Christian Clépet

(CR1) CNRS
clepet@evry.inra.fr
+33 1 60 87 45 12

Christian Clepet Portrait

Crop Functional Genomics

Scientific Career

2000 CNRS, Senior Research Scientist. URGV. Full-length cDNA Inventories in plants.
1997 - 2000 Aventis Pharma, Genomics department. Characterization of new therapeutic targets.
1994 - 1997 Genethon, Team of J. Weissenbach.
  • construction of a 10-cM physical map spanning the glaucoma GLC1A locus.
  • cloning of the familial mediterranean fever MEFV gene.
  • genetic and physical mapping of the lamellar ichthyosis ICR2B locus.
1991 - 1993 EEC postdoctoral fellow in P. Goodfellow’s laboratory. ICRF, London and Cambridge University.
Characterization of the male-sex differenciation SRY transcript.
1987 - 1991 PhD. LCB CNRS Marseille. Supervisor J-C PATTE.
Evolution of the threonine biosynthetic pathway :
characterization of the hom, thrB, thrC and thrH genes in P.aeruginosa.

 

Publications

Analysis of expressed sequence tags generated from full-length enriched cDNA libraries of melon.

BMC Genomics. 2011 May 20;12(1):252. PMID: 21599934
Clepet C, Joobeur T, Zheng Y, Jublot D, Huang M, Truniger V, Boualem A, Hernandez-Gonzalez ME, Dolcet-Sanjuan R, Portnoy V, Mascarell-Creus A, Cano-Delgado AI, Katzir N, Bendahmane A, Giovannoni JJ, Aranda MA, Garcia-Mas J, Fei Z.

ABSTRACT:
BACKGROUND: Melon (Cucumis melo), an economically important vegetable crop, belongs to the Cucurbitaceae family which includes several other important crops such as watermelon, cucumber, and pumpkin. It has served as a model system for sex determination and vascular biology studies. However, genomic resources currently available for melon are limited.
RESULTS: We constructed eleven full-length enriched and four standard cDNA libraries from fruits, flowers, leaves, roots, cotyledons, and calluses of four different melon genotypes, and generated 71,577 and 22,179 ESTs from full-length enriched and standard cDNA libraries, respectively. These ESTs, together with ~35,000 ESTs available in public domains, were assembled into 24,444 unigenes, which were extensively annotated by comparing their sequences to different protein and functional domain databases, assigning them Gene Ontology (GO) terms, and mapping them onto metabolic pathways. Comparative analysis of melon unigenes and other plant genomes revealed that 75% to 85% of melon unigenes had homologs in other dicot plants, while approximately 70% had homologs in monocot plants. The analysis also identified 6,972 gene families that were conserved across dicot and monocot plants, and 181, 1,192, and 220 gene families specific to fleshy fruit-bearing plants, the Cucurbitaceae family, and melon, respectively. Digital expression analysis identified a total of 175 tissue-specific genes, which provides a valuable gene sequence resource for future genomics and functional studies. Furthermore, we identified 4,068 simple sequence repeats (SSRs) and 3,073 single nucleotide polymorphisms (SNPs) in the melon EST collection. Finally, we obtained a total of 1,382 melon full-length transcripts through the analysis of full-length enriched cDNA clones that were sequenced from both ends. Analysis of these full-length transcripts indicated that sizes of melon 5' and 3' UTRs were similar to those of tomato, but longer than many other dicot plants. Codon usages of melon full-length transcripts were largely similar to those of Arabidopsis coding sequences.
CONCLUSION: The collection of melon ESTs generated from full-length enriched and standard cDNA libraries is expected to play significant roles in annotating the melon genome. The ESTs and associated analysis results will be useful resources for gene discovery, functional analysis, marker-assisted breeding of melon and closely related species, comparative genomic studies and for gaining insights into gene expression patterns.


RNA captor: a tool for RNA characterization.

21533245

PLoS One. 2011 Apr 13;6(4):e18445. PMID: 21533245
Clepet C.
URGV Plant Genomics, INRA UMR1165 UEVE/CNRS ERL 8196, Evry, France.

Abstract
BACKGROUND: In the genome era, characterizing the structure and the function of RNA molecules remains a major challenge. Alternative transcripts and non-protein-coding genes are poorly recognized by the current genome-annotation algorithms and efficient tools are needed to isolate the less-abundant or stable RNAs.
RESULTS: A universal RNA-tagging method using the T4 RNA ligase 2 and special adapters is reported. Based on this system, protocols for RACE PCR and full-length cDNA library construction have been developed. The RNA tagging conditions were thoroughly optimized and compared to previous methods by using a biochemical oligonucleotide tagging assay and RACE PCRs on a range of transcripts. In addition, two large-scale full-length cDNA inventories relying on this method are presented.
CONCLUSION: The RNA Captor is a straightforward and accessible protocol. The sensitivity of this approach was shown to be higher compared to previous methods, and applicable on messenger RNAs, non-protein-coding RNAs, transcription-start sites and microRNA-directed cleavage sites of transcripts. This strategy could also be used to study other classes of RNA and in deep sequencing experiments.


The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla

Nature 449, 463-467 (27 Sept. 2007) | doi:10.1038/nature06148; PMID: 17721507
Olivier Jaillon, Jean-Marc Aury, Benjamin Noel, Alberto Policriti, Christian Clepet, Alberto Casagrande, Nathalie Choisne, Sébastien Aubourg, Nicola Vitulo, Claire Jubin, Alessandro Vezzi, Fabrice Legeai, Philippe Hugueney, Corinne Dasilva, David Horner, Erica Mica, Delphine Jublot, Julie Poulain, Clémence Bruyère, Alain Billault, Béatrice Segurens, Michel Gouyvenoux, Edgardo Ugarte, Federica Cattonaro, Véronique Anthouard, Virginie Vico, Cristian Del Fabbro, Michaël Alaux, Gabriele Di Gaspero, Vincent Dumas, Nicoletta Felice, Sophie Paillard, Irena Juman, Marco Moroldo, Simone Scalabrin, Aurélie Canaguier, Isabelle Le Clainche, Giorgio Malacrida, Eléonore Durand, Graziano Pesole, Valérie Laucou, Philippe Chatelet, Didier Merdinoglu, Massimo Delledonne, Mario Pezzotti, Alain Lecharny, Claude Scarpelli, François Artiguenave, M. Enrico Pè, Giorgio Valle, Michele Morgante, Michel Caboche, Anne-Françoise Adam-Blondon, Jean Weissenbach, Francis Quétier & Patrick Wincker

Figure 2 Schematic representation of paralogous regions derived from the three ancestral genomes in the karyotypes of V. vinifera, P. trichocarpa and A. thaliana.

The analysis of the first plant genomes provided unexpected evidence for genome duplication events in species that had previously been considered as true diploids on the basis of their genetics. These polyploidization events may have had important consequences in plant evolution, in particular for species radiation and adaptation and for the modulation of functional capacities. Here we report a high-quality draft of the genome sequence of grapevine (Vitis vinifera) obtained from a highly homozygous genotype. The draft sequence of the grapevine genome is the fourth one produced so far for flowering plants, the second for a woody species and the first for a fruit crop (cultivated for both fruit and beverage). Grapevine was selected because of its important place in the cultural heritage of humanity beginning during the Neolithic period. Several large expansions of gene families with roles in aromatic features are observed. The grapevine genome has not undergone recent genome duplication, thus enabling the discovery of ancestral traits and features of the genetic organization of flowering plants. This analysis reveals the contribution of three ancestral genomes to the grapevine haploid content. This ancestral arrangement is common to many dicotyledonous plants but is absent from the genome of rice, which is a monocotyledon. Furthermore, we explain the chronology of previously described whole-genome duplication events in the evolution of flowering plants.


Characterization of Arabidopsis thaliana mismatch specific endonucleases: application to mutation discovery by TILLING in pea.

Plant J. 2007 Sep;51(6):1116-25. Epub 2007 Jul 25. PMID: 17651368
Triques K, Sturbois B, Gallais S, Dalmais M, Chauvin S, Christian Clepet, Sébastien Aubourg, Rameau C, Michel Caboche, Abdel Ihafid Bendahmane
URGV, UMR INRA CNRS. Evry, France.

Scanning DNA sequences for mutations and polymorphisms has become one of the most challenging, often expensive and time-consuming obstacles in many molecular genetic applications, including reverse genetic and clinical diagnostic applications. Enzymatic mutation detection methods are based on the cleavage of heteroduplex DNA at the mismatch sites. These methods are often limited by the availability of a mismatch-specific endonuclease, their sensitivity in detecting one allele in a pool of DNA and their costs. Here, we present detailed biochemical analysis of five Arabidopsis putative mismatch-specific endonucleases. One of them, ENDO1, is presented as the first endonuclease that recognizes and cleaves all types of mismatches with high efficiency. We report on a very simple protocol for the expression and purification of ENDO1. The ENDO1 system could be exploited in a wide range of mutation diagnostic tools. In particular, we report the use of ENDO1 for discovery of point mutations in the gibberellin 3beta-hydrolase gene of Pisum sativum. Twenty-one independent mutants were isolated, five of these were characterized and two new mutations affecting internodes length were identified. To further evaluate the quality of the mutant population we screened for mutations in four other genes and identified 5-21 new alleles per target. Based on the frequency of the obtained alleles we concluded that the pea population described here would be suitable for use in a large reverse-genetics project.


An eIF4E allele confers resistance to an uncapped and non-polyadenylated RNA virus in melon.

Plant J. 2006 Nov;48(3):452-62. Epub 2006 Oct 5. PMID: 17026540
Nieto C, Morales M, Orjeda G, Clepet C., Monfort A, Sturbois B., Puigdomenech P, Pitrat M., Caboche M, Dogimont C., Garcia-Mas J, Aranda M., and A. Bendahmane (2006).
Centro de Edafología y Biología Aplicada del Segura (CEBAS)- CSIC, Apdo. correos 164, 30100 Espinardo, Murcia, Spain.

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The characterization of natural recessive resistance genes and virus-resistant mutants of Arabidopsis have implicated translation initiation factors of the 4E family [eIF4E and eIF(iso)4E] as susceptibility factors required for virus multiplication and resistance expression. To date, viruses controlled by these genes mainly belong to the family Potyviridae. Melon necrotic spot virus (MNSV) belongs to the family Tombusviridae (genus Carmovirus) and is an uncapped and non-polyadenylated RNA virus. In melon, nsv-mediated resistance is a natural source of recessive resistance against all strains of MNSV except MNSV-264. Analyses of chimeras between non-resistance-breaking and resistance-breaking strains have shown that the avirulence determinant maps to the 3′-untranslated region (3′-UTR) of the viral genome. Using a combination of positional cloning and microsynteny analysis between Arabidopsis thaliana and melon, we genetically and physically delimited the nsv locus to a single bacterial artificial chromosome clone and identified the melon eukaryotic translation initiation factor 4E (Cm-eIF4E) as a candidate gene. Complementation analysis using a biolistic transient expression assay, confirmed Cm-eIF4E as the product of nsv. A single amino acid change at position 228 of the protein led to the resistance to MNSV. Protein expression and cap-binding analysis showed that Cm-eIF4E encoded by a resistant plant was not affected in it's cap-binding activity. The Agrobacterium-mediated transient expression of the susceptibility allele of Cm-eIF4E in Nicotiana benthamiana enhanced MNSV-264 accumulation. Based on these results, a model to explain melon resistance to MNSV is proposed. These data, and data from other authors, suggest that translation initiation factors of the eIF4E family are universal determinants of plant susceptibility to RNA viruses.


Whole genome sequence comparisons and "full-length" cDNA sequences: a combined approach to evaluate and improve Arabidopsis genome annotation.

Genome Res. 2004 Mar;14(3):406-13. PMID: 14993207
Castelli, V., Aury, J-M, Jaillon, O, Wincker, P., Clepet, C., Menard, M., Cruaud, C., Schachter, V., Temple, G., Caboche, M., Weissenbach, J., and Salanoubat, M.
Genoscope-Centre National de Séquençage and Centre National de la Recherche Scientifique Unité Mixte de Recherche-3080, 91000 Evry, France.

To evaluate the existing annotation of the Arabidopsis genome further, we generated a collection of evolutionary conserved regions (ecores) between Arabidopsis and rice. The ecore analysis provides evidence that the gene catalog of Arabidopsis is not yet complete, and that a number of these annotations require re-examination. To improve the Arabidopsis genome annotation further, we used a novel "full-length" enriched cDNA collection prepared from several tissues. An additional 1931 genes were covered by new "full-length" cDNA sequences, raising the number of annotated genes with a corresponding "full-length" cDNA sequence to about 14,000. Detailed comparisons between these "full-length" cDNA sequences and annotated genes show that this resource is very helpful in determining the correct structure of genes, in particular, those not yet supported by "full-length" cDNAs. In addition, a total of 326 genomic regions not included previously in the Arabidopsis genome annotation were detected by this cDNA resource, providing clues for new gene discovery. Because, as expected, the two data sets only partially overlap, their combination produces very useful information for improving the Arabidopsis genome annotation.


Improved full-length cDNA production based on RNA tagging by T4 DNA ligase.

Nucleic Acids Res. 2004 Jan 2;32(1):e6. PMID: 14704363
Christian Clepet, Isabelle Le Clainche and Michel Caboche (2004).
Unité de Recherches en Génomique Végétale, INRA/CNRS, 2 Rue Gaston-Crémieux, F-91057 Evry Cedex, France. clepet@evry.inra.fr

Second-strand cDNA priming is a central problem for full-length characterization of transcripts. A new strategy using bacteriophage T4 DNA ligase and partially degenerate adapters is proposed for grafting a sequence tag to the end of polyribonucleotides. Based on this RNA tagging system and previously described protocols, a new method for full-length cDNA production has been implemented. Validation of the method is shown in Arabidopsis thaliana by the construction of a full-length cDNA library and the analysis of 154 clones and by 5'-RACE-PCR run on a documented experimental system.


ThrH, a homoserine kinase isozyme with in vivo phosphoserine phosphatase activity in Pseudomonas aeruginosa.

Microbiology. 1999 Apr;145 ( Pt 4):845-53. PMID: 10220164
Patte J-C., C. Clepet, M. Bally, F. Borne, V. Mejean and M. Foglino.
LCB, Centre National de la Recherche Scientifique, Marseille, France. patte@ibsm.cnrs-mrs.fr

Homoserine kinase, the product of the thrB gene, catalyses an obligatory step of threonine biosynthesis. In Pseudomonas aeruginosa, unlike Escherichia coli, inactivation of the previously identified thrB gene does not result in threonine auxotrophy. A new gene, named thrH, was isolated that, when expressed in E. coli thrB mutant strains, results in complementation of the mutant phenotype. In P. aeruginosa, threonine auxotrophy is observed only when both thrB and thrH are simultaneously inactivated. Thus, thrH encodes a protein with an in vivo homoserine-kinase-like activity. Surprisingly, thrH overexpression allows complementation of serine auxotrophy of E. coli and P. aeruginosa serB mutants. These mutants are affected in the phosphoserine phosphatase protein, an enzyme involved in serine biosynthesis. Comparison analysis revealed sequence homology between ThrH and the SerB proteins from different organisms. This could explain the in vivo phosphoserine phosphatase activity of ThrH when overproduced. ThrH differs from the protein encoded by the serB gene which was identified in P. aeruginosa. Thus, two SerB-like proteins co-exist in P. aeruginosa, a situation also found in Mycobacterium tuberculosis.


Lamellar ichthyosis: further narrowing, physical and expression mapping of the chromosome 2 candidate locus.

European Journal of Human Genetics (1999) vol. 7 (1) ; p 77-87. PMID: 10094194
154 Parmentier L., C. Clépet, O. Boughdene-Stambouli, H. Lakhdar, C. Blanchet-Bardon, L. Dubertret, E. Wunderle, F. Pulcini, C. Fizames, J. Weissenbach.
CNRS URA 1922, Généthon, Evry, France.

Lamellar ichthyosis (LI) is an autosomal recessive genodermatosis which has been shown to be both clinically and genetically heterogeneous. Keratinocyte transglutaminase (or transglutaminase 1: TGM1) has been demonstrated to be the disease-causing gene in some families, whilst in others, a second unidentified LI gene was mapped to chromosome 2q33-35 (ICR2B locus). In this study, we present a physical map that encompasses the ICR2B locus, including the mapping of new microsatellite markers. Based on this new map, genotyping additional families highly suggests a reduction in size of the candidate interval. The final interval is covered by a single yeast artificial chromosome (937-H-3) which is 2.2Mb in length. Fine mapping of potential candidate transcripts was also focused on this region.


Homozygotes carrying an autosomal dominant TIGR mutation do not manifest glaucoma.

Nat Genet. 1998 Aug;19(4):319-21. PMID: 9697688
J. Morissette, C. Clépet, S. Moisan, S. Dubois, E. Winstall, D. Vermeeren, T. D. Nguyen, J. R. Polansky, G. Côté, J. L. Anctil, M. Amyot, M. Plante, P. Falardeau & V. Raymont.


A transcriptional Map of the FMF region.

Genomics. 1998 Jun 1;50(2):147-60. PMID: 9653642
Bernot, R. Heilig, C. Clépet, N Smaoui, C. Da Silva, J-L. Petit, C. Devaud, N. Chiannilkulchai, C. Fizames, D. Samson, C. Cruaud, C. Caloustian, G. Gyapay, M. Delpech, and J. Weissenbach.
Généthon, CNRS-URA 1922, Evry, France. bernot@genos cope.cns.fr

Familial Mediterranean fever (FMF) is a recessively inherited disorder characterized by attacks of fever and serositis, which affects primarily non-Ashkenazi Jews, Armenians, Turks, and Arabs. We present here a transcriptional map covering the FMF locus that we constructed in the course of the positional cloning of the gene responsible for this disease. This map was established from a contig constructed with YAC, BAC, and cosmid clones and covers about 500 kb of 16p13.3. It contains nine transcriptional units corresponding to known genes or to genes belonging to known gene families, 23 gene fragments characterized by partial sequences, and an endogenous retrovirus sequence. It thus considerably increases the number of genes in this interval and improves our knowledge concerning some of the genes or gene families present in this region. Data accumulated in this region were also used in a comparative study of different methods of exon detection.


Genotypic diagnosis of familial Mediterranean fever (FMF) using new microsatellite markers: example of two extensive non-Ashkenazi Jewish pedigrees.

J Med Genet. 1997 May;34(5):375-81. PMID: 9152834
Dupont M, Dross C, Smaoui N, Nedelec B, Grateau G, Clépet C, Gourdier I, Koné-Paut I, Delpech M, Demaille J, Touitou I.
Laboratoire de Génétique Moléculaire et Chromosomique, Hôpital A de Villeneuve, Montpellier, France.

Familial Mediterranean fever is an autosomal recessive disease characterised by multiple attacks of serosal inflammation in the absence of treatment. In the absence of timely diagnosis, renal amyloidosis is a life threatening complication. The diagnosis is often missed because no specific test is available. Early colchicine treatment prevents attacks and renal complications. The FMF gene (MEF) has been mapped to chromosome 16p 13.3 but has not yet been identified. We compared the suitability of a series of microsatellite markers (four of them were new) and propose the routine use of seven of these markers, exhibiting alleles in strong linkage disequilibrium with the disease and informative in 100% of diagnosed patients. Moreover, the discovery of a homozygous status for the 3-3-9 (or 3-3-18) haplotype at the core loci (D16S3070, D16S3082, and D16S3275), which was found in 73% non-Ashkenazi Jewish patients, points to a diagnosis of FMF, even in sporadic cases, with a risk of error of only 2.10(-5). Two extensive pedigrees covering most indications for genetic counselling are presented, showing that it is now possible both prospectively and retrospectively to identify members likely to have MEF mutations. With the help of this accurate test, colchicine treatment can be better targeted, especially where the symptomatology is mild or atypical.


" Haplotype analysis and mapping of the familial mediterranean fever MEFV locus in a 60 kb interval "

N. Smaoui, A. Bernot, R. Heilig, I. Touitou, B. Nédélec, G. Grateau, M. Rosenbaum, J. Medaxian, J-C. Kouyoumdjan, M. Delpech, J. Weissenbach and C. Clépet.
Familial Mediterranean Fever, Editors: E. Sohar, J. Gafni, M. Pras; Freund Publishing House Ltd, London and Tel Aviv, 1997.


"Identification of marenostrin, the gene responsible for familial mediterranean fever"

Bernot A., C. Clépet, R. Heilig, N. Smaoui, B. Nédelec, M. Delpech, G. Grateau, J. Demaille, J. Weissenbach and I. Touitou.
Familial Mediterranean Fever, Editors: E. Sohar, J. Gafni, M. Pras; Freund Publishing House Ltd, London and Tel Aviv, 1997.


" A candidate gene for familial Mediterranean fever "

Nature Genetics (1997) vol. 17 ; p 25- 31.
004 The French FMF Consortium, à Généthon : A. Bernot, C. Clépet, C. Dasilva, C. Devaud, JL. Petit, C. Caloustian, C. Cruaud, D. Samson, F. Pulcini, J. Weissenbach and R. Heilig.


"A 10-cM YAC contig spanning GLC1A, the primary open-angle locus at 1q23- q25 "

European Journal of Human Genetics (1996) ; vol. 4 ; p 250-259.
003 Clepet C., H.J.G. Dauwerse, C. Desmaze, G.J.B. van Ommen, J. Weissenbach and J. Morissette.


" Localization of the familial mediterranean fever gene (FMF) to a 250-kb interval in non-ashkenazi jewish founder haplotypes "

American Journal of Human Genetics (1996) vol. 59 ; p 603-612.
163 The French FMF Consortium, à Généthon : S. Fauré, J-F Prud'homme, C. Clépet and J. Weissenbach.


" The human SRY transcript "

Human Molecular Genetics (1993) vol. 2(12) ; p 2007-2012.
253 Clepet C, A. Shaeffer, A. Sinclair, M. Palmer, R. Lovell-Badge and P.N. Goodfellow.


" Evolutionary comparisons of three enzymes of the threonine biosynthetic pathway among several microbial species "

Biochimie (1993) ; vol. 75 ; p 487-495.
095 Cami B., C. Clepet, J.C. Patte.


" Isolation, organisation and expression of the Pseudomonas aeruginosa threonine genes "

Molecular Microbiology (1992) ; vol. 6 (21) ; p 3109-3119.
150 Clepet C., F. Borne, V. Krishnapillai, C. Baird, J. C. Patte and B. Cami.


Patents

WO 2005/045031 24 October 2003, CNRS. Christian Clépet
« Method for the production of full-length cDNAs by ligaturation of an adapter with a cohesive end »

WO 00/71710, 25 May 1999, AVENTIS Pharma
« EXPRESSION PRODUCTS OF GENES INVOLVED IN DESEASES RELATED TO CHOLESTEROL METABOLISM »

WO 99/09059, 13 August 1998, Généthon II,
« Familial mediterranean fever gene »