Identification of 28 novel mutations in the Bardet-Biedl syndrome genes: the burden of private mutations in an extensively heterogeneous disease.

Bardet-Biedl syndrome (BBS), an emblematic disease in the rapidly evolving field of ciliopathies, is characterized by pleiotropic clinical features and extensive genetic heterogeneity. To date, 14 BBS genes have been identified, 3 of which have been found mutated only in a single BBS family each (BBS11/TRIM32, BBS13/MKS1 and BBS14/MKS4/NPHP6). Previous reports of systematic mutation detection in large cohorts of BBS families (n > 90) have dealt only with a single gene, or at most small subsets of the known BBS genes. Here we report extensive analysis of a cohort of 174 BBS families for 12/14 genes, leading to the identification of 28 novel mutations. Two pathogenic mutations in a single gene have been found in 117 families, and a single heterozygous mutation in 17 families (of which 8 involve the BBS1 recurrent mutation, M390R). We confirm that BBS1 and BBS10 are the most frequently mutated genes, followed by BBS12. No mutations have been found in BBS11/TRIM32, the identification of which as a BBS gene only relies on a single missense mutation in a single consanguineous family. While a third variant allele has been observed in a few families, they are in most cases missenses of uncertain pathogenicity, contrasting with the type of mutations observed as two alleles in a single gene. We discuss the various strategies for diagnostic mutation detection, including homozygosity mapping and targeted arrays for the detection of previously reported mutations.

Décrypthon grid - grid resources dedicated to neuromuscular disorders.

Thanks to the availability of computational grids and their middleware, a seamless access to computation and storage resources is provided to application developers and scientists. The Décrypthon project is one example of such a high performance platform. In this paper, we present the architecture of the platform, the middleware developed to facilitate access to several servers deployed in France, and the data center for integrating large biological datasets over multiple sites, supported by a new query language and integration of various tools. The SM2PH project represents an example of a biological application that exploits the capacities of the Décrypthon grid. The goal of SM2PH is a better understanding of mutations involved in human monogenic diseases, their impact on the 3D structure of the protein and the subsequent consequences for the pathological phenotypes.

Time-resolved analysis of transcriptional events during SNAI1-triggered epithelial to mesenchymal transition.

The transcription regulator SNAI1 triggers a transcriptional program leading to epithelial to mesenchymal transition (EMT), providing epithelial cells with mesenchymal features and invasive properties during embryonic development and tumor progression. To identify early transcriptional changes occurring during SNAI1-induced EMT, we performed a time-resolved genome-scale study using human breast carcinoma cells conditionally expressing SNAI1. The approach we developed for microarray data analysis, allowed identifying three distinct EMT stages and the temporal classification of genes. Importantly, we identified unexpected, biphasic expression profiles of EMT-associated genes, supporting their pivotal role during this process. Finally, we established early EMT gene networks by identifying transcription factors and their potential targets which may orchestrate early events of EMT. Collectively, our work provides a framework for the identification and future systematic analysis of novel genes which contribute to SNAI1-triggered EMT.

The archaeal homolog of the Imp4 protein, a eukaryotic U3 snoRNP component.

Homologs of the Imp4 protein, a component specific to the eukaryotic U3 snoRNP complex, have been found in all archaeal genomes. The archaeal and eukaryotic Imp4 proteins that are related to four other protein families, the Imp4-like, the SSF1 homologs and two sets of hypothetical proteins, are characterized by the Imp4 signature pattern. These findings, together with the presence of other snoRNPs homologs in Archaea, provide evidence for similar RNA processing and folding in Eukarya and Archaea.

The TBP-like factor: an alternative transcription factor in metazoa?

Protein sequence analysis has revealed a family of TATA-binding-protein (TBP)-like factors (TLFs) in metazoan organisms. Modelling of the three-dimensional structure of these TLFs suggests that they form an asymmetric saddle-like structure and that, unlike TBP, TLFs might bind to DNA sequences other than classical TATA boxes. Thus, the existence of TLFs presents a challenge to the doctrine that TBP is a universal regulator of transcription in metazoans.

Mu-opioid receptor activation induces transcriptional plasticity in the central extended amygdala.

Addiction develops from the gradual adaptation of the brain to chronic drug exposure, and involves genetic reprogramming of neuronal function. The central extended amygdala (EAc) is a network formed by the central amygdala and the bed nucleus of the stria terminalis. This key site controls drug craving and seeking behaviors, and has not been investigated at the gene regulation level. We used Affymetrix microarrays to analyze transcriptional activity in the murine EAc, with a focus on mu-opioid receptor-associated events because these receptors mediate drug reward and dependence. We identified 132 genes whose expression is regulated by a chronic escalating morphine regimen in the EAc from wild-type but not mu-opioid receptor knockout mice. These modifications are mostly EAc-specific. Gene ontology analysis reveals an overrepresentation of neurogenesis, cell growth and signaling protein categories. A separate quantitative PCR analysis of genes in the last of these groups confirms the dysregulation of both orphan (Gpr88) and known (DrD1A, Adora2A, Cnr1, Grm5, Gpr6) G protein-coupled receptors, scaffolding (PSD95, Homer1) and signaling (Sgk, Cap1) proteins, and neuropeptides (CCK, galanin). These transcriptional modifications do not occur following a single morphine injection, and hence result from long-term adaptation to excessive mu receptor activation. Proteins encoded by these genes are classically associated with spine modules function in other brain areas, and therefore our data suggest a remodeling of EAc circuits at sites where glutamatergic and monoaminergic afferences interact. Together, mu receptor-dependent genes identified in this study potentially contribute to drug-induced neural plasticity, and provide a unique molecular repertoire towards understanding drug craving and relapse.

Transcriptome analysis identifies genes with enriched expression in the mouse central extended amygdala.

The central extended amygdala (EAc) is an ensemble of highly interconnected limbic structures of the anterior brain, and forms a cellular continuum including the bed nucleus of the stria terminalis (BNST), the central nucleus of the amygdala (CeA) and the nucleus accumbens shell (AcbSh). This neural network is a key site for interactions between brain reward and stress systems, and has been implicated in several aspects of drug abuse. In order to increase our understanding of EAc function at the molecular level, we undertook a genome-wide screen (Affymetrix) to identify genes whose expression is enriched in the mouse EAc. We focused on the less-well known BNST-CeA areas of the EAc, and identified 121 genes that exhibit more than twofold higher expression level in the EAc compared with whole brain. Among these, 43 genes have never been described to be expressed in the EAc. We mapped these genes throughout the brain, using non-radioactive in situ hybridization, and identified eight genes with a unique and distinct rostro-caudal expression pattern along AcbSh, BNST and CeA. Q-PCR analysis performed in brain and peripheral organ tissues indicated that, with the exception of one (Spata13), all these genes are predominantly expressed in brain. These genes encode signaling proteins (Adora2, GPR88, Arpp21 and Rem2), a transcription factor (Limh6) or proteins of unknown function (Rik130, Spata13 and Wfs1). The identification of genes with enriched expression expands our knowledge of EAc at a molecular level, and provides useful information to toward genetic manipulations within the EAc.

Gene expression is altered in the lateral hypothalamus upon activation of the mu opioid receptor.

The lateral hypothalamus (LH) is a brain structure that controls hedonic properties of both natural rewards and drugs of abuse. Mu opioid receptors are known to mediate drug reward, but whether overstimulation of these receptors impacts on LH function has not been studied. Here we have used a genome-wide microarray approach to identify LH responses to chronic mu opioid receptor activation at the transcriptional level. We have subjected wild-type and mu opioid receptor knockout mice to an escalating morphine regimen, which produces severe physical dependence in wild-type but not mutant animals. We have analyzed gene profiles in LH samples using the 430A.2 Affymetrix array and identified a set of 25 genes whose expression is altered by morphine in wild-type mice only. The regulation was confirmed for a subset of these genes using real-time quantitative PCR on samples from independent treatments. Altered expression of aquaporin 4, apolipoprotein D, and prostaglandin synthase is indicative of modified LH physiology. The regulation of two signaling genes (the serum glucocorticoid kinase and the regulator of G protein signaling 4) suggests that neurotransmission is altered in LH circuitry. Finally, the downregulation of apelin may indicate a potential role for this neuropeptide in opioid signaling and hedonic homeostasis. Altogether, our study shows that chronic mu opioid receptor stimulation induces gene expression plasticity in the LH and provides a unique collection of mu opioid receptor-dependent genes that potentially contribute to alter reward processes in addictive diseases.

Head and neck squamous cell carcinoma transcriptome analysis by comprehensive validated differential display.

Head and neck squamous cell carcinoma (HNSCC) is common worldwide and is associated with a poor rate of survival. Identification of new markers and therapeutic targets, and understanding the complex transformation process, will require a comprehensive description of genome expression, that can only be achieved by combining different methodologies. We report here the HNSCC transcriptome that was determined by exhaustive differential display (DD) analysis coupled with validation by different methods on the same patient samples. The resulting 820 nonredundant sequences were analysed by high throughput bioinformatics analysis. Human proteins were identified for 73% (596) of the DD sequences. A large proportion (>50%) of the remaining unassigned sequences match ESTs (expressed sequence tags) from human tumours. For the functionally annotated proteins, there is significant enrichment for relevant biological processes, including cell motility, protein biosynthesis, stress and immune responses, cell death, cell cycle, cell proliferation and/or maintenance and transport. Three of the novel proteins (TMEM16A, PHLDB2 and ARHGAP21) were analysed further to show that they have the potential to be developed as therapeutic targets.

MAGOS: multiple alignment and modelling server.

UNLABELLED: MAGOS is a web server allowing automated protein modelling coupled to the creation of a hierarchical and annotated multiple alignment of complete sequences. MAGOS is designed for an interactive approach of structural information within the framework of the evolutionary relevance of mined and predicted sequence information. AVAILABILITY: The web server is freely available at http://pig-pbil.ibcp.fr/magos.

The human TFIID components TAF(II)135 and TAF(II)20 and the yeast SAGA components ADA1 and TAF(II)68 heterodimerize to form histone-like pairs.

It has been previously proposed that the transcription complexes TFIID and SAGA comprise a histone octamer-like substructure formed from a heterotetramer of H4-like human hTAF(II)80 (or its Drosophila melanogaster dTAF(II)60 and yeast [Saccharomyces cerevisiae] yTAF(II)60 homologues) and H3-like hTAF(II)31 (dTAF(II)40 and yTAF(II)17) along with two homodimers of H2B-like hTAF(II)20 (dTAF(II)30alpha and yTAF(II)61/68). However, it has not been formally shown that hTAF(II)20 heterodimerizes via its histone fold. By two-hybrid analysis with yeast and biochemical characterization of complexes formed by coexpression in Escherichia coli, we showed that hTAF(II)20 does not homodimerize but heterodimerizes with hTAF(II)135. Heterodimerization requires the alpha2 and alpha3 helices of the hTAF(II)20 histone fold and is abolished by mutations in the hydrophobic face of the hTAF(II)20 alpha2 helix. Interaction with hTAF(II)20 requires a domain of hTAF(II)135 which shows sequence homology to H2A. This domain also shows homology to the yeast SAGA component ADA1, and we show that yADA1 heterodimerizes with the histone fold region of yTAF(II)61/68, the yeast hTAF(II)20 homologue. These results are indicative of a histone fold type of interaction between hTAF(II)20-hTAF(II)135 and yTAF(II)68-yADA1, which therefore constitute novel histone-like pairs in the TFIID and SAGA complexes.

Synergistic transcriptional activation by TATA-binding protein and hTAFII28 requires specific amino acids of the hTAFII28 histone fold.

Coexpression of the human TATA-binding protein (TBP)-associated factor 28 (hTAFII28) with the altered-specificity mutant TBP spm3 synergistically enhances transcriptional activation by the activation function 2 of the nuclear receptors (NRs) for estrogen and vitamin D3 from a reporter plasmid containing a TGTA element in mammalian cells. This synergy is abolished by mutation of specific amino acids in the alpha2-helix of the histone fold in the conserved C-terminal region of hTAFII28. Critical amino acids are found on both the exposed hydrophilic face of this helix and the hydrophobic interface with TAFII18. This alpha-helix of hTAFII28 therefore mediates multiple interactions required for coactivator activity. We further show that mutation of specific residues in the H1' alpha-helix of TBP either reduces or increases interactions with hTAFII28. The mutations which reduce interactions with hTAFII28 do not affect functional synergy, whereas the TBP mutation which increases interaction with hTAFII28 is defective in its ability to synergistically enhance activation by NRs. However, this TBP mutant supports activation by other activators and is thus specifically defective for its ability to synergize with hTAFII28.

DbClustal: rapid and reliable global multiple alignments of protein sequences detected by database searches.

DbClustal addresses the important problem of the automatic multiple alignment of the top scoring full-length sequences detected by a database homology search. By combining the advantages of both local and global alignment algorithms into a single system, DbClustal is able to provide accurate global alignments of highly divergent, complex sequence sets. Local alignment information is incorporated into a ClustalW global alignment in the form of a list of anchor points between pairs of sequences. The method is demonstrated using anchors supplied by the Blast post-processing program, Ballast. The rapidity and reliability of DbClustal have been demonstrated using the recently annotated Pyrococcus abyssi proteome where the number of alignments with totally misaligned sequences was reduced from 20% to <2%. A web site has been implemented proposing BlastP database searches with automatic alignment of the top hits by DbClustal.

BAliBASE (Benchmark Alignment dataBASE): enhancements for repeats, transmembrane sequences and circular permutations.

BAliBASE is specifically designed to serve as an evaluation resource to address all the problems encountered when aligning complete sequences. The database contains high quality, manually constructed multiple sequence alignments together with detailed annotations. The alignments are all based on three-dimensional structural superpositions, with the exception of the transmembrane sequences. The first release provided sets of reference alignments dealing with the problems of high variability, unequal repartition and large N/C-terminal extensions and internal insertions. Here we describe version 2.0 of the database, which incorporates three new reference sets of alignments containing structural repeats, trans-membrane sequences and circular permutations to evaluate the accuracy of detection/prediction and alignment of these complex sequences. BAliBASE can be viewed at the web site http://www-igbmc.u-strasbg. fr/BioInfo/BAliBASE2/index.html or can be downloaded from ftp://ftp-igbmc.u-strasbg.fr/pub/BAliBASE2 /.

Oxidants at the Surface of Mars: A Review in Light of Recent Exploration Results.

In 1976, the Viking landers carried out the most comprehensive search for organics and microbial life in the martian regolith. Their results indicate that Mars' surface is lifeless and, surprisingly, depleted in organics at part-per-billion levels. Several biology experiments on the Viking landers gave controversial results that have since been explained by the presence of oxidizing agents on the surface of Mars. These oxidants may degrade abiotic or biological organics, resulting in their nondetection in the regolith. As several exploration missions currently focus on the detection of organics on Mars (or will do so in the near future), knowledge of the oxidative state of the surface is fundamental. It will allow for determination of the capability of organics to survive on a geological timescale, the most favorable places to seek them, and the best methods to process the samples collected at the surface. With this aim, we review the main oxidants assumed to be present on Mars, their possible formation pathways, and those laboratory studies in which their reactivity with organics under Mars-like conditions has been evaluated. Among the oxidants assumed to be present on Mars, only four have been detected so far: perchlorate ions (ClO4-) in salts, hydrogen peroxide (H2O2) in the atmosphere, and clays and metal oxides composing surface minerals. Clays have been suggested as catalysts for the oxidation of organics but are treated as oxidants in the following to keep the structure of this article straightforward. This work provides an insight into the oxidizing potential of the surface of Mars and an estimate of the stability of organic matter in an oxidizing environment. Key Words: Mars surface-Astrobiology-Oxidant-Chemical reactions. Astrobiology 16, 977-996.

Towards a reliable objective function for multiple sequence alignments.

Multiple sequence alignment is a fundamental tool in a number of different domains in modern molecular biology, including functional and evolutionary studies of a protein family. Multiple alignments also play an essential role in the new integrated systems for genome annotation and analysis. Thus, the development of new multiple alignment scores and statistics is essential, in the spirit of the work dedicated to the evaluation of pairwise sequence alignments for database searching techniques. We present here norMD, a new objective scoring function for multiple sequence alignments. NorMD combines the advantages of the column-scoring techniques with the sensitivity of methods incorporating residue similarity scores. In addition, norMD incorporates ab initio sequence information, such as the number, length and similarity of the sequences to be aligned. The sensitivity and reliability of the norMD objective function is demonstrated using structural alignments in the SCOP and BAliBASE databases. The norMD scores are then applied to the multiple alignments of the complete sequences (MACS) detected by BlastP with E-value<10, for a set of 734 hypothetical proteins encoded by the Vibrio cholerae genome. Unrelated or badly aligned sequences were automatically removed from the MACS, leaving a high-quality multiple alignment which could be reliably exploited in a subsequent functional and/or structural annotation process. After removal of unreliable sequences, 176 (24 %) of the alignments contained at least one sequence with a functional annotation. 103 of these new matches were supported by significant hits to the Interpro domain and motif database.

Analysis of the structural diversity of monoclonal antibodies to cyclosporine.

The immunosuppressive cyclic undecapeptide cyclosporine (Cs) represents a useful model for studying the molecular basis of antibody-antigen interactions. The three-dimensional structure of the Cs molecule is known and a large panel of monoclonal antibodies (mAbs) to Cs has been well characterized by cross-reactivity studies with numerous Cs analogs. In the present study, the sequences of the variable regions of seven mAbs to Cs were determined and a striking relationship was found between the expressed variable region genes and the Cs recognition pattern. An analysis of the length and hydrophobic content of the hypervariable regions and sequence similarities suggested that the heavy chain plays a major role in Cs recognition. Different fine specificities were observed for mAbs exhibiting identical light chains, while two antibodies differed by only a single amino acid located in the heavy chain. The presence of a duplication of 12 nucleotides within the heavy chain third hypervariable region of two antibodies suggests the existence of an additional mechanism for creating antibody diversity.

Conservation of a putative inhibitory domain in the GAL4 family members.

The GAL4 family members are fungal transcriptional activators composed of several functional domains: a characteristic cysteine-rich DNA-binding domain common to all members, a dimerization domain, various transactivation domains generally exhibiting a high acidic content and a highly variable central region supposed to be involved in regulation and in effector recognition. We report here that the central region of the GAL4 family members share eight conserved motifs embedded in a large functional domain of 225 up to 405 residues. This domain may also be present in four proteins belonging to another family of transcriptional activators sharing a C2H2-type zinc finger. Analysis of the biochemical data available on the well-studied GAL4 protein suggests that this domain may be involved in the regulation of the activity of the protein, particularly in an inhibitory function. This hypothesis is further supported by deletion and site-directed mutagenesis experiments on other GAL4 family members. The mean secondary structure prediction performed on the eight motifs strongly suggests that the inhibitory activity may be mediated by hydrophobic interactions linked to the presence of amphipathic alpha-helices.

Multiple alignment of complete sequences (MACS) in the post-genomic era.

Multiple alignment, since its introduction in the early seventies, has become a cornerstone of modern molecular biology. It has traditionally been used to deduce structure / function by homology, to detect conserved motifs and in phylogenetic studies. There has recently been some renewed interest in the development of multiple alignment techniques, with current opinion moving away from a single all-encompassing algorithm to iterative and / or co-operative strategies. The exploitation of multiple alignments in genome annotation projects represents a qualitative leap in the functional analysis process, opening the way to the study of the co-evolution of validated sets of proteins and to reliable phylogenomic analysis. However, the alignment of the highly complex proteins detected by today's advanced database search methods is a daunting task. In addition, with the explosion of the sequence databases and with the establishment of numerous specialized biological databases, multiple alignment programs must evolve if they are to successfully rise to the new challenges of the post-genomic era. The way forward is clearly an integrated system bringing together sequence data, knowledge-based systems and prediction methods with their inherent unreliability. The incorporation of such heterogeneous, often non-consistent, data will require major changes to the fundamental alignment algorithms used to date. Such an integrated multiple alignment system will provide an ideal workbench for the validation, propagation and presentation of this information in a format that is concise, clear and intuitive.

Sequence of the Kluyveromyces lactis beta-galactosidase: comparison with prokaryotic enzymes and secondary structure analysis.

The LAC4 gene encoding the beta-galactosidase (beta Gal) of the yeast, Kluyveromyces lactis, was cloned on a 7.2-kb fragment by complementation of a lacZ-deficient Escherichia coli strain. The nucleotide sequence of the structural gene, with 42 bp and 583 bp of the 5'- and 3'-flanking sequences, respectively, was determined. The deduced amino acid (aa) sequence of the K. lactis beta Gal predicts a 1025-aa polypeptide with a calculated M(r) of 117618 and reveals extended sequence homologies with all the published prokaryotic beta Gal sequences. This suggests that the eukaryotic beta Gal is closely related, evolutionarily and structurally, to the prokaryotic beta Gal's. In addition, sequence similarities were observed between the highly conserved N-terminal two-thirds of the beta Gal and the entire length of the beta-glucuronidase (beta Glu) polypeptides, which suggests that beta Glu is clearly related, structurally and evolutionarily, to the N-terminal two-thirds of the beta Gal. The structural analysis of the beta Gal alignment, performed by mean secondary structure prediction, revealed that most of the invariant residues are located in turn or loop structures. The location of the invariant residues is discussed with respect to their accessibility and their possible involvement in the catalytic process.