1. Alvinella pompejana cDNA collection Gagnière, N. 1, Bigot, Y. 2, Brelivet, Y. 1, Busso, D. 3, Chénais, B. 4, Gaill, F. 5, Higuet, D. 6, Jollivet, D. 7, Leize, E. 8, Rees, J.F. 9, Thierry, J.C. 1, Weissenbach, J. 10, Zal, F. 11, Moras, D. 12, Poch, O. 1, Lecompte, O. 1 Semi automated cDNA sequence analysis protocol AbstractAbstract Alvinella pompejana, the « pompeii worm », is a Polychaete Annelid discovered in 1980. This tubiculous worm colonizes hydrothermal Vents where it is faced with extreme and variable physico-chemical conditions including very high temperatures (from 20 to over 80°C), anoxic conditions, low pH, high concentration of heavy metals and sulfide…This environment makes A. pompejana an ideal model for studies aimed at deciphering adaptation in general as well as a unique source of thermostable proteins of eukaryotic origin for structural studies. To obtain phylogenetic and adaptative data as well as a pool of thermotolerant proteins with potential biotechnology implications, a massive cDNA sequencing project has been initiated. Here we describe the cDNA libraries constructed for this project, the semi automated sequence analysis protocol for the first 70,000 reads, and the preliminary results that highlight Alvinella as a model organism for eukaryotic protein studies. Phare 2002,IFREMER© gills dorsal face with epibiotic bacteria pygidium Available cDNA libraries An ideal model for eukaryotic proteins production Full-length enriched cDNA libraries have been generated at the Genoscope (http://www.genoscope.cns.fr/) for: whole animal (Cloneminer method) gills (Oligo-capping method) ventral tissue (Oligo-capping method) pygidium (Cloneminer method, sequencing in progress) Whole animals as well as dissected tissues were been collected during the oceanographic Biospeedo cruise on the Pacific Ridge in 2004. The sequencing of the 5’ ends is ongoing at Genoscope on a ABI 3730 sequencer using dye- terminator fluorescent DNA sequencing technology. A total of 200,000 reads will be achieved. We will select about 10,000 full-length cDNA using the sequence data and the entire sequence of the selected clones will be determined. Cleaning and assembling process chromatograms PHRED: sequence and quality extraction Cross-match: vector masking ad hoc script: polyA masking PHRED: low-quality region trimming File synchronization eliminated sequences (<100 bp, chimera) ad hoc scripts: sequence trimming and parsing 0.1 TAF10_ARATH TAF10_ORYSA 100 TAF10_CRYNE TAF10_SCHPO TAF10_NEUCR TAF10_CANAL TAF10_CANGA TAF10_YEAST 100 86 TAF10_ENCCU TAF10_SCHJA TAF10_CAEEL TAF10_CAEBR 100 TAF10_DROME TAF10_ANOGA TAFAB_DROME ALVINELLA TAF10_TETNG TAF10_HUMAN TAF10_MOUSE 100 98 87 88 References Bianchetti L, Thompson JD, Lecompte O, Plewniak F, Poch O. vALId: validation of protein sequence quality based on multiple alignment data. J Bioinform Comput Biol. 2005 Chalmel F, Lardenois A, Thompson JD, Muller J, Sahel JA, Leveillard T, PochO. GOAnno: GO annotation based on multiple alignment. Bioinformatics. 2005 Ewing B, Hillier L, Wendl MC, Green P. Base-calling of automated sequencer traces using phred. Genome Res. 1998 Huang X, Madan A. CAP3: A DNA sequence assembly program. Genome Res. 1999 Lecompte O, Thompson JD, Plewniak F, Thierry J, Poch O. Multiple alignment of complete sequences (MACS) in the post-genomic era.Gene. 2001 Plewniak F, Bianchetti L, Brelivet Y, Carles A, Chalmel F, Lecompte O,Mochel T, Moulinier L, Muller A, Muller J, Prigent V, Ripp R, Thierry JC,Thompson JD, Wicker N, Poch O. PipeAlign: A new toolkit for protein family analysis.Nucleic Acids Res. 2003 Thompson JD, Muller A, Waterhouse A, Procter J, Barton GJ, Plewniak F, Poch O. MACSIMS: multiple alignment of complete sequences information management system. BMC Bioinformatics. 2006 Thompson JD, Plewniak F, Thierry J, Poch O. DbClustal: rapid and reliable global multiple alignments of protein sequences detected by database searches. Nucleic Acids Res. 2000 1 CNRS-INSERM-ULP, UMR7104/U596 – Laboratoire de Biologie et Génomique Intégratives 2 CNRS-UFR: FRE 2535- Laboratoire d’Etude des Parasites Génétiques 3 CNRS-INSERM-ULP, UMR7104/U596 – Plate-forme technologique de Biologie et Génomique structurales 4 Université du Maine, EA 3265 - Laboratoire de Biologie et Génétique Evolutive 5 CNRS-UPMC-MNHN-IRD, UMR 7138 – Systématique, Adaptation, Evolution 6 CNRS-UPMC-MNHN-IRD, UMR 7138 – Génétique et Evolution 7 CNRS-UPMC, UMR 7144 - Evolution et Génétique des Populations Marines 8 CNRS-ULP, UMR 7512 - Laboratoire de Spectrométrie de masse BioOrganique 9 ISV-UCL, Laboratoire de Biologie cellulaire (Belgium) 10 GENOSCOPE 11 CNRS-UPMC Equipe Ecophysiologie : Adaptation et Evolution Moléculaires 12 CNRS-INSERM-ULP, UMR7104/U596 – Institut de Génétique et de Biologie Moléculaire et Cellulaire For the 70,000 available reads, base-calling and low-quality (Q≤13) region trimming were performed using the Phred program. Vector sequences and other contaminants were masked using Cross-match. Poly(A/T) regions as well as repetitive sequences were masked using ad hoc scripts. After sequence trimming and masking, sequences with fewer than 100 unmasked bases were excluded from further processing. Cleaned sequences of each library were assembled separately using Cap3, leading to a total of 15,000 contigs and singlets. Mean contig length is > 900 bp and the library redundancy ranges from 53 to 79%. Annotation by the GScope platform Contigs and singlets are annotated by the genomic software platform, GScope, developed at the Laboratory of Integrative Bioinformatics and Genomics (R. Ripp, manuscript in preparation). GScope is dedicated to the integration, validation and analysis of high-throughput information. It allows management and visualisation of data (genome sequences, transcriptomic data, proteins…) through a user-friendly interface. Classical tools such as similarity search, gene prediction, codon usage determination are implemented as well as in-house programs for specialised analysis (validation of start codon, frameshift detection, oligonucleotide design, target characterisation, phylogenetic distribution…). Most of these specialised programs rely on high quality clustered multiple alignments generated by the PipeAlign (http://bips.u-strasbg.fr/PipeAlign/) protein analysis toolkit. This allows the reliable characterisation of a target protein sequence in its evolutionary context. (A) In particular, we use MACSIMS ( http://bips.u- strasbg.fr/MACSIMS/ ) to propagate structural and functional information mined from the public databases to Alvinella sequences. (B) We also use the GoAnno program ( http://bips.u-strasbg.fr/GOAnno/) to automatically annotate proteins according to the Gene Ontology.http://bips.u-strasbg.fr/PipeAlign/ http://bips.u- strasbg.fr/MACSIMS/ http://bips.u-strasbg.fr/GOAnno/ (A) Propagation of functional and structural information using MACSIMS (Multiple Alignment of Complete Sequences Information Management System) (B) Display of GOAnno results for the whole animal cDNA library Alvinella, a model for Vertebrate proteome analysis More than 50% of the Alvinella CDS exhibit a close relationship to vertebrate proteins. These results confirm the phylogenetic position of annelid and highlight Alvinella as a valuable model for studies of the vertebrate proteome at the functional, structural and evolutionary level. Phylogenetic tree (neighbour-joining method) of the transcription initiation factor TFIID subunit 10 (TAF10). Bootstrap values > 85 are indicated (100 replicates). Sequence ID are coloured according to the phylogenetic origin of the sequence: plants in green, fungi in violet, invertebrates in blue and vertebrates in red. High-throughput proteins production In order to develop a reliable experimental protocol for high throughput production of Alvinella target proteins, we collaborated with the Structural Biology and Genomics platform of Strasbourg on a test case of 53 targets. The test set comprises informational, house-keeping as well as oxidative stress proteins. E. coli expression vectors are constructed using Gateway ® technology with in-house modified vectors. Gateway ® cloning was 92% successful. Protein expression in total extracts and soluble fraction have been compared. Thermostability assaysOngoing developments To ease and speed up oligo design for protein expression tests, we have developed a new program called OliDA (Oligo Design Automatization) to automatically determine optimized cDNAs and protein boundaries through MACSIMS results analysis. Boundary determination combines PFAM-A domains or PDB structure boundaries with phylogenetic distribution and conservation patterns. This program is integrated into the GScope platform upstream to oligo ordering for PCR and will be available as a web application. WEB site showing a read chromatogram and a contig alignment Data availability All steps of assembly processes and annotation results can be viewed with the help of the secured web site interface (http://www-alvinella.u-strasbg.fr/Alvinella/). Textual and BLAST searches allow users to find potential targets. Remarkably, contig alignments and their schematic representations, as well as reads chromatograms, can be displayed.http://www-alvinella.u-strasbg.fr/Alvinella/ SDS-PAGE of soluble fraction after affinity chromatography enrichment. (*) Lanes with visible expression Beta version of OliDA web results page. The red lines indicate the proposed boundaries. User can correct cloning boundaries by clicking on the alignment. Complementary to previous experiments showing an increased thermostability of Alvinella enzymes or processes compared to human ones (table below, and K.L. Henscheid et al. 2005 for U2AF65 splicing factor which shows an increase of 6°C), we have initiated thermostability studies through the analysis of ThermoFluor kinetics. Here a transcriptional factor : the Alvinella homologue of the ERR3 human nuclear receptor. Parameter measuredMax. TAuthors Mitochondrial respiration (Arrhenius break temperature)49°CDahlhoff et al., 1991 Hemoglobin dissociation50°CTerwilliger and Terwilliger, 1984 Kinetics of cytosolic malate dehydrogenases (cMDHs)31°CDahlhoff and Somero, 1991 Thermal stability of aspartate-amino transferase61°CJollivet et al., 1995 Thermal stability of glucose-6-phosphate isomerase52°CJollivet et al., 1995 rDNA denaturation87°CDixon et al., 1992 Cuticle collagen denaturation45°CGaill et al., 1995 Interstitial collagen denaturation46°CGaill et al., 1995 Maximal functional temperature of some Alvinella enzymes and biological processes ThermoFluor ® kinetic on a Ligand Binding Domain of ERR3 nuclear receptor (collaboration with Y. Brelivet). Fluorescence before 47°C is artefactual. Maximal activity is reach at 57°C. 57°C Ladder **** Proposed boundary Propagated strand Propagated helix 25 50 75