Next-generation sequencing of experimental mouse strains.

Since the turn of the century the complete genome sequence of just one mouse strain, C57BL/6J, has been available. Knowing the sequence of this strain has enabled large-scale forward genetic screens to be performed, the creation of an almost complete set of embryonic stem (ES) cell lines with targeted alleles for protein-coding genes, and the generation of a rich catalog of mouse genomic variation. However, many experiments that use other common laboratory mouse strains have been hindered by a lack of whole-genome sequence data for these strains. The last 5 years has witnessed a revolution in DNA sequencing technologies. Recently, these technologies have been used to expand the repertoire of fully sequenced mouse genomes. In this article we review the main findings of these studies and discuss how the sequence of mouse genomes is helping pave the way from sequence to phenotype. Finally, we discuss the prospects for using de novo assembly techniques to obtain high-quality assembled genome sequences of these laboratory mouse strains, and what advances in sequencing technologies may be required to achieve this goal.

Genomic analysis of a nontoxigenic, invasive Corynebacterium diphtheriae strain from Brazil

We report the complete genome sequence and analysis of an invasive Corynebacterium diphtheriae strain that caused endocarditis in Rio de Janeiro, Brazil. It was selected for sequencing on the basis of the current relevance of nontoxigenic strains for public health. The genomic information was explored in the context of diversity, plasticity and genetic relatedness with other contemporary strains.

Isolation of a novel monkey adenovirus reveals a new phylogenetic clade in the evolutionary history of simian adenoviruses

Adenoviruses of primates include human (HAdV) and simian (SAdV) isolates classified into 8 species (Human Adenovirus A to G, and Simian Adenovirus A). In this study, a novel adenovirus was isolated from a colony of cynomolgus macaques (Macaca fascicularis) and subcultured in VERO cells. Its complete genome was purified and a region encompassing the hexon gene, the protease gene, the DNA binding protein (DBP) and the 100 kDa protein was amplified by PCR and sequenced by primer walking. Sequence analysis of these four genes showed that the new isolate had 80% identity to other primate adenoviruses and lacked recombination events. The study of the evolutionary relationships of this new monkey AdV based on the combined sequences of the four genes supported a close relationship to SAdV-3 and SAdV-6, lineages isolated from Rhesus monkeys. The clade formed by these three types is separated from the remaining clades and establishes a novel branch that is related to species HAdV-A, F and G. However, the genetic distance corresponding to the newly isolated monkey AdV considerably differs from these as to belong to a new, not yet established species. Results presented here widen our knowledge on SAdV and represents an important contribution to the understanding of the evolutionary history of primate adenoviruses.

Isolation of a novel monkey adenovirus reveals a new phylogenetic clade in the evolutionary history of simian adenoviruses

Adenoviruses of primates include human (HAdV) and simian (SAdV) isolates classified into 8 species (Human Adenovirus A to G, and Simian Adenovirus A). In this study, a novel adenovirus was isolated from a colony of cynomolgus macaques (Macaca fascicularis) and subcultured in VERO cells. Its complete genome was purified and a region encompassing the hexon gene, the protease gene, the DNA binding protein (DBP) and the 100 kDa protein was amplified by PCR and sequenced by primer walking. Sequence analysis of these four genes showed that the new isolate had 80% identity to other primate adenoviruses and lacked recombination events. The study of the evolutionary relationships of this new monkey AdV based on the combined sequences of the four genes supported a close relationship to SAdV-3 and SAdV-6, lineages isolated from Rhesus monkeys. The clade formed by these three types is separated from the remaining clades and establishes a novel branch that is related to species HAdV-A, F and G. However, the genetic distance corresponding to the newly isolated monkey AdV considerably differs from these as to belong to a new, not yet established species. Results presented here widen our knowledge on SAdV and represents an important contribution to the understanding of the evolutionary history of primate adenoviruses.

Comparative genomic and phylogeographic analysis of Mycobacterium leprae.

Reductive evolution and massive pseudogene formation have shaped the 3.31-Mb genome of Mycobacterium leprae, an unculturable obligate pathogen that causes leprosy in humans. The complete genome sequence of M. leprae strain Br4923 from Brazil was obtained by conventional methods (6x coverage), and Illumina resequencing technology was used to obtain the sequences of strains Thai53 (38x coverage) and NHDP63 (46x coverage) from Thailand and the United States, respectively. Whole-genome comparisons with the previously sequenced TN strain from India revealed that the four strains share 99.995% sequence identity and differ only in 215 polymorphic sites, mainly SNPs, and by 5 pseudogenes. Sixteen interrelated SNP subtypes were defined by genotyping both extant and extinct strains of M. leprae from around the world. The 16 SNP subtypes showed a strong geographical association that reflects the migration patterns of early humans and trade routes, with the Silk Road linking Europe to China having contributed to the spread of leprosy.

BadiRate: Estimating Family Turnover Rates by Likelihood-Based Methods

Motivation: The comparative analysis of gene gain and loss rates is critical for understanding the role of natural selection and adaptation in shaping gene family sizes. Studying complete genome data from closely related species allows accurate estimation of gene family turnover rates. Current methods and software tools, however, are not well designed for dealing with certain kinds of functional elements, such as microRNAs or transcription factor binding sites. Results: Here, we describe BadiRate, a new software tool to estimate family turnover rates, as well as the number of elements in internal phylogenetic nodes, by likelihood-based methods and parsimony. It implements two stochastic population models, which provide the appropriate statistical framework for testing hypothesis, such as lineage-specific gene family expansions or contractions. We have assessed the accuracy of BadiRate by computer simulations, and have also illustrated its functionality by analyzing a representative empirical dataset.

BadiRate: estimating family turnover rates by likelihood-based methods

Motivation: The comparative analysis of gene gain and loss rates is critical for understanding the role of natural selection and adaptation in shaping gene family sizes. Studying complete genome data from closely related species allows accurate estimation of gene family turnover rates. Current methods and software tools, however, are not well designed for dealing with certain kinds of functional elements, such as microRNAs or transcription factor binding sites. Results: Here, we describe BadiRate, a new software tool to estimate family turnover rates, as well as the number of elements in internal phylogenetic nodes, by likelihood-based methods and parsimony. It implements two stochastic population models, which provide the appropriate statistical framework for testing hypothesis, such as lineage-specific gene family expansions or contractions. We have assessed the accuracy of BadiRate by computer simulations, and have also illustrated its functionality by analyzing a representative empirical dataset.

Breeding, genetic and genomic of citrus for disease resistance

Although the citriculture is one of the most important economic activities in Brazil, it is based on a small number of varieties. This fact has contributed for the vulnerability of the culture regarding the phytosanitary problems. A higher number of varieties/genotypes with potential for commercial growing, either for the industry or fresh market, has been one of the main objectives of citrus breeding programs. The genetic breeding of citrus has improved, in the last decades, due to the possibility of an association between biotechnological tools and classical methods of breeding. The use of molecular markers for early selection of zygotic seedlings from controlled crosses resulted in the possibility of selection of a high number of new combination and, as a consequence, the establishment of a great number of hybrids in field experiments. The faster new tools are incorporated in the program, the faster is possibility to reach new genotypes that can be tested as a new variety. Good traits should be kept or incorporate, whereas bad traits have to be excluded or minimized in the new genotype. Scion and rootstock can not be considered separately, and graft compatibility, fruit quality and productivity are essential traits to be evaluated in the last stages of the program. The mapping of QTLs has favored breeding programs of several perennial species and in citrus it was possible to map several characteristics with qualitative and quantitative inheritance. The existence of linkage maps and QTLs already mapped, the development of EST and BAC library and the sequencing of the Citrus complete genome altogether make very demanding and urgent the exploration of such data to launch a wider genetic study of citrus. The rising of information on genome of several organisms has opened new approaches looking for integration between breeding, genetic and genome. Genome assisted selection (GAS) involves more than gene or complete genome sequencing and is becoming an import support in breeding programs of annual and perennial species. An huge information amount can be derivate from genome analysis. The use and benefit of such informations will depend on the genetic basis of the breeding program.

Genomic analysis of the blood attributed to Louis XVI (1754-1793), king of France.

A pyrographically decorated gourd, dated to the French Revolution period, has been alleged to contain a handkerchief dipped into the blood of the French king Louis XVI (1754-1793) after his beheading but recent analyses of living males from two Bourbon branches cast doubts on its authenticity. We sequenced the complete genome of the DNA contained in the gourd at low coverage (similar to 2.5x) with coding sequences enriched at a higher similar to 7.3x coverage. We found that the ancestry of the gourd's genome does not seem compatible with Louis XVI's known ancestry. From a functional perspective, we did not find an excess of alleles contributing to height despite being described as the tallest person in Court. In addition, the eye colour prediction supported brown eyes, while Louis XVI had blue eyes. This is the first draft genome generated from a person who lived in a recent historical period; however, our results suggest that this sample may not correspond to the alleged king.

Evolutionary analysis of mitogenomes from parasitic and free-living flatworms

Mitochondrial genomes (mitogenomes) are useful and relatively accessible sources of molecular data to explore and understand the evolutionary history and relationships of eukaryotic organisms across diverse taxonomic levels. The availability of complete mitogenomes from Platyhelminthes is limited; of the 40 or so published most are from parasitic flatworms (Neodermata). Here, we present the mitogenomes of two free-living flatworms (Tricladida): the complete genome of the freshwater species Crenobia alpina (Planariidae) and a nearly complete genome of the land planarian Obama sp. (Geoplanidae). Moreover, we have reanotated the published mitogenome of the species Dugesia japonica (Dugesiidae). This contribution almost doubles the total number of mtDNAs published for Tricladida, a species-rich group including model organisms and economically important invasive species. We took the opportunity to conduct comparative mitogenomic analyses between available free-living and selected parasitic flatworms in order to gain insights into the putative effect of life cycle on nucleotide composition through mutation and natural selection. Unexpectedly, we did not find any molecular hallmark of a selective relaxation in mitogenomes of parasitic flatworms; on the contrary, three out of the four studied free-living triclad mitogenomes exhibit higher A+T content and selective relaxation levels. Additionally, we provide new and valuable molecular data to develop markers for future phylogenetic studies on planariids and geoplanids.

Isolation of DNA sequences with homology to a degenerate FaRP oligonucleotide from the crayfish Procambarus clarkii

The neuropeptide Th1RFamide with the sequence Phe-Met-Arg-Phe-amide was originally isolated in the clam Macrocallista nimbosa (price and Greenberg, 1977). Since its discovery, a large family ofFl\1RFamide-related peptides termed FaRPs have been found to be present in all major animal phyla with functions ranging from modulation of neuronal activity to alteration of muscular contractions. However, little is known about the genetics encoding these peptides, especially in invertebrates. As FaRP-encoding genes have yet to be investigated in the invertebrate Malacostracean subphylum, the isolation and characterization ofFaRP-encoding DNA and mRNA was pursued in this project. The immediate aims of this thesis were: (1) to amplify mRNA sequences of Procambarus clarkii using a degenerate oligonucleotide primer deduced from the common amino acid sequence ofisolated Procambarus FaRPS, (2) to determine if these amplification products encode FaRP gene sequences, and (3) to create a selective cDNA library of sequences recognized by the degenerate oligonucleotide primer. The polymerase chain reaction - rapid amplification of cDNA ends (PCR-RACE) is a procedure in which a single gene-specific primer is used in conjunction with a generalized 3' or 5' primer to amplify copies ofthe region between a single point in the transcript and the 3' or 5' end of cDNA of interest (Frohman et aI., 1988). PCRRACE reactions were optimized with respect to primers used, buffer composition, cycle number, nature ofgenetic substrate to be amplified, annealing, extension and denaturation temperatures and times, and use of reamplification procedures. Amplification products were cloned into plasmid vectors and recombinant products were isolated, as were the recombinant plaques formed in the selective cDNA library. Labeled amplification products were hybridized to recombinant bacteriophage to determine ligated amplification product presence. When sequenced, the five isolated PCR-RACE amplification products were determined not to possess FaRP-encoding sequences. The 200bp, 450bp, and 1500bp sequences showed homology to the Caenorhabditis elegans cosmid K09A11, which encodes for cytochrome P450; transfer-RNA; transposase; and tRNA-Tyr, while the 500bp and 750bp sequences showed homology with the complete genome of the Vaccinia virus. Under the employed amplification conditions the degenerate oligonucleotide primer was observed to bind to and to amplify sequences with either 9 or 10bp of 17bp identity. The selective cDNA library was obselVed to be of extremely low titre. When library titre was increased, white. plaques were isolated. Amplification analysis of eight isolated Agt11 sequences from these plaques indicated an absence of an insertion sequence. The degenerate 17 base oligonucleotide primer synthesized from the common amino acid sequence ofisolated Procambarus FaRPs was thus determined to be non-specific in its binding under the conditions required for its use, and to be insufficient for the isolation and identification ofFaRP-encoding sequences. A more specific primer oflonger sequence, lower degeneracy, and higher melting temperature (TJ is recommended for further investigation into the FaRP-encoding genes of Procambarlls clarkii.

New scientific paradigms for probiotics and prebiotics

The inaugural meeting of the International Scientific Association for Probiotics and Prebiotics (ISAPP) was held May 3 to May 5 2002 in London, Ontario, Canada. A group of 63 academic and industrial scientists from around the world convened to discuss current issues in the science of probiotics and prebiotics. ISAPP is a non-profit organization comprised of international scientists whose intent is to strongly support and improve the levels of scientific integrity and due diligence associated with the study, use, and application of probiotics and prebiotics. In addition, ISAPP values its role in facilitating communication with the public and healthcare providers and among scientists in related fields on all topics pertinent to probiotics and prebiotics. It is anticipated that such efforts will lead to development of approaches and products that are optimally designed for the improvement of human and animal health and well being. This article is a summary of the discussions, conclusions, and recommendations made by 8 working groups convened during the first ISAPP workshop focusing on the topics of: definitions, intestinal flora, extra-intestinal sites, immune function, intestinal disease, cancer, genetics and genomics, and second generation prebiotics. Humans have evolved in symbiosis with an estimated 1014 resident microorganisms. However, as medicine has widely defined and explored the perpetrators of disease, including those of microbial origin, it has paid relatively little attention to the microbial cells that constitute the most abundant life forms associated with our body. Microbial metabolism in humans and animals constitutes an intense biochemical activity in the body, with profound repercussions for health and disease. As understanding of the human genome constantly expands, an important opportunity will arise to better determine the relationship between microbial populations within the body and host factors (including gender, genetic background, and nutrition) and the concomitant implications for health and improved quality of life. Combined human and microbial genetic studies will determine how such interactions can affect human health and longevity, which communication systems are used, and how they can be influenced to benefit the host. Probiotics are defined as live microorganisms which, when administered in adequate amounts confer a health benefit on the host.1 The probiotic concept dates back over 100 years, but only in recent times have the scientific knowledge and tools become available to properly evaluate their effects on normal health and well being, and their potential in preventing and treating disease. A similar situation exists for prebiotics, defined by this group as non-digestible substances that provide a beneficial physiological effect on the host by selectively stimulating the favorable growth or activity of a limited number of indigenous bacteria. Prebiotics function complementary to, and possibly synergistically with, probiotics. Numerous studies are providing insights into the growth and metabolic influence of these microbial nutrients on health. Today, the science behind the function of probiotics and prebiotics still requires more stringent deciphering both scientifically and mechanistically. The explosion of publications and interest in probiotics and prebiotics has resulted in a body of collective research that points toward great promise. However, this research is spread among such a diversity of organisms, delivery vehicles (foods, pills, and supplements), and potential health targets such that general conclusions cannot easily be made. Nevertheless, this situation is rapidly changing on a number of important fronts. With progress over the past decade on the genetics of lactic acid bacteria and the recent, 2,3 and pending, 4 release of complete genome sequences for major probiotic species, the field is now armed with detailed information and sophisticated microbiological and bioinformatic tools. Similarly, advances in biotechnology could yield new probiotics and prebiotics designed for enhanced or expanded functionality. The incorporation of genetic tools within a multidisciplinary scientific platform is expected to reveal the contributions of commensals, probiotics, and prebiotics to general health and well being and explicitly identify the mechanisms and corresponding host responses that provide the basis for their positive roles and associated claims. In terms of human suffering, the need for effective new approaches to prevent and treat disease is paramount. The need exists not only to alleviate the significant mortality and morbidity caused by intestinal diseases worldwide (especially diarrheal diseases in children), but also for infections at non-intestinal sites. This is especially worthy of pursuit in developing nations where mortality is too often the outcome of food and water borne infection. Inasmuch as probiotics and prebiotics are able to influence the populations or activities of commensal microflora, there is evidence that they can also play a role in mitigating some diseases. 5,6 Preliminary support that probiotics and prebiotics may be useful as intervention in conditions including inflammatory bowel disease, irritable bowel syndrome, allergy, cancer (especially colorectal cancer of which 75% are associated with diet), vaginal and urinary tract infections in women, kidney stone disease, mineral absorption, and infections caused by Helicobacter pylori is emerging. Some metabolites of microbes in the gut may also impact systemic conditions ranging from coronary heart disease to cognitive function, suggesting the possibility that exogenously applied microbes in the form of probiotics, or alteration of gut microecology with prebiotics, may be useful interventions even in these apparently disparate conditions. Beyond these direct intervention targets, probiotic cultures can also serve in expanded roles as live vehicles to deliver biologic agents (vaccines, enzymes, and proteins) to targeted locations within the body. The economic impact of these disease conditions in terms of diagnosis, treatment, doctor and hospital visits, and time off work exceeds several hundred billion dollars. The quality of life impact is also of major concern. Probiotics and prebiotics offer plausible opportunities to reduce the morbidity associated with these conditions. The following addresses issues that emerged from 8 workshops (Definitions, Intestinal Flora, Extra-Intestinal Sites, Immune Function, Intestinal Disease, Cancer, Genomics, and Second Generation Prebiotics), reflecting the current scientific state of probiotics and prebiotics. This is not a comprehensive review, however the study emphasizes pivotal knowledge gaps, and recommendations are made as to the underlying scientific and multidisciplinary studies that will be required to advance our understanding of the roles and impact of prebiotics, probiotics, and the commensal microflora upon health and disease management.

alpha-Hydroxynitrile lyase protein from Xylella fastidiosa: Cloning, expression, and characterization

Xylella fastidiosa is a xylem-restricted plant pathogen that causes a range of diseases in several and important crops. Through comparative genomic sequence analysis many genes were identified and, among them, several potentially involved in plant-pathogen interaction. The experimental determination of the primary sequence of some markedly expressed proteins for X fastidiosa and the comparison with the nucleic acids sequence of genome identified one of them as being SCJ21.16 (XFa0032) gene product. The comparative analysis of this protein against SWISSPROT database, in special, resulted in similarity with a-hydroxynitrile lyase enzyme (HNL) from Arabidopsis thaliana, causing interest for being one of the most abundant proteins both in the whole cell extract as well as in the extracellular protein fraction. It is known that HNL enzyme are involved in a process termed ""cyanogenesis"", which catalyzes the dissociation of alpha-hydroxinitrile into carbonyle and HCN when plant tissue is damaged. Although the complete genome sequences of X.fastidiosa are available and the cyanogenesis process is well known, the biological role of this protein in this organism is not yet functionally characterized. In this study we presented the cloning, expression, characterization of recombinant HNL from X fastidiosa, and its probable function in the cellular metabolism. The successful cloning and heterologous expression in Escherichia coli resulted in a satisfactory amount of the recombinant HNL expressed in a soluble, and active form giving convenient access to pure enzyme for biochemical and structural studies. Finally, our results confirmed that the product of the gene XFa0032 can be positively assigned as FAD-independent HNLs. (C) 2009 Elsevier Ltd. All rights reserved.

Estudo do genoma do vírus causador da mionecrose infecciosa em camarões e desenvolvimento de métodos para detecção de diferentes variantes

Shrimp farming is one of the activities that contribute most to the growth of global aquaculture. However, this business has undergone significant economic losses due to the onset of viral diseases such as Infectious Myonecrosis (IMN). The IMN is already widespread throughout Northeastern Brazil and affects other countries such as Indonesia, Thailand and China. The main symptom of disease is myonecrosis, which consists of necrosis of striated muscles of the abdomen and cephalothorax of shrimp. The IMN is caused by infectious myonecrosis virus (IMNV), a non-enveloped virus which has protrusions along its capsid. The viral genome consists of a single molecule of double-stranded RNA and has two Open Reading Frames (ORFs). The ORF1 encodes the major capsid protein (MCP) and a potential RNA binding protein (RBP). ORF2 encodes a probable RNA-dependent RNA polymerase (RdRp) and classifies IMNV in Totiviridae family. Thus, the objective of this research was study the IMNV complete genome and encoded proteins in order to develop a system differentiate virus isolates based on polymorphisms presence. The phylogenetic relationship among some totivirus was investigated and showed a new group to IMNV within Totiviridae family. Two new genomes were sequenced, analyzed and compared to two other genomes already deposited in GenBank. The new genomes were more similar to each other than those already described. Conserved and variable regions of the genome were identified through similarity graphs and alignments using the four IMNV sequences. This analyze allowed mapping of polymorphic sites and revealed that the most variable region of the genome is in the first half of ORF1, which coincides with the regions that possibly encode the viral protrusion, while the most stable regions of the genome were found in conserved domains of proteins that interact with RNA. Moreover, secondary structures were predicted for all proteins using various softwares and protein structural models were calculated using threading and ab initio modeling approaches. From these analyses was possible to observe that the IMNV proteins have motifs and shapes similar to proteins of other totiviruses and new possible protein functions have been proposed. The genome and proteins study was essential for development of a PCR-based detection system able to discriminate the four IMNV isolates based on the presence of polymorphic sites

Xylella fastidiosa: An in vivo system to study possible survival strategies within citrus xylem vessels based on global gene expression analysis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)