Predição computacional de promotores em Xanthomonas axonopodis pv. citri

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Pesquisa de genomas virais em primatas não humanos do novo mundo por metagenômica

Pós-graduação em Medicina Veterinária - FMVZ

Organização genômima e análise da expressão do gene hnRNP Q-like (Heterogeneous nuclear ribonucleoprotein A-like) retroinserido no cromossomo B de Astatotilapia latifasciata

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

A step to the gigantic genome of the desert locust: chromosome sizes and repeated DNAs

The desert locust (Schistocerca gregaria) has been used as material for numerous cytogenetic studies. Its genome size is estimated to be 8.55 Gb of DNA comprised in 11 autosomes and the X chromosome. Its X0/XX sex chromosome determinism therefore results in females having 24 chromosomes whereas males have 23. Surprisingly, little is known about the DNA content of this locust's huge chromosomes. Here, we use the Feulgen Image Analysis Densitometry and C-banding techniques to respectively estimate the DNA quantity and heterochromatin content of each chromosome. We also identify three satellite DNAs using both restriction endonucleases and next-generation sequencing. We then use fluorescent in situ hybridization to determine the chromosomal location of these satellite DNAs as well as that of six tandem repeat DNA gene families. The combination of the results obtained in this work allows distinguishing between the different chromosomes not only by size, but also by the kind of repetitive DNAs that they contain. The recent publication of the draft genome of the migratory locust (Locusta migratoria), the largest animal genome hitherto sequenced, invites for sequencing even larger genomes. S. gregaria is a pest that causes high economic losses. It is thus among the primary candidates for genome sequencing. But this species genome is about 50 % larger than that of L. migratoria, and although next-generation sequencing currently allows sequencing large genomes, sequencing it would mean a greater challenge. The chromosome sizes and markers provided here should not only help planning the sequencing project and guide the assembly but would also facilitate assigning assembled linkage groups to actual chromosomes.

Capacitive DNA detection driven by electronic charge fluctuations in a graphene nanopore

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

Complete genome sequence and structural characterization of a novel iflavirus isolated from Opsiphanes invirae (Lepidoptera: Nymphalidae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

P-220-Escherichia coli displaying virulence features of multiple diarrheogenic pathotypes detected in a Crohn's disease patient

Background: The number of Escherichia coli in the gut of Crohn's disease (CD) patients is higher than that of normal subjects, but the virulence potential of these bacteria is not fully known. Previous studies have shown that these E. coli are closely related to extraintestinal pathogenic categories (ExPEC), are able to invade epithelial cells, and usually do not produce exotoxins. We report here the detection, in a CD patient, of an E. coli which belongs to a classical enteropathogenic (EPEC) serotype and displays virulence markers of enteroinvasive (EIEC), enteroaggregative (EAEC) and enterohemorrhagic (EHEC) pathotypes. Methods: The E. coli strain was isolated, in 2009, by classical bacteriological procedures from a 56 year old woman who underwent ileo-terminal resection 1 year before, due to intestinal obstruction. The bacterial characterization was carried out by in vitro adhesion and invasion assays to cultured epithelial cells and macrophages and screening by PCR to identify virulence genetic markers of diarrheogenic E. coli (DEC) and to detect one of the gene combinations which define the phylogroups of the E. coli reference (EcoR) collection. The strain was also tested for the ability to produce biofilm and shiga cytotoxins and had its whole genome sequenced by Ion Torrent Sequencing Technology. Results: The studied strain, which was detected both in ileum biopsies and the stools of the patient, displayed the aggregative adherence (AA) phenotype to Hep-2 cells and an ability to enter Caco-2 cells 3x as high as that of EIEC reference strain and 89% of that of the prototype AIEC LF82 strain. Although it could invade cultured macrophages, the strain was unable to replicate inside these cells. PCR screening revealed the presence of eae, aggR and stx1. Tests with bacterial culture supernatants in Vero cells demonstrating cytotoxicity suggested the production of Stx1. In addition, the strain revealed to be a strong biofilm producer, belonged to the B2 EcoR phylogroup, to the O126:H27 serogroup and to the multilocus sequencing type (MLST) ST3057. The 2 later features were deduced from the whole genome sequence of the strain. Conclusions: The characterization of this E. coli isolate from a CD patient revealed a combination of virulence markers of distinct DEC pathotypes, namely eae and stx1 of EHEC, AA, aggR and biofilm formation of EAEC, and invasiveness of EIEC. These features along with its serotype and phylogroup identity seem to suggest a potential to be involved in CD, an observation which should be tested with additional studies.

Identificação de genes de predisposição à síndrome dos carcinomas de mama e tireoide por sequenciamento total do exoma

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Assessing Pathogenicity for Novel Mutation/Sequence Variants: The Value of Healthy Older Individuals

Improvement in DNA technology is increasingly revealing unexpected/unknown mutations in healthy persons and generating anxiety due to their still unknown health consequences. We report a 44-year-old healthy father of a 10-year-old daughter with bilateral coloboma and hearing loss, but without muscle weakness, in whom a whole-genome CGH revealed a deletion of exons 38-44 in the dystrophin gene. This mutation was inherited from her asymptomatic father, who was further clinically and molecularly evaluated for prognosis and genetic counseling (GC). This deletion was never identified by us in 982 Duchenne/Becker patients. To assess whether the present case represents a rare case of non-penetrance, and aiming to obtain more information for prognosis and GC, we suggested that healthy older relatives submit their DNA for analysis, to which several complied. Mutation analysis revealed that his mother, brother, and 56-year-old maternal uncle also carry the 38-44 deletion, suggesting it an unlikely cause of muscle weakness. Genome sequencing will disclose mutations and variants whose health impact are still unknown, raising important problems in interpreting results, defining prognosis, and discussing GC. We suggest that, in addition to family history, keeping the DNA of older relatives could be very informative, in particular for those interested in having their genome sequenced.

Mutations in SRCAP, Encoding SNF2-Related CREBBP Activator Protein, Cause Floating-Harbor Syndrome

Floating-Harbor syndrome (FHS) is a rare condition characterized by short stature, delayed osseous maturation, expressive-language deficits, and a distinctive facial appearance. Occurrence is generally sporadic, although parent-to-child transmission has been reported on occasion. Employing whole-exome sequencing, we identified heterozygous truncating mutations in SRCAP in five unrelated individuals with sporadic MS. Sanger sequencing identified mutations in SRCAP in eight more affected persons. Mutations were de novo in all six instances in which parental DNA was available. SRCAP is an SNF2-related chromatin-remodeling factor that serves as a coactivator for CREB-binding protein (CREBBP, better known as CBP, the major cause of Rubinstein-Taybi syndrome [RTS]). Five SRCAP mutations, two of which are recurrent, were identified; all are tightly clustered within a small (111 codon) region of the final exon. These mutations are predicted to abolish three C-terminal AT-hook DNA-binding motifs while leaving the CBP-binding and ATPase domains intact. Our findings show that SRCAP mutations are the major cause of FHS and offer an explanation for the clinical overlap between FHS and RTS.

Haploinsufficiency of a Spliceosomal GTPase Encoded by EFTUD2 Causes Mandibulofacial Dysostosis with Microcephaly

Mandibulofacial dysostosis with microcephaly (MFDM) is a rare sporadic syndrome comprising craniofacial malformations, microcephaly, developmental delay, and a recognizable dysmorphic appearance. Major sequelae, including choanal atresia, sensorineural hearing loss, and cleft palate, each occur in a significant proportion of affected individuals. We present detailed clinical findings in 12 unrelated individuals with MFDM; these 12 individuals compose the largest reported cohort to date. To define the etiology of MFDM, we employed whole-exome sequencing of four unrelated affected individuals and identified heterozygous mutations or deletions of EFTUD2 in all four. Validation studies of eight additional individuals with MFDM demonstrated causative EFTUD2 mutations in all affected individuals tested. A range of EPTUD2-mutation types, including null alleles and frameshifts, is seen in MFDM, consistent with haploinsufficiency; segregation is de novo in all cases assessed to date. U5-116kD, the protein encoded by EFTUD2, is a highly conserved spliceosomal GTPase with a central regulatory role in catalytic splicing and post-splicing-complex disassembly. MFDM is the fast multiple-malformation syndrome attributed to a defect of the major spliceosome. Our findings significantly extend the range of reported spliceosomal phenotypes in humans and pave the way for further investigation in related conditions such as Treacher Collins syndrome.

Genome wide scan for quantitative trait loci affecting tick resistance in cattle (Bos taurus × Bos indicus)

Abstract Background In tropical countries, losses caused by bovine tick Rhipicephalus (Boophilus) microplus infestation have a tremendous economic impact on cattle production systems. Genetic variation between Bos taurus and Bos indicus to tick resistance and molecular biology tools might allow for the identification of molecular markers linked to resistance traits that could be used as an auxiliary tool in selection programs. The objective of this work was to identify QTL associated with tick resistance/susceptibility in a bovine F2 population derived from the Gyr (Bos indicus) × Holstein (Bos taurus) cross. Results Through a whole genome scan with microsatellite markers, we were able to map six genomic regions associated with bovine tick resistance. For most QTL, we have found that depending on the tick evaluation season (dry and rainy) different sets of genes could be involved in the resistance mechanism. We identified dry season specific QTL on BTA 2 and 10, rainy season specific QTL on BTA 5, 11 and 27. We also found a highly significant genome wide QTL for both dry and rainy seasons in the central region of BTA 23. Conclusions The experimental F2 population derived from Gyr × Holstein cross successfully allowed the identification of six highly significant QTL associated with tick resistance in cattle. QTL located on BTA 23 might be related with the bovine histocompatibility complex. Further investigation of these QTL will help to isolate candidate genes involved with tick resistance in cattle.

Genome analysis of DNA repair genes in the alpha proteobacterium Caulobacter crescentus

Abstract Background The integrity of DNA molecules is fundamental for maintaining life. The DNA repair proteins protect organisms against genetic damage, by removal of DNA lesions or helping to tolerate them. DNA repair genes are best known from the gamma-proteobacterium Escherichia coli, which is the most understood bacterial model. However, genome sequencing raises questions regarding uniformity and ubiquity of these DNA repair genes and pathways, reinforcing the need for identifying genes and proteins, which may respond to DNA damage in other bacteria. Results In this study, we employed a bioinformatic approach, to analyse and describe the open reading frames potentially related to DNA repair from the genome of the alpha-proteobacterium Caulobacter crescentus. This was performed by comparison with known DNA repair related genes found in public databases. As expected, although C. crescentus and E. coli bacteria belong to separate phylogenetic groups, many of their DNA repair genes are very similar. However, some important DNA repair genes are absent in the C. crescentus genome and other interesting functionally related gene duplications are present, which do not occur in E. coli. These include DNA ligases, exonuclease III (xthA), endonuclease III (nth), O6-methylguanine-DNA methyltransferase (ada gene), photolyase-like genes, and uracil-DNA-glycosylases. On the other hand, the genes imuA and imuB, which are involved in DNA damage induced mutagenesis, have recently been described in C. crescentus, but are absent in E. coli. Particularly interesting are the potential atypical phylogeny of one of the photolyase genes in alpha-proteobacteria, indicating an origin by horizontal transfer, and the duplication of the Ada orthologs, which have diverse structural configurations, including one that is still unique for C. crescentus. Conclusion The absence and the presence of certain genes are discussed and predictions are made considering the particular aspects of the C. crescentus among other known DNA repair pathways. The observed differences enlarge what is known for DNA repair in the Bacterial world, and provide a useful framework for further experimental studies in this organism.

Theoretical studies of Membrane Proteins : Properties, Prediction Methods and Genome-wide analysis

Membrane proteins are a large and important class of proteins. They are responsible for several of the key functions in a living cell, e.g. transport of nutrients and ions, cell-cell signaling, and cell-cell adhesion. Despite their importance it has not been possible to study their structure and organization in much detail because of the difficulty to obtain 3D structures. In this thesis theoretical studies of membrane protein sequences and structures have been carried out by analyzing existing experimental data. The data comes from several sources including sequence databases, genome sequencing projects, and 3D structures. Prediction of the membrane spanning regions by hydrophobicity analysis is a key technique used in several of the studies. A novel method for this is also presented and compared to other methods. The primary questions addressed in the thesis are: What properties are common to all membrane proteins? What is the overall architecture of a membrane protein? What properties govern the integration into the membrane? How many membrane proteins are there and how are they distributed in different organisms? Several of the findings have now been backed up by experiments. An analysis of the large family of G-protein coupled receptors pinpoints differences in length and amino acid composition of loops between proteins with and without a signal peptide and also differences between extra- and intracellular loops. Known 3D structures of membrane proteins have been studied in terms of hydrophobicity, distribution of secondary structure and amino acid types, position specific residue variability, and differences between loops and membrane spanning regions. An analysis of several fully and partially sequenced genomes from eukaryotes, prokaryotes, and archaea has been carried out. Several differences in the membrane protein content between organisms were found, the most important being the total number of membrane proteins and the distribution of membrane proteins with a given number of transmembrane segments. Of the properties that were found to be similar in all organisms, the most obvious is the bias in the distribution of positive charges between the extra- and intracellular loops. Finally, an analysis of homologues to membrane proteins with known topology uncovered two related, multi-spanning proteins with opposite predicted orientations. The predicted topologies were verified experimentally, providing a first example of "divergent topology evolution".

Design and implementation of bioinformatics tools for large scale genome annotation

The continuous increase of genome sequencing projects produced a huge amount of data in the last 10 years: currently more than 600 prokaryotic and 80 eukaryotic genomes are fully sequenced and publically available. However the sole sequencing process of a genome is able to determine just raw nucleotide sequences. This is only the first step of the genome annotation process that will deal with the issue of assigning biological information to each sequence. The annotation process is done at each different level of the biological information processing mechanism, from DNA to protein, and cannot be accomplished only by in vitro analysis procedures resulting extremely expensive and time consuming when applied at a this large scale level. Thus, in silico methods need to be used to accomplish the task. The aim of this work was the implementation of predictive computational methods to allow a fast, reliable, and automated annotation of genomes and proteins starting from aminoacidic sequences. The first part of the work was focused on the implementation of a new machine learning based method for the prediction of the subcellular localization of soluble eukaryotic proteins. The method is called BaCelLo, and was developed in 2006. The main peculiarity of the method is to be independent from biases present in the training dataset, which causes the over‐prediction of the most represented examples in all the other available predictors developed so far. This important result was achieved by a modification, made by myself, to the standard Support Vector Machine (SVM) algorithm with the creation of the so called Balanced SVM. BaCelLo is able to predict the most important subcellular localizations in eukaryotic cells and three, kingdom‐specific, predictors were implemented. In two extensive comparisons, carried out in 2006 and 2008, BaCelLo reported to outperform all the currently available state‐of‐the‐art methods for this prediction task. BaCelLo was subsequently used to completely annotate 5 eukaryotic genomes, by integrating it in a pipeline of predictors developed at the Bologna Biocomputing group by Dr. Pier Luigi Martelli and Dr. Piero Fariselli. An online database, called eSLDB, was developed by integrating, for each aminoacidic sequence extracted from the genome, the predicted subcellular localization merged with experimental and similarity‐based annotations. In the second part of the work a new, machine learning based, method was implemented for the prediction of GPI‐anchored proteins. Basically the method is able to efficiently predict from the raw aminoacidic sequence both the presence of the GPI‐anchor (by means of an SVM), and the position in the sequence of the post‐translational modification event, the so called ω‐site (by means of an Hidden Markov Model (HMM)). The method is called GPIPE and reported to greatly enhance the prediction performances of GPI‐anchored proteins over all the previously developed methods. GPIPE was able to predict up to 88% of the experimentally annotated GPI‐anchored proteins by maintaining a rate of false positive prediction as low as 0.1%. GPIPE was used to completely annotate 81 eukaryotic genomes, and more than 15000 putative GPI‐anchored proteins were predicted, 561 of which are found in H. sapiens. In average 1% of a proteome is predicted as GPI‐anchored. A statistical analysis was performed onto the composition of the regions surrounding the ω‐site that allowed the definition of specific aminoacidic abundances in the different considered regions. Furthermore the hypothesis that compositional biases are present among the four major eukaryotic kingdoms, proposed in literature, was tested and rejected. All the developed predictors and databases are freely available at: BaCelLo http://gpcr.biocomp.unibo.it/bacello eSLDB http://gpcr.biocomp.unibo.it/esldb GPIPE http://gpcr.biocomp.unibo.it/gpipe