Jean-Christophe Aude

La deuxième moitié du siècle dernier a vu l'émergence et le succès de l'approche réductionniste en physiologie rénale, approche qui a permis de caractériser les mécanismes cellulaires de transport tubulaire et de leur régulation, et d'en déter-miner le support moléculaire. Il s'est agi d'une approche essentiellement déductive de type gène cible : ayant caractérisé une protéine d'intérêt dans un processus phy-siologique, on a recherché celles qui interagissent en amont et en aval, et par ité-rations successives on a pu, dans le meilleur des cas, déterminer toute la chaîne d'intermédiaires moléculaires qui sous-tendent la régulation d'un effecteur. Cette approche est très performante pour analyser le fonctionnement d'architectures quasi linéaires. Cependant, l'approche réductionniste a mis en évidence la complexité des processus de régulation des fonctions tubulaires rénales, complexité qui résulte de plusieurs phénomènes : a) une régulation phy...

As an approach to understanding how mammals regulate H(2)O(2) toxicity, intracellular concentration to prevent its we analyzed the genome-wide mRNA profile changes of human cells after treatment with a non-toxic H(2)O(2) concentration. We identified a large and essentially late H(2)O(2) response of induced and repressed genes that unexpectedly comprise few or no antioxidants but mostly apoptosis and cell cycle control activities. The requirement of the p53 regulator for regulating about a third of this H(2)O(2) stimulon and the lack of an associated enhancement of total cellular H(2)O(2) scavenging activity further suggest that H(2)O(2) elicits a stress antiproliferative/repair response that does not increase antioxidant defenses. We conclude that mammalian antioxidant defenses are constitutive, a finding that contrasts with the oxidant-inducibility of such defenses in microorganisms. This finding might be important in understanding the role of H(2)O(2) as a key signaling molecule in mammals.

The Z-value (Comput. Chem. 23 (1999) 333) is an extension of the Z-score that is classically used to compare sets of biological sequences. The Z-value has been successfully used to handle complete genome studies as well as analyze large sets of proteins. The Z-value computation is based on a Monte Carlo approach to estimate the statistical significance of a Smith & Waterman alignment score. Comet et al. (Comput. Chem. 23 (1999) 333) have shown that, in contrast to the alignment score, the Z-value largely reduces the bias due to the lengths and compositions of the sequences. They also described an estimator of the deviation of Z-values, that we extend in this paper in order to optimize Z-values computation. The incremental algorithm described here provides two characteristics which are usually incompatible: (i) it improves the accuracy of Z-values calculation; (ii) it reduces the time complexity (this algorithm has been named incremental because it iteratively adds random sequences t...

Characterization of tissue-specific gene expression profiles, or transcriptomes, may serve two purposes: a) establishing relationships between cell transcriptomes and functions (i.e. molecular and physiological phenotypes) under physiological and pathophysiological conditions serves to elucidate gene functions, and b) determination of the totality of genes expressed in a cell seems a prerequisite for understanding cell functions, because the properties of proteins vary with their environment. Sophisticated methods are now available for transcriptome analysis. They are based on serial, partial sequencing of cDNAs (sequencing of expressed sequenced tags (ESTs) and serial analysis of gene expression (SAGE)), or on parallel hybridization of labeled cDNAs to specific probes immobilized on a grid (macro- and microarrays and DNA chips). Some methods were designed specifically to compare gene expression under different conditions (substractive hybridization, glass microarrays). However, all...

Progress accomplished by complete genomes and cDNA-sequencing projects calls for methods that fully use these resources to study gene expression patterns in characterized cell populations. However, since the number of functional genes cannot be readily inferred from the genomic sequence, it is highly desirable to make use of methods enabling to study both known and unknown genes. The method of serial analysis of gene expression provides short diagnostic cDNA tags without bias towards known genes. In addition, the frequency of each tag in the library conveys quantitative information on gene expression. A microassay was set-up to perform serial analysis of gene expression in minute samples such as those obtained by microdissecting nephron segments. Studies carried out in the thick ascending limb of Henle's loop and the collecting duct of the mouse kidney provided expression data for several thousand genes. Known markers were found appropriately enriched, and several of the thick ascending limb or collecting duct specific transcripts had no database match. The microassay for serial analysis of gene expression makes possible large-scale quantitative measurements of mRNA levels in nephron segments. The comprehensive picture generated by analyzing both known and unknown transcripts in defined cell populations should help to discover genes with dedicated functions.

ATP-dependent chromatin remodellers allow access to DNA for transcription factors and the general transcription machinery, but whether mammalian chromatin remodellers target specific nucleosomes to regulate transcription is unclear. Here we present genome-wide remodeller-nucleosome interaction profiles for the chromatin remodellers Chd1, Chd2, Chd4, Chd6, Chd8, Chd9, Brg1 and Ep400 in mouse embryonic stem (ES) cells. These remodellers bind one or both full nucleosomes that flank micrococcal nuclease (MNase)-defined nucleosome-free promoter regions (NFRs), where they separate divergent transcription. Surprisingly, large CpG-rich NFRs that extend downstream of annotated transcriptional start sites are nevertheless bound by non-nucleosomal or subnucleosomal histone variants (H3.3 and H2A.Z) and marked by H3K4me3 and H3K27ac modifications. RNA polymerase II therefore navigates hundreds of base pairs of altered chromatin in the sense direction before encountering an MNase-resistant nucleosome at the 3' end of the NFR. Transcriptome analysis after remodeller depletion reveals reciprocal mechanisms of transcriptional regulation by remodellers. Whereas at active genes individual remodellers have either positive or negative roles via altering nucleosome stability, at polycomb-enriched bivalent genes the same remodellers act in an opposite manner. These findings indicate that remodellers target specific nucleosomes at the edge of NFRs, where they regulate ES cell transcriptional programs.

We investigated the relationship between unfolded proteins, silica nanoparticles and chaperonin to determine whether unfolded proteins could stick to silica surfaces and how this process could impair heat shock protein activity. The HSP60 catalyzed green fluorescent protein (GFP) folding was used as a model system. The adsorption isotherms and adsorption kinetics of denatured GFP were measured, showing that denaturation increases GFP affinity for silica surfaces. This affinity is maintained even if the surfaces are covered by a protein corona and allows silica NPs to interfere directly with GFP folding by trapping it in its unstructured state. We determined also the adsorption isotherms of HSP60 and its chaperonin activity once adsorbed, showing that SiO2 NP can interfere also indirectly with protein folding through chaperonin trapping and inhibition. This inhibition is specifically efficient when NPs are covered first with a layer of unfolded proteins. These results highlight for the first time the antichaperonin activity of silica NPs and ask new questions about the toxicity of such misfolded proteins/nanoparticles assembly toward cells.

The Z-value is an attempt to estimate the statistical significance of a Smith-Waterman dynamic alignment score (SW-score) through the use of a Monte-Carlo process. It partly reduces the bias induced by the composition and length of the sequences. This paper is not a theoretical study on the distribution of SW-scores and Z-values. Rather, it presents a statistical analysis of Z-values on large datasets of protein sequences, leading to a law of probability that the experimental Z-values follow. First, we determine the relationships between the computed Z-value, an estimation of its variance and the number of randomizations in the Monte-Carlo process. Then, we illustrate that Z-values are less correlated to sequence lengths than SW-scores. Then we show that pairwise alignments, performed on 'quasi-real' sequences (i.e., randomly shuffled sequences of the same length and amino acid composition as the real ones) lead to Z-value distributions that statistically fit the extreme val...

In conventional hierarchical clustering methods, any object can belong to only one class or cluster. We present here an application of the pyramidal classification method to biological objects, which illustrates the intuitively appealing idea that some objects may belong simultaneously to two classes. In a first step, we performed an all-by-all comparison of all the open reading frames in the genomes from S. cerevisiae, M. jannaschii, E. coli, H. influenzae and Synechocystis. In a second step, a series of connex classes was built, each connex class containing all those sequences that were linked by a Z-value (obtained after 100 sequence shufflings) greater than a given threshold. Finally, each connex class was submitted to a pyramidal classification. Three examples of such classifications are given, concerning two sets of multi-domains protein sequences and a family of aminoacyl-tRNA synthetases. They make it clear that the linear order among the classified objects that results from the pyramidal classification is useful in deciphering the multiple relationships that can exist between the objects under study. A program for calculating and displaying a pyramidal classification from a dissimilarity matrix is available from http:/(/)genome.genetique.uvsq.fr/Pyramids. The pyramidal classifications of the connex classes from the five organisms (intra- and inter-genomic comparisons) are available from http:/(/)www.gene-it.com under the family item.

Protein-protein interaction networks provide insights into the relationships between the proteins of an organism thereby contributing to a better understanding of cellular processes. Nevertheless, large-scale interaction networks are available for only a few model organisms. Thus, interologs are useful for a systematic transfer of protein interaction networks between organisms. However, no standard tool is available so far for that purpose. In this study, we present an automated prediction tool developed for all sequenced genomes available in Integr8. We also have developed a second method to predict protein-protein interactions in the widely used cyanobacterium Synechocystis. Using these methods, we have constructed a new network of 8783 inferred interactions for Synechocystis. InteroPORC is open-source, downloadable and usable through a web interface at http://biodev.extra.cea.fr/interoporc/.

To identify genetic conservation relative to precise aspects of developmental diversity, an essential question in computational biology, we developed a new comparative method that allows conserved modules for the best balance between protein sequence similarity and gene co-expression to be constructed, in invertebrates. Our method, referred to as the best-balance constraint procedure (BBCP), yielded 719 functionally conserved modules (FCMs) comprising 2-23 gene pairs. These modules were consistent with the developmental roles of orthologues as inferred from Gene Ontology, RNAi knockouts, InterPro and process-specific microarray data. New relationships were defined between genetic conservation and developmental diversity. Novel gene associations were indeed found in 94% of the FCMs, 150 modules being completely new. A significant proportion of the FCMs (18%, 132 modules) described cell type-specific mechanisms, comprising neuronal, muscle and germ cell signaling, new associations being found in 125 modules. Also found were gene associations for cell fate specification activities previously not highlighted by computational means, e.g. in FCMs containing homeogenes. These data indicate that highly discriminative description of genetic conservation can be deduced using BBCP, and reveal new correlations between cellular and developmental diversity and gene essentiality in invertebrates. christian.neri@broca.inserm.fr For supplementary information, please refer to Bioinformatics online.