Finding candidate genes under positive selection in Non-model species: examples of genes involved in host specialization in pathogens.

Numerous genes in diverse organisms have been shown to be under positive selection, especially genes involved in reproduction, adaptation to contrasting environments, hybrid inviability, and host-pathogen interactions. Looking for genes under positive selection in pathogens has been a priority in efforts to investigate coevolution dynamics and to develop vaccines or drugs. To elucidate the functions involved in host specialization, here we aimed at identifying candidate sequences that could have evolved under positive selection among closely related pathogens specialized on different hosts. For this goal, we sequenced c. 17,000-32,000 ESTs from each of four Microbotryum species, which are fungal pathogens responsible for anther smut disease on host plants in the Caryophyllaceae. Forty-two of the 372 predicted orthologous genes showed significant signal of positive selection, which represents a good number of candidate genes for further investigation. Sequencing 16 of these genes in 9 additional Microbotryum species confirmed that they have indeed been rapidly evolving in the pathogen species specialized on different hosts. The genes showing significant signals of positive selection were putatively involved in nutrient uptake from the host, secondary metabolite synthesis and secretion, respiration under stressful conditions and stress response, hyphal growth and differentiation, and regulation of expression by other genes. Many of these genes had transmembrane domains and may therefore also be involved in pathogen recognition by the host. Our approach thus revealed fruitful and should be feasible for many non-model organisms for which candidate genes for diversifying selection are needed.

Assessing the performance of single-copy genes for recovering robust phylogenies.

Phylogenies involving nonmodel species are based on a few genes, mostly chosen following historical or practical criteria. Because gene trees are sometimes incongruent with species trees, the resulting phylogenies may not accurately reflect the evolutionary relationships among species. The increase in availability of genome sequences now provides large numbers of genes that could be used for building phylogenies. However, for practical reasons only a few genes can be sequenced for a wide range of species. Here we asked whether we can identify a few genes, among the single-copy genes common to most fungal genomes, that are sufficient for recovering accurate and well-supported phylogenies. Fungi represent a model group for phylogenomics because many complete fungal genomes are available. An automated procedure was developed to extract single-copy orthologous genes from complete fungal genomes using a Markov Clustering Algorithm (Tribe-MCL). Using 21 complete, publicly available fungal genomes with reliable protein predictions, 246 single-copy orthologous gene clusters were identified. We inferred the maximum likelihood trees using the individual orthologous sequences and constructed a reference tree from concatenated protein alignments. The topologies of the individual gene trees were compared to that of the reference tree using three different methods. The performance of individual genes in recovering the reference tree was highly variable. Gene size and the number of variable sites were highly correlated and significantly affected the performance of the genes, but the average substitution rate did not. Two genes recovered exactly the same topology as the reference tree, and when concatenated provided high bootstrap values. The genes typically used for fungal phylogenies did not perform well, which suggests that current fungal phylogenies based on these genes may not accurately reflect the evolutionary relationships among species. Analyses on subsets of species showed that the phylogenetic performance did not seem to depend strongly on the sample. We expect that the best-performing genes identified here will be very useful for phylogenetic studies of fungi, at least at a large taxonomic scale. Furthermore, we compare the method developed here for finding genes for building robust phylogenies with previous ones and we advocate that our method could be applied to other groups of organisms when more complete genomes are available.

PERMANENT GENETIC RESOURCES: Isolation of 60 polymorphic microsatellite loci in EST libraries of four sibling species of the phytopathogenic fungal complex Microbotryum.

We report the development of 60 microsatellite markers on four species of the fungal complex Microbotryum, causing anther smut of the Caryophyllaceae. Microsatellites were found in four expressed sequence tag (EST) libraries, built from isolates of M. lychnis-dioicae, M. violaceum sensus stricto, M. lagerheimii and M. dianthorum, collected, respectively, from the plants Silene latifolia, S. nutans, S. vulgaris and Dianthus carthusianorum. Intrapopulation polymorphism was investigated using 24 isolates, and cross-amplification was explored using 23 isolates belonging to at least 10 different Microbotryum species. This study provides numerous microsatellite markers for population genetics and mapping studies.

Systematic determination of the mosaic structure of bacterial genomes: species backbone versus strain-specific loops.

BACKGROUND: Public databases now contain multitude of complete bacterial genomes, including several genomes of the same species. The available data offers new opportunities to address questions about bacterial genome evolution, a task that requires reliable fine comparison data of closely related genomes. Recent analyses have shown, using pairwise whole genome alignments, that it is possible to segment bacterial genomes into a common conserved backbone and strain-specific sequences called loops. RESULTS: Here, we generalize this approach and propose a strategy that allows systematic and non-biased genome segmentation based on multiple genome alignments. Segmentation analyses, as applied to 13 different bacterial species, confirmed the feasibility of our approach to discern the 'mosaic' organization of bacterial genomes. Segmentation results are available through a Web interface permitting functional analysis, extraction and visualization of the backbone/loops structure of documented genomes. To illustrate the potential of this approach, we performed a precise analysis of the mosaic organization of three E. coli strains and functional characterization of the loops. CONCLUSION: The segmentation results including the backbone/loops structure of 13 bacterial species genomes are new and available for use by the scientific community at the URL: http://genome.jouy.inra.fr/mosaic.

Deciphering Arabidopsis thaliana gene neighborhoods through bibliographic co-citations.

In the framework of genome annotation, scientific literature is obviously the major source of biological knowledge. The aim of the work described in this paper is to exploit this source of data for the model plant Arabidopsis thaliana. The first step has consisted in constituting a relevant bibliographic references dataset for plant genomic research. Genes co-citations have then been systematically annotated in this reference dataset, starting from the simple idea that if genes are cited in the same publication, they must probably share some related functional properties. In order to deal with the synonymous gene name problem, a gene name reference list has been constituted starting from A. thaliana SwissProt entries. This list was used to build clusters of co-cited genes by a single linkage procedure such that any gene in a given cluster possesses at least one co-cited partner in the same cluster. Analysis of the clusters demonstrate the biological consistency of this approach, with only very few fortuitous links. As an example, a cluster including genes related to flowering time is more deeply described in the paper. Finally, a graphical representation of each cluster was performed, which provides a convenient way to retrieve the genes (the nodes of the graphs) and the references in which they were co-cited (the edges of the graphs). All the results can be accessed at the URL http://chlora.Igi.infobiogen.fr:1234/bib_arath/.

SPiD: a subtilis protein interaction database.

MOTIVATION: Protein-protein interactions are a potential source of valuable clues in determining the functional role of as yet uncharacterized gene products in metabolic pathways. Graph-like structures emerging from the accumulation of interaction data make it difficult to maintain a consistent and global overview by hand. Bioinformatics tools are needed to perform this graph visualization while maintaining a link to the experimental data. RESULTS: "SPiD" is an online database for exploring networks of interacting proteins in Bacillus subtilis characterized by the two-hybrid system. Graphical displays of interaction networks are created dynamically as users interactively navigate through these networks. Third party applications can interface the database through a Common Object Request Broker Architecture (CORBA) tier. AVAILABILITY: SPiD is available through its web site at http://www-mig.versailles.inra.fr/bdsi/SPiD, and through an Interoperable Object Reference (IOR) and its associated Interface Definition Language (IDL). CONTACT: hoebeke@versailles.inra.fr

Tnt1 transposition events are induced by in vitro transformation of Arabidopsis thaliana, and transposed copies integrate into genes.

Tissue culture has been shown to induce the transposition of plant transposable elements; their insertion at novel sites results in somaclonal variation. Introduction of the tobacco retrotransposon Tnt1 into Arabidopsis thaliana by co-cultivation of root explants with Agrobacterium tumefaciens induces its transposition at a high frequency, but no transposed copies are found in plants transformed by the in planta procedure. Transposition occurs in the transformed root cells or in the calli derived from them, allowing the regeneration of transformed plants with up to 26 transposed copies of Tnt1. Analysis of Tnt1 integration sites in Arabidopsis shows that the Tnt1 endonuclease does not show any cleavage-site specificity at the sequence level. The insertion sites are unlinked and distributed on all five Arabidopsis chromosomes. The fact that the majority of the integration sites are located in coding regions, and none in repeated sequences, demonstrates the potential of Tnt1 as a tool for gene tagging.

Detecting localized repeats in genomic sequences: a new strategy and its application to Bacillus subtilis and Arabidopsis thaliana sequences.

A new method for the search of local repeats in long DNA sequences, such as complete genomes, is presented. It detects a large variety of repeats varying in length from one to several hundred bases, which may contain many mutations. By mutations we mean substitutions, insertions or deletions of one or more bases. The method is based on counting occurrences of short words (3-12 bases) in sequence fragments called windows. A score is computed for each window, based on calculating exact word occurrence probabilities for all the words of a given length in the window. The probabilities are defined using a Bernoulli model (independent letters) for the sequence, using the actual letter frequencies from each window. A plot of the probabilities along the sequence for high-scoring windows facilitates the identification of the repeated patterns. We applied the method to the 1.87 Mb sequence of chromosome 4 of Arabidopsis thaliana and to the complete genome of Bacillus subtilis (4.2 Mb). The repeats that we found were classified according to their size, number of occurrences, distance between occurrences, and location with respect to genes. The method proves particularly useful in detecting long, inexact repeats that are local, but not necessarily tandem. The method is implemented as a C program called EXCEP, which is available on request from the authors.

Codon usage as a tool to predict the cellular location of eukaryotic ribosomal proteins and aminoacyl-tRNA synthetases.

In spite of many efforts, the prediction of the location of proteins in eukaryotic cells (cytoplasm, mitochondrion or chloroplast) is still far from straightforward. In some cases (e.g. ribosomal proteins and aminoacyl-tRNA synthetases) both the cytoplasmic proteins and their organellar counterparts are encoded by the nuclear genome. A factorial correspondence analysis of the codon usage in yeast and Caenorhabditis elegans shows that the codon usage of those nuclear genes encoding ribosomal proteins or aminoacyl-tRNA synthetases is markedly different, depending on the final location of the proteins (cytoplasmic or mitochondrial). As a consequence, the location of such proteins-whose sequences are now frequently determined by systematic genomic sequencing-can be easily and quickly predicted. A WWW interface has been developed, aimed at providing a user-friendly tool for codon usage pattern analysis. It is available from http://www.genetique.uvsq.fr/afc.html

Indigo: a World-Wide-Web review of genomes and gene functions.

The present article describes a genome database reviewing gene-related knowledge of two model bacteria, Bacillus subtilis and Escherichia coli. The database, Indigo, is open through the World-Wide Web (http://indigo.genetique.uvsq.fr). The concept used for organising the data, the concept of neighbourhood, allows one to explore the database content in an efficient although somewhat unusual way. Here, genes are related to each other by a variety of neighbourhoods, including proximity in the chromosome, phylogenetic kinship, participation in a common metabolic pathway, common presence in an article of the literature, or similar use of the genetic code. Several examples illustrate how this concept of neighbourhood permits one to review the available knowledge about a given gene or gene family, and elaborate unexpected, but revealing, analyses about gene functions.

Codon usage and gene function are related in sequences of Arabidopsis thaliana.

In this paper, the relationship between codon usage and the physiological pattern of expression of a gene is investigated while considering a dataset of 815 nuclear genes of Arabidopsis thaliana. Factorial Correspondence Analysis, a commonly used multivariate statistical approach in codon usage analysis, was used in order to analyse codon usage bias gene by gene. The analysis reveals a single major trend in codon usage among genes in Arabidopsis. At one end of the trend lie genes with a highly G/C biased codon usage. This group contains mainly photosynthetic and housekeeping genes which are known to encode the most abundant proteins of the vegetal cell. At the other extreme lie genes with a weaker A/T-biased codon usage. This group contain genes with various functions which exhibits most of the time a strong tissue-specific pattern of expression in relation, for example, to stress conditions. These observations were confirmed by the detailed analysis of codon usage in the multigene family of tubulins and appear to be general in plant species, even as distant from Arabidopsis thaliana as a monocotyledonous plant such as maize.

Evolution of genes, evolution of species: the case of aminoacyl-tRNA synthetases.

All of the aminoacyl-tRNA synthetase (aaRS) sequences currently available in the data banks have been subjected to a systematic analysis aimed at finding gene duplications, genetic recombinations, and horizontal transfers. Evidence is provided for the occurrence (or probable occurrence) of such phenomena within this class of enzymes. In particular, it is suggested that the monomeric PheRS from the yeast mitochondrion is a chimera of the alpha and beta chains of the standard tetrameric protein. In addition, it is proposed that the dimeric and tetrameric forms of GlyRS are the result of a double and independent acquisition of the same specificity within two different subclasses of aaRS. The phylogenetic reconstructions of the evolutionary histories of the genes encoding aaRS are shown to be extremely diverse. While large segments of the population are consistent with the broad grouping into the three Woesian domains, some phylogenetic reconstructions do not place the Archae and the Eucarya as sister groups but, rather, show a gram-negative bacteria/eukaryote clustering. In addition, many individual genes pose difficulties that preclude any simple evolutionary scheme. Thus, aaRS's are clearly a paradigm of F. Jacob's "odd jobs of evolution" but, on the whole, do not call into question the evolutionary scenario originally proposed by Woese and subsequently refined by others.

An inventory of 1152 expressed sequence tags obtained by partial sequencing of cDNAs from Arabidopsis thaliana.

As part of the goal to generate a detailed transcript map for Arabidopsis thaliana, 1152 single run sequences (expressed sequence tags or ESTs) have been determined from cDNA clones taken at random in libraries prepared from different sources of plant material: developing siliques, etiolated seedlings, flower buds, and cultured cells. Eight hundred and ninety-five different genes could be identified, 32% of which showed significant similarity to existing sequences in Arabidopsis and an array of other organisms. These sequences in combination with their positioning on the Arabidopsis genetic map will not only constitute a new set of molecular markers for genome analysis in Arabidopsis but also provide a direct route for the in vivo analysis of their gene products. The sequences have been made available to the public databases.

[Urea cycle and purinolysis in the embryos and pregnant females of the teleost fish Poecilia reticulata]. / Cycle de l'urée et purinolyse dans les embryons et les femelles gravides du poisson téléostéen Poecilia reticulata.

The presence of a functional ornithine-urea cycle is evidenced in the embryos of the viviparous Teleost Poecilia reticulata. The activity of the cycle decreases during developmental period but persists in adult tissues. Purinolytic (uricolytic) pathway is another source of urea in both embryos and adults. The implications of these findings are briefly discussed in relation to the problems of ureogenic and ureosmotic phenomena in Teleostean Fishes.