The poliovirus 2C cis-acting replication element-mediated uridylylation of VPg is not required for synthesis of negative-sense genomes

Nucleotides in the terminal loop of the poliovirus 2C cis-acting replication element (2C(CRE)), a 61 nt structured RNA, function as the template for the addition of two uridylate (U) residues to the viral protein VPg. This uridylylation reaction leads to the formation of VPgpUpU, which is used by the viral RNA polymerase as a nucleotide-peptide primer for genome replication. Although VPg primes both positive- and negative-strand replication, the specific requirement for 2C(CRE)-mediated uridylylation for one or both events has not been demonstrated. We have used a cell-free in vitro translation and replication reaction to demonstrate that 2C(CRE) is not required for the initiation of the negative-sense strand, which is synthesized in the absence of 2C(CRE)-mediated VPgpUpU formation. We propose that the 3' poly(A) tail could serve as the template for the formation of a VPg-poly(U) primer that functions in the initiation of negative-sense strands.

Sustained activation of p53 in confluent nucleotide excision repair-deficient cells resistant to ultraviolet-induced apoptosis

p53 activation is one of the main signals after DNA damage, controlling cell cycle arrest, DNA repair and apoptosis. We have previously shown that confluent nucleotide excision repair (NER)-deficient cells are more resistant to apoptosis induced by ultraviolet irradiation (UV). Here, we further investigated the effect of cell confluence on UV-induced apoptosis in normal and NER-deficient (XP-A and XP-C) cells, as well as the effects of treatments with the ATWATR inhibitor caffeine, and the patterns of p53 activation. Strong p53 activation was observed in either proliferating or confluent cells. Caffeine increased apoptosis levels and inhibited p53 activation in proliferating cells, suggesting a protective role for p53. However, in confluent NER-deficient cells no effect of caffeine was observed. Transcription recovery measurements showed decreased recovery in proliferating XPA-deficient cells, but no recovery was observed in confluent cells. The levels of the cyclin/Cdk inhibitor, p21(Waf1/Cip1), correlated well with p53 activation in proliferating cells. Surprisingly, confluent cells also showed similar activation of p21(Waf1/Cip1). These results indicate that reduced apoptosis in confluent cells is associated with the deficiency in DNA damage removal, since this effect is not clearly observed in NER-proficient cells. Moreover, the strong activation of p53 in confluent cells, which barely respond to apoptosis, suggests that this protein, under these conditions, is not linked to UV-induced cell death signaling. (c) 2008 Elsevier B.V. All rights reserved.

Alkaline phosphatase as a reporter of sigma(S) levels and rpoS polymorphisms in different E-coli strains

sigma(S) is responsible for the transcriptional regulation of genes related to protection against stresses and bacterial survival and it accumulates in the cell under conditions of stress, such as nutrient limitation. An increase in the levels of sigma(S) causes a reduction in the expression of genes that are transcribed by RNA polymerase associated with the principal sigma factor, sigma(70). phoA, that encodes alkaline phosphatase (AP) is expressed under phosphate shortage conditions, and is also repressed by sigma(S). Here we show that in a Pi-limited chemostat, accumulation of rpoS mutations is proportional to the intrinsic level of sigma(S) in the cells. Acquisition of mutations in rpoS relieves repression of the PHO genes. We also devised a non-destructive method based on the rpoS effect on AP that differentiates between rpo(S+) and rpoS mutants, as well as between high and low-sigma(S) producers. Using this method, we provide evidence that sigma(S) contributes to the repression of AP under conditions of Pi excess and that AP variation among different strains is at least partly due to intrinsic variation in sigma(S) levels. Consequently, a simple and non-destructive AP assay can be employed to differentiate between strains expressing different levels of sigma(S) on agar plates.

Alternative promoters in the pst operon of Escherichia coli

The pst operon of Escherichia coli is composed of five genes pstS, pstC, pstA, pstB and phoU, that encode a high-affinity phosphate transport system and a negative regulator of the PHO regulon. Transcription of pst is induced under phosphate shortage and is initiated at the promoter located upstream of the first gene of the operon, pstS. Here, we show by four different technical approaches the existence of additional internal promoters upstream of pstC, pstB and phoU. These promoters are not induced by Pi-limitation and do not possess PHO-box sequences. Plasmids carrying the pst internal genes partially complement chromosomal mutations in their corresponding genes, indicating that they are translated into functional proteins.

The small nuclear ribonucleoprotein U1A interacts with NS5 from yellow fever virus

The flavivirus NS5 protein is one of the most important proteins of the replication complex, and cellular proteins can interact with it. This study shows for the first time that the yellow fever virus (YFV) NS5 protein is able to interact with U1A, a protein involved in splicing and polyadenylation. We confirmed this interaction by GST-pulldown assay and by co-immunoprecipitation in YFV-infected cells. A region between amino acids 368 and 448 was identified as the site of interaction of the NS5 protein with U1A. This region was conserved among some flaviviruses of medical importance. The implications of this interaction for flavivirus replication are discussed.

The Xanthomonas citri effector protein PthA interacts with citrus proteins involved in nuclear transport, protein folding and ubiquitination associated with DNA repair

P>Xanthomonas axonopodis pv. citri utilizes the type III effector protein PthA to modulate host transcription to promote citrus canker. PthA proteins belong to the AvrBs3/PthA family and carry a domain comprising tandem repeats of 34 amino acids that mediates protein-protein and protein-DNA interactions. We show here that variants of PthAs from a single bacterial strain localize to the nucleus of plant cells and form homo- and heterodimers through the association of their repeat regions. We hypothesize that the PthA variants might also interact with distinct host targets. Here, in addition to the interaction with alpha-importin, known to mediate the nuclear import of AvrBs3, we describe new interactions of PthAs with citrus proteins involved in protein folding and K63-linked ubiquitination. PthAs 2 and 3 preferentially interact with a citrus cyclophilin (Cyp) and with TDX, a tetratricopeptide domain-containing thioredoxin. In addition, PthAs 2 and 3, but not 1 and 4, interact with the ubiquitin-conjugating enzyme complex formed by Ubc13 and ubiquitin-conjugating enzyme variant (Uev), required for K63-linked ubiquitination and DNA repair. We show that Cyp, TDX and Uev interact with each other, and that Cyp and Uev localize to the nucleus of plant cells. Furthermore, the citrus Ubc13 and Uev proteins complement the DNA repair phenotype of the yeast Delta ubc13 and Delta mms2/uev1a mutants, strongly indicating that they are also involved in K63-linked ubiquitination and DNA repair. Notably, PthA 2 affects the growth of yeast cells in the presence of a DNA damage agent, suggesting that it inhibits K63-linked ubiquitination required for DNA repair.

Identificação de genes em Chromobacterium violaceum relacionados à resposta ao estresse

The sequencing of the genome of Chromobacterium violaceum identified one single circular chromosome of 4.8 Mb, in which approximately 40% of the founded ORFs are classified as hypothetical conserved or hypothetical. Some genic regions of biotechnological and biological interest had been characterized, e. g., environmental detoxification and DNA repair genes, respectively. Given this fact, the aim of this work was to identify genes of C. violaceum related to stress response, as the ones involved with mechanisms of DNA repair and/or genomic integrity maintenance. For this, a genomic library of C. violaceum was built in Escherichia coli strain DH10B (RecA-), in which clones were tested to UVC resistance, resulting in five candidates clones. In the PLH6A clone were identified four ORFs (CV_3721 to 3724). Two ORFs, CV_3722 and CV_3724, were subcloned and a synergic complementation activity was observed. The occurrence of an operon was confirmed using cDNA from C. violaceum in a RT-PCR assay. Further, it was observed the induction of the operon after the treatment with UVC. Thus, this operon was related to the stress response in C. violaceum. The mutagenesis assay with rifampicin after the treatment with UVC light showed high frequency of mutagenicity for the ORF CV_3722 (Pol III δ subunit). In this way, we propose that the C. violaceum δ subunit can act in DH10B in the translesion synthesis using Pol IV in a RecA independent-manner pathway. In growth curve assays other four clones (PLE1G, PLE7B, PLE10B and PLE12H) were able to complement the function at the dose 5 J/m2 and in mutagenicity assays PLE7B, PLE10B and PLE12H showed frequencies of mutation with significant differences upon the control (DH10B), demonstrating that in some way they are involved with the stress response in C. violaceum. These clones appear to be interrelated, probably regulated by a messenger molecule (eg., nucleotide c-di-GMP) and/or global regulatory molecule (eg., σS subunit of RNA polymerase).The results obtained contribute for a better genetic knowledge of this specie and its response mechanisms to environmental stress.

A search for markers of sugarcane evolution

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

Predição de promotores de Bacillus subtilis usando técnicas de aprendizado de máquina

One of the most important goals of bioinformatics is the ability to identify genes in uncharacterized DNA sequences on world wide database. Gene expression on prokaryotes initiates when the RNA-polymerase enzyme interacts with DNA regions called promoters. In these regions are located the main regulatory elements of the transcription process. Despite the improvement of in vitro techniques for molecular biology analysis, characterizing and identifying a great number of promoters on a genome is a complex task. Nevertheless, the main drawback is the absence of a large set of promoters to identify conserved patterns among the species. Hence, a in silico method to predict them on any species is a challenge. Improved promoter prediction methods can be one step towards developing more reliable ab initio gene prediction methods. In this work, we present an empirical comparison of Machine Learning (ML) techniques such as Na¨ýve Bayes, Decision Trees, Support Vector Machines and Neural Networks, Voted Perceptron, PART, k-NN and and ensemble approaches (Bagging and Boosting) to the task of predicting Bacillus subtilis. In order to do so, we first built two data set of promoter and nonpromoter sequences for B. subtilis and a hybrid one. In order to evaluate of ML methods a cross-validation procedure is applied. Good results were obtained with methods of ML like SVM and Naïve Bayes using B. subtilis. However, we have not reached good results on hybrid database

Effects of mutations in acetate metabolism on high-cell-density growth of Escherichia coli

To study the role played by acetate metabolism during high-cell-density growth of Escherichia coli cells, we constructed isogenic null mutants of strain W3100 deficient for several genes involved either in acetate metabolism or the transition to stationary phase. We grew these strains under identical fed-batch conditions to the highest cell densities achievable in 8 h using a predictive-plus-feedback-controlled computer algorithm that maintained glucose at a set-point of 0.5 g/l, as previously described. Wild-type strains, as well as mutants lacking the sigma(s) subunit of RNA polymerase (rpoS), grew reproducibly to high cell densities (44-50 g/l dry cell weights, DCWs). In contrast, a strain lacking acetate kinase (ackA) failed to reach densities greater than 8 g/l. Strains lacking other acetate metabolism genes (pta, acs, poxB, iciR, and fadR) achieved only medium cell densities (15-21 g/l DCWs). Complementation of either the acs or the ackA mutant restored wild-type high-cell-density growth, on a dry weight basis, poxB and fadR strains produced approximately threefold more acetate than did the wild-type strain. In contrast, the pta, acs, or rpoS strains produced significantly less acetate per cell dry weight than did the wild-type strain. Our results show that acetate metabolism plays a critical role during growth of E. coli cultures to high cell densities. They also demonstrate that cells do not require the sigma(s) regulon to grow to high cell densities, at least not under the conditions tested.

Análise comparativa da variação entre quasiespecies do Vírus da Hepatite C genótipo 1 em amostras prétratamento de pacientes tratados com Peginterferon

Pós-graduação em Genética - IBILCE

Diversidade genética de Rhizoctonia spp. e análise de sequência multilocos

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Molecular phylogeny resolves a taxonomic misunderstanding and places Geisleria close to Absconditella s. str. (Ostropales: Stictidaceae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Vertical transmission of hepatitis C virus: A tale of multiple outcomes

Globally, hepatitis C virus (HCV) infection affects approximately 130 million people and 3 million new infections occur annually. HCV is also recognized as an important cause of chronic liver disease in children. The absence of proofreading properties of the HCV RNA polymerase leads to a highly error prone replication process, allowing HCV to escape host immune response. The adaptive nature of HCV evolution dictates the outcome of the disease in many ways. Here, we investigated the molecular evolution of HCV in three unrelated children who acquired chronic HCV infection as a result of mother-to-child transmission, two of whom were also coinfected with HIV-1. The persistence of discrete HCV variants and their population structure were assessed using median joining network and Bayesian approaches. While patterns of viral evolution clearly differed between subjects, immune system dysfunction related to HIV coinfection or persistent HCV seronegativity stand as potential mechanisms to explain the lack of molecular evolution observed in these three cases. In contrast, treatment of HCV infection with PegIFN, which did not lead to sustained virologic responses in all 3 cases, was not associated with commensurate variations in the complexity of the variant spectrum. Finally, the differences in the degree of divergence suggest that the mode of transmission of the virus was not the main factor driving viral evolution. (C) 2013 Elsevier B. V. All rights reserved.

Mechanical loading induces the expression of a Pol I regulon at the onset of skeletal muscle hypertrophy

von Walden F, Casagrande V, Ostlund Farrants AK, Nader GA. Mechanical loading induces the expression of a Pol I regulon at the onset of skeletal muscle hypertrophy. Am J Physiol Cell Physiol 302: C1523-C1530, 2012. First published March 7, 2012; doi:10.1152/ajpcell.00460.2011.-The main goal of the present study was to investigate the regulation of ribosomal DNA (rDNA) gene transcription at the onset of skeletal muscle hypertrophy. Mice were subjected to functional overload of the plantaris by bilateral removal of the synergist muscles. Mechanical loading resulted in muscle hypertrophy with an increase in rRNA content. rDNA transcription, as determined by 45S pre-rRNA abundance, paralleled the increase in rRNA content and was consistent with the onset of the hypertrophic response. Increased transcription and protein expression of c-Myc and its downstream polymerase I (Pol I) regulon (POL1RB, TIF-1A, PAF53, TTF1, TAF1C) was also consistent with the increase in rRNA. Similarly, factors involved in rDNA transcription, such as the upstream binding factor and the Williams syndrome transcription factor, were induced by mechanical loading in a corresponding temporal fashion. Chromatin immunoprecipitation revealed that these factors, together with Pol I, were enriched at the rDNA promoter. This, in addition to an increase in histone H3 lysine 9 acetylation, demonstrates that mechanical loading regulates rRNA synthesis by inducing a gene expression program consisting of a Pol I regulon, together with accessory factors involved in transcription and chromatin remodeling at the rDNA promoter. Altogether, these data indicate that transcriptional and epigenetic mechanisms take place in the regulation of ribosome production at the onset of muscle hypertrophy.