Allorecognition, defined as the ability to discriminate between self and non-self, is ubiquitous to colonial metazoans and widespread in aclonal taxa. Invertebrate allorecognition phenomena are of broad interest and have long captured the attention of geneticists by virtue of the allotypic diversity they display, marine ecologists by virtue of their control of effector mechanisms determining the outcome of intraspecific competition, evolutionary biologists by virtue of their regulation of the level at which selection acts, and immunologists by virtue of their resemblance to the allogeneic interactions that characterize pregnancy and transplantation in vertebrates. Diverse histocompatibility modes have been described in the jawed vertebrates, protochordates, and cnidarians, which are to date the only three taxa for which a genetic model to study allorecognition has been developed. Outside of the MHC-based histocompatibility of vertebrates, allorecognition determinants have been recognized in only two invertebrates. In the tunicate Botryllus, two genes involved in the histocompatibility response were characterized, FuHc and fester. In Hydractinia, the loci controlling allorecognition, alr1 and alr2, were mapped to a single chromosomal region, the allorecognition complex, and alr2 was recently identified as a polymorphic immunoglobulin superfamily (IgSF) receptor. In this study, the identification of the second Hydractinia allodeterminant, alr1, was undertaken. Chapter I briefly reviews prominent allorecognition model organisms and details the phenomenon in the model organism, Hydractinia symbiolongicarpus studied here. Chapter II describes the isolation of a 300.8 kb alr1-containing chromosomal interval by positional cloning. The analysis of that interval for its gene content and the determination of a primary alr1 candidate, CDS4, are described in Chapter III. Chapter III also reveals the existence of a complex of IgSF-like genes, to which belongs CDS4. CDS4, a novel polymorphic IgSF receptor that encodes a type I transmembrane protein with two hypervariable immunoglobulin-like extracellular domains, was confirmed to be the alr1 allodeterminant in Chapter IV, based on the investigation of natural polymorphism. CDS4 allele sequences were found to largely predict the outcome of allorecognition responses within and between laboratory lines and wild-type colonies, confirming the identity of CDS4 as the classically defined locus alr1.

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<p>Positional cloning is a method to identify genes from their position in the genome without prior knowledge about function. We used this approach to investigate the basis for three distinct genetic disorders; Obesity, Ichthyosis Prematurity Syndrome and Meniere's disease.</p><p>Obesity appears when energy intake exceeds energy expenditure which leads to an abnormal accumulation of fat in the adipocyte tissue. We have studied a family with a balanced chromosomal translocation t(4;15) segregating with severe obesity. The chromosomal breakpoints create a fusion gene involving the gene for isoform 1 of RAR-related orphan receptor A (<i>RORa1</i>) which is implicated in the regulation of adipogenesis and lipoprotein metabolism. We hypothesize that the obesity in this family is caused by haploinsufficiency of this gene or a gain of function of the fusion gene.</p><p>Ichthyosis prematurity syndrome (IPS) is a rare skin disorder belonging to a group of autosomal recessive congenital ichthyosis. We have mapped the locus for IPS to chromosome 9q34. Within the IPS locus, we identified a core haplotype with a high carrier frequency among affected, which indicate a possible founder mutation for the disease. The minimal shared region in affected patients contains seven genes which are candidates for IPS.</p><p>Meniere's disease (MD) is characterised by spontaneous attacks of vertigo, fluctuating sensorineural low frequency hearing loss, aural fullness, and tinnitus. We mapped the MD locus to chromosome 12p13 using three Swedish families. The linked region is 463 kb, containing only one gene, a phosphoinositide-3-kinase (<i>PIK3C2G</i>). Involvement of phosphatidylinositol 3-kinases (PI-3K) in the intra cellular signalling cascades of cells in mammalian balance epithelia makes this gene a good candidate gene for MD.</p>

Positional cloning is a method to identify genes from their position in the genome without prior knowledge about function. We used this approach to investigate the basis for three distinct genetic disorders; Obesity, Ichthyosis Prematurity Syndrome and Meniere's disease. Obesity appears when energy intake exceeds energy expenditure which leads to an abnormal accumulation of fat in the adipocyte tissue. We have studied a family with a balanced chromosomal translocation t(4;15) segregating with severe obesity. The chromosomal breakpoints create a fusion gene involving the gene for isoform 1 of RAR-related orphan receptor A (RORa1) which is implicated in the regulation of adipogenesis and lipoprotein metabolism. We hypothesize that the obesity in this family is caused by haploinsufficiency of this gene or a gain of function of the fusion gene. Ichthyosis prematurity syndrome (IPS) is a rare skin disorder belonging to a group of autosomal recessive congenital ichthyosis. We have mapped the locus for IPS to chromosome 9q34. Within the IPS locus, we identified a core haplotype with a high carrier frequency among affected, which indicate a possible founder mutation for the disease. The minimal shared region in affected patients contains seven genes which are candidates for IPS. Meniere's disease (MD) is characterised by spontaneous attacks of vertigo, fluctuating sensorineural low frequency hearing loss, aural fullness, and tinnitus. We mapped the MD locus to chromosome 12p13 using three Swedish families. The linked region is 463 kb, containing only one gene, a phosphoinositide-3-kinase (PIK3C2G). Involvement of phosphatidylinositol 3-kinases (PI-3K) in the intra cellular signalling cascades of cells in mammalian balance epithelia makes this gene a good candidate gene for MD.

Hereditary Motor and Sensory Neuropathy-Russe (HMSNR) is a rare recessive form of Charcot-Marie-Tooth disease (CMT) that has been identified in the European Gypsy (Roma) population. Clinically, HMSNR manifests with typical CMT symptoms, while no associated features have been detected. Distinct neuropathological features of HMSNR include the presence of numerous clusters of thinly myelinated fibres originating from regenerative activity. HMSNR has been previously mapped to chromosome 10q using a large Bulgarian Gypsy kindred. Subsequent identification of related chromosome 10q haplotypes in Spanish and Romanian Gypsy families suggested a founder mutation in the Gypsy population as the cause of HMSNR. This thesis describes the refined mapping of the HMSNR gene by generating a high-density physical-genetic map of the HMSNR region containing 20 microsatellite markers and 229 SNPs and insertion/deletions which allowed meticulous mapping of recombination breakpoints resulting in a reduction of the HMSNR gene region from 1 Mb to just 63.8 kb. Analysis of positional candidates by direct sequencing included 14 known genes, 7 predicted genes and 42 expressed sequence tags (ESTs) nonoverlapping with the genes. 78 putative HMSNR mutations were identified, two of which exhibit complete segregation with the HMSNR phenotype. Both are located in the so-called testis-specific part of unexpected candidate gene hexokinase 1 (HK1), in a rare alternative untranslated 5’ exon of HK1 and in the adjacent downstream intron. Expression analysis of transcripts containing the alternative exon suggests that the exon is not confined to testis but may be expressed in the nervous system. It remains to be speculated how a gene that functions in the fundamental process of energy generation might be involved in a neuropathy. Further investigations are likely to expand the knowledge about the importance of HK1 in the peripheral nervous system and may elucidate new roles of HK1

Allorecognition, defined as the ability to discriminate between self and non-self, is ubiquitous to colonial metazoans and widespread in aclonal taxa. Invertebrate allorecognition phenomena are of broad interest and have long captured the attention of geneticists by virtue of the allotypic diversity they display, marine ecologists by virtue of their control of effector mechanisms determining the outcome of intraspecific competition, evolutionary biologists by virtue of their regulation of the level at which selection acts, and immunologists by virtue of their resemblance to the allogeneic interactions that characterize pregnancy and transplantation in vertebrates. Diverse histocompatibility modes have been described in the jawed vertebrates, protochordates, and cnidarians, which are to date the only three taxa for which a genetic model to study allorecognition has been developed. Outside of the MHC-based histocompatibility of vertebrates, allorecognition determinants have been recognized in only two invertebrates. In the tunicate Botryllus, two genes involved in the histocompatibility response were characterized, FuHc and fester. In Hydractinia, the loci controlling allorecognition, alr1 and alr2, were mapped to a single chromosomal region, the allorecognition complex, and alr2 was recently identified as a polymorphic immunoglobulin superfamily (IgSF) receptor. In this study, the identification of the second Hydractinia allodeterminant, alr1, was undertaken. Chapter I briefly reviews prominent allorecognition model organisms and details the phenomenon in the model organism, Hydractinia symbiolongicarpus studied here. Chapter II describes the isolation of a 300.8 kb alr1-containing chromosomal interval by positional cloning. The analysis of that interval for its gene content and the determination of a primary alr1 candidate, CDS4, are described in Chapter III. Chapter III also reveals the existence of a complex of IgSF-like genes, to which belongs CDS4. CDS4, a novel polymorphic IgSF receptor that encodes a type I transmembrane protein with two hypervariable immunoglobulin-like extracellular domains, was confirmed to be the alr1 allodeterminant in Chapter IV, based on the investigation of natural polymorphism. CDS4 allele sequences were found to largely predict the outcome of allorecognition responses within and between laboratory lines and wild-type colonies, confirming the identity of CDS4 as the classically defined locus alr1. / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished

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Les interactions plante-virus diffèrent des autres interactions plante-pathogènes du fait de la nature des virus qui sont des parasites intracellulaires obligatoires. Plus spécifiquement, l’interaction haricot commun (Phaseolus vulgaris L.)-Bean pod mottle virus (BPMV) a été étudiée en mettant l’accent à la fois sur la résistance de la plante mais aussi sur la virulence du virus dans l’objectif de mieux comprendre et d’identifier les facteurs intervenant dans le dialogue moléculaire entre plante et virus. Ces deux partenaires interagissent selon le modèle «gène-à-gène» de Flor. 1) Côté plante, nous avons identifié un gène de résistance dominant vis-à-vis du BPMV chez BAT93, le gène R-BPMV. Ce gène est localisé à l’extrémité du chromosome Pv02, dans la région du locus I, un locus de résistance multi-parasitaire vis-à-vis de différents virus, bactérie et champignon. La cartographie fine du gène R-BPMV suivie du séquençage de la région à partir d’un contig de clones BACs chez BAT93 a permis d’identifier des séquences codant pour des protéines NB-LRR qui pourraient correspondre au gène R-BPMV. Des études de microsynténie et de phylogénie ont été réalisées afin de mieux comprendre l’évolution des gènes présents dans cette région. L’étude au niveau cellulaire du phénotype associé à la résistance a permis de montrer que le gène R-BPMV bloque le mouvement de cellule à cellule du virus et que le phénotype associé est température-dépendant. 2) Côté virus, le clonage de toutes les ORFs du BPMV associé à des expériences d’agroinfiltration sur P. vulgaris et Nicotiana benthamiana ont permis d’identifier deux facteurs viraux importants dans le dialogue moléculaire plante-virus : la protéine VPg du BPMV correspond à la protéine d’avirulence agissant en interaction avec le produit du gène R-BPMV dans le cadre du modèle «gène-à-gène», et l’ARN polymérase ARN-dépendante virale correspond à un suppresseur de silencing à effet faible. 3) A ce jour, la transformation génétique stable n’est pas applicable en routine chez les légumineuses. Un objectif de la thèse est de développer des outils de validation fonctionnelle pouvant s’appliquer à des gènes d’intérêt agronomique, dont des gènes de résistance aux maladies. L’approche VIGS basée sur un vecteur viral dérivé du BPMV, déjà utilisée chez le soja, a ainsi été adaptée sur haricot et pois (Pisum sativum), une légumineuse économiquement importante en Europe. / Plant-virus interactions differ from other plant-pathogen interactions because viruses are obligate intracellular parasites. More specifically, common bean (Phaseolus vulgaris L.)-Bean pod mottle virus (BPMV) interaction was studied by focusing both on the plant resistance and on the virus virulence in order to highlight and identify factors involved in the molecular dialog between plant and virus. These two partners interact according to the “gene-for-gene” model described by Flor. 1) On the plant side, we identified a dominant resistance gene against BPMV in cv. BAT93, the R-BPMV gene. This gene is located at one end of chromosome Pv02 in the I locus region, a multi-parasitic resistance locus involved in resistance to different viruses, bacteria and fungi. Fine mapping of R-BPMV followed by sequencing of the region from a BACs contig in BAT93 allowed us to identify sequences encoding NB-LRR proteins that could correspond to R-BPMV. Microsynteny and phylogeny studies were performed to understand the evolution of genes present in this region. When resistance phenotype was studied at the cellular level, we found that R-BPMV blocks BPMV cell-to-cell movement and that resistance phenotype is temperature-dependent. 2) On the virus side, cloning of all BPMV ORFs in association with agroinfiltration assays in P. vulgaris and Nicotiana benthamiana allowed us to identify two important factors involved in plant-virus molecular dialog: the BPMV VPg acting as an avirulence factor in interaction with the product of R-BPMV in the “gene-for-gene” model, and the viral RNA-dependent RNA polymerase that corresponds to a weak RNA silencing suppressor. 3) To date, stable genetic transformation is not routinely feasible in legumes. One objective of this thesis was to develop news tools for functional validation studies for genes of agronomic interest, including disease resistance genes. The VIGS approach based on the viral BPMV vector, first used in soybean, was adapted to common bean and pea (Pisum sativum), a legume species of high economic importance in Europe.