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Nucleotide or amino-acid sequences are interpreted as successions of words of length k (k-tuples) the frequencies of which are highly variable in different statistical populations of genes or proteins. After building k-tuple reference tables from coherent subsets or entire data banks, the local information content profile of individual sequences is drawn. Anomalous regions (peaks or depressions) of such a profile can lead to the discovery and identification of specific sequence patterns. Along the same principle, the simultaneous use of two reference statistical populations and the computation of an index combining the two information profiles lead to a general and powerful discriminant analysis methods. The identification of a "signal" associated with gene conversion, the introns/exons discrimination and the location of function specific patterns in proteins are given as examples of successful applications of this heuristic informational approach.

The hypothesis that self as well as foreign proteins are processed into peptides and presented by major histocompatibility complex antigens leads to a set of working principles that could govern cellular interactions in immune responses. In particular, "idiopeptides," derived from immunoglobulins and T-cell receptors and recognized by appropriate T cells, are expected to play an important regulatory role. We show here that these speculations fit into a consistent view of the immune system.

In Chinese hamster somatic cells, the spontaneous change of phcnotype from 2-deoxygalactose sensitivity to resistance was studied using fluctuation test experiments à la LURIA and DELBRUCK (1943) for four Chinese hamster cell strains derived from V79. The results are consistent with true mutational events. The mutation rates are in the range of 1 to 3.5 × 10-5 per cell per generation. The relationship between the 2-deoxygalactose resistance and the galactokinase markers is discussed.

Publisher Summary It was recognized that in most cases the antigen is processed by the antigen-presenting cells (APC), such that the fragments of antigen, rather than antigen per se, are presented by major histocompatibility complex (MHC) molecules and are recognized by the T cells (TcRs). There are numerous examples of synthetic peptides corresponding to defined portions of protein antigens that actually substitute for antigen in well-defined immunological reactions involving T cells. In several instances, it was shown directly that such peptides bind to MHC molecules. So far, however, such biochemical data are rather scarce and the binding of the processed antigen to the MHC molecules is often inferred from functional assays involving recognition by TcRs. It is well known that most MHC molecules are highly polymorphic. Polymorphism of MHC molecules may influence both the antigen presentation and recognition by the TcRs in many ways. Polymorphic residues from the peptide-binding site may influence the selection of a given set of fragments from the native antigen as well as their conformation and orientation. Other polymorphic residues may positively or negatively interfere with the recognition of the bound peptide with a given TcR. The purpose of this chapter is to survey the recent data and to critically analyze their interpretation. The chapter shows that data can fit within a consistent, though hypothetical, framework of MHC-antigen-TcR interactions.

The relationship between the tumorigenic potential and the cloning efficiency of T lymphoma BW5147 in semi-solid media has been studied. Two stable variants exhibiting a 30-fold decrease of their cloning efficiencies in agarose an methylcellulose media were independently isolated by negative selection with FuDr. These variants show no alteration of their growth properties in liquid medium and are still able to proliferate in liquid suspension over a bottom layer of agarose. This new phenotype is not correlated with any decreased tumorigenicity in syngeneic AKR/J mice.

Four major protein sequence data collections (NBRF-PIR, PSD-Kyoto, PGtrans, and NEWAT) have been merged into a single nonredundant data bank called PseqIP. The data bank entries were automatically matched by a heuristic computer program relying on the fast computation of the number of tetrapeptides shared by two sequences. PseqIP 1.0 includes 6,068 different protein sequences for a total of 1,357,067 residues, representing most of the available sequence information to date. During the course of this work, we found about 600 occurrences course of a protein sequence recorded with a one-amino-acid variation in at least two different data banks. A flat file (ASCII computer-readable format) version of PseqIP 1.0, well-suited for exhaustive homology searches and statistical sequence analysis, is available from our laboratory.

A very general approach to the chemical equilibria between many interacting molecules during sedimentation (boundary, band, or active enzyme) taking into account boundary conditions, cell geometry, equilibrium constants, diffusion, enzyme kinetics, etc., is presented. Through a Fortran program, the method has been applied to two very simple but typical cases. With only minor adjustments, the method presented here for sedimentation studies can be extended to all sorts of problems in which “pools” of various species are interacting with each other.

A coding region homologous to the sequence for essential eukaryotic enzyme dUTPase has been identified in different genomic regions of several viral lineages. Unlike the nonprimate lentiviruses (caprine arthritis- encephalitis virus, equine infectious anemia virus, feline immunodeficiency virus, and visna virus), where dUTPase is integrated into the pol coding region, this enzyme has never been demonstrated to be present in the primate lentivirus genomes (human immunodeficiency virus type 1 [HIV-1], HIV-2, or the related simian immunodeficiency virus). A novel approach allowed us to identify a weak but significant sequence similarity between HIV-1 gp120 and the human dUTPase. This finding was then extended to all of the primate lentivirus lineages. Together with the recently reported fragmentary structural similarity between the V3 loop region and the Escherichia coli dUTPase (P. D. Kwong, R. Wyatt, J. Robinson, R. W. Sweet, J. Sodroski, and W. A. Hendrickson, Nature 393:648–659, 1998), our results strongly suggest that an ancestral dUTPase gene has evolved into the present primate lentivirus CD4 and cytokine receptor interacting region of gp120.

biology. Nature 2002; 420: 629-35. 3. Burridge K, Wennerberg K. Rho and Rac take center stage. Cell 2004; 116: 167-79. 4. West MA, Wallin RP, Matthews SP, et al. Enhanced dendritic cell antigen capture via toll-like receptor-induced actin remodeling. Science 2004; 305: 1153-7. 5. Benvenuti F, Hugues S, Walmsley M, et al. Requirement of Rac1 and Rac2 expression by mature dendritic cells for T cell priming. Science 2004; 305: 1150-3. 6. West MA, Antoniou AN, Prescott AR, et al. Membrane ruffling, macropinocytosis and antigen presentation in the absence of gelsolin in murine dendritic cells. Eur J Immunol 1999; 29: 3450-5. 7. Burns S, Thrasher AJ, Blundell MP, et al. Configuration of human dendritic cell cytoskeleton by Rho GTPases, the WAS protein, and differentiation. Blood 2001; 98: 1142-9.

The human pathogen Tropheryma whipplei is the only known reduced genome species (T. whipplei Twist strain, encoding 808 predicted protein-coding genes. Specific genome features include deficiencies in amino acid metabolisms, the lack of clear thioredoxin and thioredoxin reductase homologs, and a mutation in DNA gyrase predicting a resistance to quinolone antibiotics. Moreover, the alignment of the two available T. whipplei genome sequences (Twist vs. TW08/27) revealed a large chromosomal inversion the extremities of which are located within two paralogous genes. These genes belong to a large cell-surface protein family defined by the presence of a common repeat highly conserved at the nucleotide level. The repeats appear to trigger frequent genome rearrangements in T. whipplei, potentially resulting in the expression of different subsets of cell surface proteins. This might represent a new mechanism for evading host defenses. The T. whipplei genome sequence was also compared to other reduced bacterial genomes to examine the generality of previously detected features. The analysis of the genome sequence of this previously largely unknown human pathogen is now guiding the development of molecular diagnostic tools and more convenient culture conditions.

Alternate polyadenylation is an important post-transcriptional regulatory process now open to large-scale analysis by use of cDNA databases. We clustered 164,000 expressed sequence tags (ESTs) into ∼15,000 groups and aligned each group to a putative mRNA 3′ end. By use of stringent criteria to discard artifactual mRNA extremities, clear evidence for alternate polyadenylation was obtained in 189 of the 1000 EST clusters studied. A number of previously unreported polyadenylation sites were identified, together with possible instances of tissue-specific differential polyadenylation. This study demonstrates that, besides quantitative aspects of gene expression, the distribution of alternate mRNA forms can be analyzed through EST sampling.

Background Giant viruses in the genus Chlorovirus (family Phycodnaviridae) infect eukaryotic green microalgae. The prototype member of the genus, Paramecium bursaria chlorella virus 1, was sequenced more than 15 years ago, and to date there are only 6 fully sequenced chloroviruses in public databases. Presented here are the draft genome sequences of 35 additional chloroviruses (287 – 348 Kb/319 – 381 predicted protein encoding genes) collected across the globe; they infect one of three different green algal species. These new data allowed us to analyze the genomic landscape of 41 chloroviruses, which revealed some remarkable features about these viruses. Results Genome colinearity, nucleotide conservation and phylogenetic affinity were limited to chloroviruses infecting the same host, confirming the validity of the three previously known subgenera. Clues for the existence of a fourth new subgenus indicate that the boundaries of chlorovirus diversity are not completely determined. Comparison of the chlorovirus phylogeny with that of the algal hosts indicates that chloroviruses have changed hosts in their evolutionary history. Reconstruction of the ancestral genome suggests that the last common chlorovirus ancestor had a slightly more diverse protein repertoire than modern chloroviruses. However, more than half of the defined chlorovirus gene families have a potential recent origin (after Chlorovirus divergence), among which a portion shows compositional evidence for horizontal gene transfer. Only a few of the putative acquired proteins had close homologs in databases raising the question of the true donor organism(s). Phylogenomic analysis identified only seven proteins whose genes were potentially exchanged between the algal host and the chloroviruses. Conclusion The present evaluation of the genomic evolution pattern suggests that chloroviruses differ from that described in the related Poxviridae and Mimiviridae. Our study shows that the fixation of algal host genes has been anecdotal in the evolutionary history of chloroviruses. We finally discuss the incongruence between compositional evidence of horizontal gene transfer and lack of close relative sequences in the databases, which suggests that the recently acquired genes originate from a still largely un-sequenced reservoir of genomes, possibly other unknown viruses that infect the same hosts.

Background Mimivirus, a giant dsDNA virus infecting Acanthamoeba, is the prototype of the mimiviridae family, the latest addition to the family of the nucleocytoplasmic large DNA viruses (NCLDVs). Its 1.2 Mb-genome was initially predicted to encode 917 genes. A subsequent RNA-Seq analysis precisely mapped many transcript boundaries and identified 75 new genes. Findings We now report a much deeper analysis using the SOLiD™ technology combining RNA-Seq of the Mimivirus transcriptome during the infectious cycle (202.4 Million reads), and a complete genome re-sequencing (45.3 Million reads). This study corrected the genome sequence and identified several single nucleotide polymorphisms. Our results also provided clear evidence of previously overlooked transcription units, including an important RNA polymerase subunit distantly related to Euryarchea homologues. The total Mimivirus gene count is now 1018, 11% greater than the original annotation. Conclusions This study highlights the huge progress brought about by ultra-deep sequencing for the comprehensive annotation of virus genomes, opening the door to a complete one-nucleotide resolution level description of their transcriptional activity, and to the realistic modeling of the viral genome expression at the ultimate molecular level. This work also illustrates the need to go beyond bioinformatics-only approaches for the annotation of short protein and non-coding genes in viral genomes.

Background Little is known about the mechanisms of adaptation of life to the extreme environmental conditions encountered in polar regions. Here we present the genome sequence of a unicellular green alga from the division chlorophyta, Coccomyxa subellipsoidea C-169, which we will hereafter refer to as C-169. This is the first eukaryotic microorganism from a polar environment to have its genome sequenced. Results The 48.8 Mb genome contained in 20 chromosomes exhibits significant synteny conservation with the chromosomes of its relatives Chlorella variabilis and Chlamydomonas reinhardtii. The order of the genes is highly reshuffled within synteny blocks, suggesting that intra-chromosomal rearrangements were more prevalent than inter-chromosomal rearrangements. Remarkably, Zepp retrotransposons occur in clusters of nested elements with strictly one cluster per chromosome probably residing at the centromere. Several protein families overrepresented in C. subellipsoidae include proteins involved in lipid metabolism, transporters, cellulose synthases and short alcohol dehydrogenases. Conversely, C-169 lacks proteins that exist in all other sequenced chlorophytes, including components of the glycosyl phosphatidyl inositol anchoring system, pyruvate phosphate dikinase and the photosystem 1 reaction center subunit N (PsaN). Conclusions We suggest that some of these gene losses and gains could have contributed to adaptation to low temperatures. Comparison of these genomic features with the adaptive strategies of psychrophilic microbes suggests that prokaryotes and eukaryotes followed comparable evolutionary routes to adapt to cold environments.

International audience; Genome data, with underlying new knowledge, are accumulating at exponential rate thanks to ever-improving sequencing technologies and the parallel development of dedicated efficient Bioinformatics methods and tools. Advanced Education in Bioinformatics and Genome Analyses is to a large extent not accessible to students in developing countries where endeavors to set up Bioinformatics courses concern most often only basic levels. Here, we report a pioneering pilot experience concerning the design and implementation, from scratch, of a three-months advanced and extensive course in Bioinformatics and Genome Analyses in the Institut Pasteur de Tunis. Most significantly the outcome of the course was upgrading the participants’ skills in Bioinformatics and Genome Analyses to recognized international standards. Here we detail the different steps involved in the implementation of this course as well as the topics covered in the program. The description of this pilot experience might be helpful for the implementation of other similar educational projects, notably in developing countries, aiming to go beyond basics and providing young researchers with high-level skills.

Nucleo Cytoplasmic Large DNA Viruses (NCLDV) form a very diverse group of double-stranded DNA viruses exclusively infecting eukaryotes. The NCLDVs contain the so-called "giant" viruses visible under light microscope, the first specimen of which, discovered in 2003, opened an unexplored field of viral biodiversity. Some families of NCLDVs could be as old as the emergence of contemporary cellular domains. These viruses inhabit a wide variety of environments (soils, oceans, etc.) where they contribute to the control of eukaryotic microbial populations (e.g., protists, phytoplankton). Moreover, some of these viruses are parasitized by smaller viruses, the virophages, that use the giant virus machinery for their own replication in a common cellular host. Understanding NCLDVs’ evolution, biodiversity and host / virus/virophage interactions is now one of the major areas of virology. Using genomics and bioinformatics approaches, I pursued two objectives in this thesis: (1) Characterize the biodiversity and evolution within the taxonomic family of Mimiviridae (NCLDV) through the description and comparative analysis of the genome of the virus "CeV" which infects the unicellular alga haptophyte Haptolina (ex Chrysochromulina) ericina; (2) Characterize the co-evolution of NCLDVs and virophages with their eukaryotic hosts. This second part was addressed by studying horizontal transfers of viral DNA in the available sequences of eukaryotic genomes. The icosahedral particle of the CeV virus has a diameter of 160 nm and contains a 474-kb genome. The analysis of the latter confirmed that CeV is related to the viruses of the Mimiviridae family, originally defined around the giant viruses prototypes Mimivirus and Megavirus which infect the amoebae. Other members of the Mimiviridae, closer to CeV, also infect unicellular algae. Together with CeV, the latter form a subgroup that we proposed to cluster into the subfamily Mesomimivirinae. Mesomimivirinae possess exclusive genomic characters: a second copy of the RNA polymerase gene, the presence of a gene coding for a second capsid protein, and 7 other groups of orthologous genes absent in other Mimiviridae. Despite their relatedness, each of these viruses possesses a majority of unique genes, the origin of which is unknown. On the other hand, several cases of parallel acquisitions by horizontal transfer of the same gene by CeV and other unrelated viruses were analyzed. This manuscript also documents genetic events that have occurred only in CeV, such as unique gene fusions. Bioinformatic screening of the sequence databases revealed DNA fragments of varying sizes (up to ~ 500Kb) integrated into the genomes of many eukaryotes, originally belonging to NCLDVs or virophages. Their analysis allowed to widen the spectrum of known NCLDVs’ hosts and suggest the existence of new viral families that remain to be discovered. Also, these inserts contain genes that are not found in the sequenced NCLDVs genomes, and could encode novel functionalities of the donor virus. Copies of virophage genomes integrated into the Bigelowiella natans algae genome suggest a new strategy for co-infection and dissemination of virophage, which may also constitute a defense mechanism against NCLDVs for the eukaryotic host., Les Nucleo Cytoplasmic Large DNA virus (NCLDV) forment un groupe très divers de virus à ADN double brin infectant exclusivement des eucaryotes. Les NCLDVs contiennent les-dits virus « géants » visibles au microscope optique, dont le premier spécimen découvert en 2003 a ouvert un champ inexploré de la biodiversité virale. Certaines familles de NCLDVs pourraient être aussi anciennes que l’émergence des domaines cellulaires contemporains. Ces virus habitent une grande variété d’environnements (sols, océans, etc.) dans lesquels ils concourent au contrôle des populations microbiennes eucaryotes (e.g., protistes, phytoplancton). Par ailleurs, certains de ces virus sont parasités par d’autres virus de taille plus modeste, les virophages. Ces derniers utilisent la machinerie de réplication des virus géants pour leur propre réplication dans un hôte cellulaire commun. La compréhension de l’évolution, la biodiversité et des interactions hôte/virus/virophage chez les NCLDVs est aujourd’hui un des grands chantiers de la virologie.A l’aide d’approches génomiques et bioinformatiques, deux objectifs ont été poursuivis dans ce travail de thèse: (1) Caractériser la biodiversité et l’évolution au sein de la famille taxonomique des Mimiviridae (NCLDV) à travers la description et l’analyse comparative du génome du virus « CeV » qui infecte l’algue unicellulaire haptophyte Haptolina (ex Chrysochromulina) ericina; (2) Caractériser la co-évolution des NCLDVs et des virophages avec leurs hôtes eucaryotes. Ce deuxième volet a été adressé en étudiant les transferts horizontaux d’ADNs viraux dans les génomes eucaryotes séquencés.La particule icosahédrique du virus CeV a un diamètre de 160 nm et contient un génome de 474-kb dont l’analyse a confirmé que CeV est apparenté aux virus de la famille des Mimiviridae, initialement définie autour des virus géant prototypes Mimivirus et Megavirus infectant des amibes. D’autres membres des Mimiviridae, plus proches de CeV, infectent aussi des espèces d’algues unicellulaires. Ces derniers forment un sous-groupe, que nous proposons d’élever au rang d’une sous-famille, les Mesomimivirinae. Les Mesomimivirinae possèdent des caractères génomiques exclusifs: une deuxième copie du gène de l’ARN polymérase, la présence d’un gène codant pour seconde protéine de capside, ainsi que 7 autres groupes de gènes orthologues absents chez les autres Mimiviridae. En dépit de leurs liens de parenté, chacun de ces virus possède une majorité de gènes qui lui est unique, et dont l’origine est inconnue. D’autre part, plusieurs cas indépendants d’acquisition par transfert horizontal du même gène par CeV et d’autres virus non-apparentés ont été analysés. Cette thèse documente également des évènements génétiques qui se sont produits dans la seule lignée de CeV, comme par exemple des fusions de gènes inédites.Le criblage bioinformatique des bases de données de séquences a mis en évidence des fragments d’ADNs de tailles variables (jusqu’à ~500Kb) intégrés au sein des génomes de nombreux eucaryotes, ayant originellement appartenus à des NCLDVs ou des virophages. Leur analyse a permis d’élargir le spectre d’hôtes connus des NCLDVs et suggèrent l’existence de nouvelles familles virales qui restent à découvrir. Egalement, ces insertions contiennent des gènes que l’on ne retrouve pas dans les génomes de NCLDVs séquencés, et pourraient coder pour des fonctionnalités inédites du virus donneur. Des copies de génomes de virophages intégrées au génome de l’algue Bigelowiella natans suggèrent une nouvelle stratégie de co-infection et de dissémination du virophage, qui pourrait aussi constituer un mécanisme de défense contre les NCLDVs pour l’hôte eucaryote.

Downloadable manuscript in PDF format: http://igs-server.cnrs-mrs.fr/suhre/HDR.pdf