of the M. leprae hsp65 are now under investigation in our laboratory.

Characterization of suramin binding sites on the human group IIA secreted phospholipase A(2) by site-directed mutagenesis and molecular dynamics simulation

Suramin is a polysulphonated naphthylurea with inhibitory activity against the human secreted group IIA phospholipase A(2) (hsPLA2GIIA), and we have investigated suramin binding to recombinant hsPLA2GIIA using site-directed mutagenesis and molecular dynamics (MD) simulations. The changes in suramin binding affinity of 13 cationic residue mutants of the hsPLA2GIIA was strongly correlated with alterations in the inhibition of membrane damaging activity of the protein. Suramin binding to hsPLA2GIIA was also studied by MD simulations, which demonstrated that altered intermolecular potential energy of the suramin/mutant complexes was a reliable indicator of affinity change. Although residues in the C-terminal region play a major role in the stabilization of the hsPLA2GIIA/suramin complex, attractive and repulsive hydrophobic and electrostatic interactions with residues throughout the protein together with the adoption of a bent suramin conformation, all contribute to the stability of the complex. Analysis of the h5PLA2GIIA/suramin interactions allows the prediction of the properties of suramin analogues with improved binding and higher affinities which may be candidates for novel phospholipase A(2) inhibitors. (C) 2012 Elsevier Inc. All rights reserved.

SmTRC1, a novel Schistosoma mansoni DNA transposon, discloses new families of animal and fungi transposons belonging to the CACTA superfamily

Abstract Background The CACTA (also called En/Spm) superfamily of DNA-only transposons contain the core sequence CACTA in their Terminal Inverted Repeats (TIRs) and so far have only been described in plants. Large transcriptome and genome sequence data have recently become publicly available for Schistosoma mansoni, a digenetic blood fluke that is a major causative agent of schistosomiasis in humans, and have provided a comprehensive repository for the discovery of novel genes and repetitive elements. Despite the extensive description of retroelements in S. mansoni, just a single DNA-only transposon belonging to the Merlin family has so far been reported in this organism. Results We describe a novel S. mansoni transposon named SmTRC1, for S. mansoni Transposon Related to CACTA 1, an element that shares several characteristics with plant CACTA transposons. Southern blotting indicates approximately 30–300 copies of SmTRC1 in the S. mansoni genome. Using genomic PCR followed by cloning and sequencing, we amplified and characterized a full-length and a truncated copy of this element. RT-PCR using S. mansoni mRNA followed by cloning and sequencing revealed several alternatively spliced transcripts of this transposon, resulting in distinct ORFs coding for different proteins. Interestingly, a survey of complete genomes from animals and fungi revealed several other novel TRC elements, indicating new families of DNA transposons belonging to the CACTA superfamily that have not previously been reported in these kingdoms. The first three bases in the S. mansoni TIR are CCC and they are identical to those in the TIRs of the insects Aedes aegypti and Tribolium castaneum, suggesting that animal TRCs may display a CCC core sequence. Conclusion The DNA-only transposable element SmTRC1 from S. mansoni exhibits various characteristics, such as generation of multiple alternatively-spliced transcripts, the presence of terminal inverted repeats at the extremities of the elements flanked by direct repeats and the presence of a Transposase_21 domain, that suggest a distant relationship to CACTA transposons from Magnoliophyta. Several sequences from other Metazoa and Fungi code for proteins similar to those encoded by SmTRC1, suggesting that such elements have a common ancestry, and indicating inheritance through vertical transmission before separation of the Eumetazoa, Fungi and Plants.

Analysing the possible influence of transposon TCl4.7 insertion on the function of the genome of Cydia pomonella granulovirus

CpGV-MCp5 is a natural mutant of the Cydia pomonella Granulovirus (Mexican isolate) (CpGV-M) that harbors an insect host transposon termed TCl4.7 in its genome. TCl4.7 is located between the open reading frames Cp15 and Cp16 and separates two homologous regions hr3 and hr4, which have been recently shown to be origins of replication of CpGV-M. The MCp5 has a significant replication disadvantage in the presence of the wild-type CpGV-M. In this study, the possible effects of TCl4.7 transposon insertion on the genome function of its insertion site has been analysed. The role of Cp15 and Cp16 in the context of the virus infection cycle was examined by generating a CpGV-Bacmid (CpBAC) and Cp15 knock-out (CpBACCp15KO) and Cp16 knock-out (CpBACCp16KO) mutants. The mutant CpBACCp15KO was not able to replicate in CM larvae suggesting that Cp15 was essential for virus replication. In contrast, the mutant CpBACCp16KO infected CM larvae and produced viable occlusion bodies (OBs) demonstrating that Cp16 is a non-essential gene for virus in vivo infection of C. pomonella. The temporal transcription of Cp15 and Cp16, as well as of Cp31 (F protein) as a control, was analysed using RT-PCR and quantitative real-time PCR. It suggested a general delay or reduction of gene transcription of MCp5 compared to the parental CpGV-M. Western blot analyses using anti-Cp15 and anti-Cp16 polyclonal antibodies, however, did not show any immuno-reactive response. Thus, a direct influence of TCl4.7 on the expression of Cp15 and Cp16 could not be substantiated. To investigate whether the interruption of hr3 and hr4 palindromes affects the virus replication, two mutant bacmids with a deletion of hr3 and hr4 (CpBAChr3/hr4-KO) and another with an insertion of a Kanamycin resistance cassette between hr3 and hr4 (CpBAChr3-kan-hr4) were generated. Both mutant bacmids replicated and produced infectious virus OBs, which did not significantly differ in their median lethal concentration (LC50) and median survival time (ST50) compared to the parental CpBAC. Interestingly, the mutant CpBAChr3-kan-hr4 was very effectively out-competed by parental CpBAC, when CM larvae were co-infected with known ratios of OBs of CpBAC and the mutant CpBAChr3-kan-hr4. These observations suggested a functional co-operation between hr3 and hr4 which was interrupted by the KanR insertion in CpBAChr3-kan-hr4 and possibly by TCl4.7 transposon insertion in the mutant MCp5. This hypothesis may explain the observed replication disadvantage of the mutants MCp5 and CpBAChr3-kan-hr4 in the presence of the parental viruses CpGV-M and CpBAC, respectively.