L’ordre viral des Mononegavirales contient de nombreux virus pathogènes tels que le virus de la rougeole, le virus de la rage, le virus des oreillons ou encore le virus Ebola. Ces virus mettent tous en place des mécanismes moléculaires similaires, notamment concernant la synthèse des ARN viraux. Le complexe polymérase, composé de la polymérase virale (L) et de la phosphoprotéine (P), possède un fonctionnement encore obscur et unique dans le monde du vivant, notamment car il utilise une matrice enchâssée dans une gaine protéique. La formation de ce complexe a été étudiée et la protéine chaperon HSP90 (« Heat Shock Protein of 90 kD ») s’est révélée nécessaire à la formation du complexe. L’inhibition de l’activité d’HSP90 entraine l’ubiquitination et la dégradation de la protéine L par le protéasome. Les protéines P et HSP90 sont toutes les deux nécessaires au repliement de la protéine L et à la formation d’un complexe P-L stable, soluble et fonctionnel. Les domaines de P impliqués dans la formation du complexe, ont également été cartographiés et révèlent des interactions complexes entre P et L, mêlant liaison, stabilisation, repliement et fonction. Enfin, une interaction entre P et la protéine virale C, connue pour inhiber la synthèse des ARN viraux, a été identifiée, cartographiée et ouvre des perspectives quant aux mécanismes moléculaires sous-jacents à son effet inhibiteur. / The Mononegavirales order contains several pathogens like measles, rabies, mumps and Ebola viruses. These viruses share numerous homologous molecular mechanisms and in particular they have a highly conserved RNA synthesis machinery that is unique in the living world. Indeed, the polymerase complex, composed of the polymerase (L) and the phosphoprotein (P), uses a template of RNA recovered by a sheath made of nucleoproteins. The formation of the complex was investigated and the chaperone protein HSP90 (Heat Shock Protein of 90 kD) was shown to be required for the formation of the complex. The inhibition of HSP90 activity induces the ubiquitinylation and the degradation of the L protein by the proteasome. Both P and HSP90 are required to form stable, soluble and functional polymerase complexes. The domains of P involved in the formation of the complex have been mapped and they show that the interplay between P and L is complex with at least three identified functions: binding, folding and function of the polymerase complex. Finally, an interaction between P and the viral C protein, known to inhibit the viral RNA synthesis, have been identified, mapped and allows new perspectives concerning the molecular mechanism underlying its inhibitory effect.

Les virus Nipah (NiV) et Hendra (HeV) sont des paramyxovirus zoonotiques appartenant au genre Henipavirus. Les paramyxovirus possèdent un génome ARN simple brin de polarité négative encapsidé par la nucléoprotéine (N) au sein d’une nucléocapside hélicoïdale. Cette dernière sert de substrat pour la transcription et la réplication, réalisées par la polymérase virale qui consiste en un complexe entre la protéine L et la phosphoprotéine (P). A l’aide d’approches biophysiques, j’ai établit une cartographie de l’interaction entre la région C-terminale désordonnée de N (NTAIL) et la région C-terminale de P (PXD) chez NiV, HeV et MeV. L’observation à l’échelle atomique par RMN a confirmé l’intervention d’un élément de reconnaissance moléculaire (MoRE) qui subit un repliement α-hélical au contact de PXD. J’ai également montré la capacité des domaines NTAIL et PXD des henipavirus à former des complexes hétérologues soulignant leur proximité structurale. L’interaction NTAIL-PXD, cruciale pour le recrutement de la polymérase virale constitue une cible idéale pour des approches antivirales. Ainsi, un test de criblage à haut débit par HTRF a été mis en place dans le but d’identifier des inhibiteurs. Enfin, une approche structurale a révélé une organisation trimérique de la protéine P de NiV et HeV en solution. La résolution de la structure cristalline de la région de tétramérisation de P du virus de la rougeole montre la présence d’une région désordonnée à proximité du site putatif de recrutement de L. Collectivement, ces résultats représentent une étape clé vers l’élucidation du l’impact fonctionnel de l’oligomérisation de la protéine P sur le cycle réplicatif des paramyxovirus. / Nipah (NiV) and Hendra (HeV) viruses are zoonotic paramyxoviruses that belong to the Henipavirus genus. Paramyxoviruses possess a single-stranded negative-sense RNA genome that is encapsidated by the nucleoprotein (N) into a helical nucleocapsid. This latter is the substrate for both transcription and replication that are carried out by the polymerase, consisting of a complex between the large protein (L) and the phosphoprotein (P). Using various biophysical approaches, I was able to map the interaction between the C-terminal disordered region of N (NTAIL) and the C-terminal region of P (PXD) in NiV, HeV and MeV. Atomic resolution description of the HeV NTAIL-PXD interaction by NMR confirms the involvement of a molecular recognition element (MoRE) of α−helical nature in binding to PXD. I also showed that Henipavirus NTAIL-PXD form heterologous complexes, involving a structural similarity. As this interaction is crucial for the recruitment of the viral polymerase, it is a promising target for antiviral approaches. This prompted me to set up a protein-protein interaction (PPI) assay based on the HTRF technology to identify inhibitors. Finally, I provided the first experimental evidence of a trimeric organization of P proteins in NiV and HeV. We also solved the crystal structure of two different forms of MeV P tetramerization domain who unveiled the presence of a disordered region located near the putative L-binding site and reveal significant structural variations in coiled-coils organization. Collectively, these results represent a key step towards the elucidation of the functional impact of P protein oligomerization on the replicative cycle of paramyxoviruses.

Les rhabdovirus, dont les virus de la stomatite vésiculaire (VSV) et de la rage (RAV) constituent des prototypes, sont des virus enveloppés dont le génome est constitué d'une seule molécule d'ARN simple brin de polarité négative qui font partie de l'ordre des Mononegavirales (MNV). La machinerie de transcription/réplication de ces virus est constituée de l'ARN génomique et de trois protéines qui sont communes à tous les virus de l'ordre des MNV, la (N) qui encapside le génome viral, la grande sous-unité de l'ARN polymérase ARN dépendante (L) et la phosphoprotéine (P) qui est un cofacteur non-catalytique de la L et sert de chaperonne à la N. Le premier objectif de mon travail de thèse consistait à déterminer la structure cristallographique du domaine de dimérisation de la phosphoprotéine du virus de la rage. La P des rhabdovirus est une protéine modulaire qui contient deux régions intrinsèquement désordonnée, un domaine central responsable la dimérisation et un domaine C-terminal responsable de la fixation sur la matrice N-ARN. Le modèle atomique obtenu à une résolution de 1.5A montre que la structure est très différente de celle du domaine correspondant chez VSV. Le second objectif de mon travail était la caractérisation structurale de la grande sous-unité L de la polymérase du virus de la stomatite vésiculaire. Cette enzyme de 2109,aa, possède six régions conservées. Le domaine conservé III comprend les régions impliquées dans l'activité de polymérisation et les domaines V et VI sont responsables de la formation de la coiffe des ARNm. Plusieurs stratégies ont été envisagées successivement. (1) Sur la base de prédictions de structures secondaires et de prédictions de désordre, nous avons essayé d'exprimer différents fragments en système d'expression bactérien. Les constructions testées se sont avérées insolubles et certaines d'entre elles fixaient GroEL, indiquant un problème de repliement. (2) Nous avons alors essayé d'exprimer la L seule ou en complexe avec la P en système d'expression eucaryote. La purification s'est avérée impossible, la protéine L restant toujours associées à des protéines cellulaires visibles par coloration au bleu de Coommassie. (3) Finalement nous avons réussi à purifier la polymérase à partir de virus entier. La préparation de la polymérase était très homogène et a permis d'entreprendre une caractérisation par microscopie électronique. Une classification d'images a permis de construire un premier modèle à basse résolution. Le modèle révèle la présence d'un domaine annulaire avec plusieurs domaines structurés attachés au coeur de la polymérase. La cryo-microscopie électronique et la tomographie permettront d'obtenir plus de détails sur cette protéine. / Rhabdoviruses, including vesicular stomatitis virus (VSV) and rabies virus (RAV), are enveloped viruses which genome is made of a single molecule of negative-sense RNA and are classified in the order Mononegavirales (MNV). The transcription/replication machinery of these viruses consists of the genomic RNA and of three proteins, which are common to all other viruses of the order MNV, a nucleoprotein (N) that encapsidates the viral genome, a large subunit of the RNA-dependent RNA polymerase (L) and a phosphoprotein (P) that acts as a non-catalytic cofactor of L and a chaperone of N. The first goal of my research project was to determine the crystallographic structure of the dimerization domain of the rabies virus phosphoprotein. The P protein of the rhabdoviruses is a modular protein, which contains two intrinsically disordered regions, a central dimerization domain and a C-terminal domain involved in binding to the N-RNA template. The atomic model obtained at a resolution of 1.5 A showed that the structure is different from that of the corresponding domain of VSV. The second goal was the structural characterization of the large subunit L of VSV polymerase. The enzyme of 2109 aa has six conserved regions. Conserved region III includes the residues involved in the RNA synthesis activity, whereas domains V and VI are involved in mRNA capping formation. Three strategies were successively developed: (1) On the basis of secondary structure and disorder predictions, we tried to express different fragments in bacterial expression systems. These constructions appeared to be insoluble and some of them bound GroEL suggesting a folding problem; (2) We tried to express L alone or co-express it with P in eukaryotic expression system. The purification appeared to be impossible, the L protein always remaining associated with host-cell proteins in amounts detectable by Coommassie staining; (3) We succeeded in purifying the L protein from the virus. The L samples were homogenous and allowed a characterization by electron microscopy. Image classes allowed the reconstruction of a first low-resolution model. This model revealed the presence of a large ring-like domain and several globular domains. Cryo-electron microscopy and tomography should lead to a more detailed description of this protein.

Depuis l'avènement des techniques d'ionisation douces comme le MALDI ou l'ESI, la spectrométrie de masse est devenue un outil incontournable pour l'étude des biomolécules, et en particulier, des protéines. Le mécanisme d'ionisation spécifique des sources MALDI introduit un phénomène de discrimination spectrale généralement considéré comme un frein. Le greffage d'un motif α-cyano 4-hydroxy cinnamique ou HCCA sur un peptide permet d'en tirer parti en créant une discrimination spectrale favorable au composé marqué. Deux applications du marquage HCCA ont été développées au cours de ce travail de thèse.Une des limitations des études phosphoprotéomiques réside dans la faible ionisation des peptides phosphorylés. De nombreuses méthodes de purification ont été proposées pour contourner ce problème. Nous avons étudié une approche alternative qui consiste à greffer spécifiquement un motif HCCA sur une position phosphorylée afin d'amplifier les signaux des peptides d'intérêt. Nous avons développé en parallèle une méthodologie pour l'étude de la liaison d'un ligand peptidique à son récepteur ne nécessitant pas de radioactivité. Le greffage covalent d'HCCA sur un ligand nous a permis de le détecter et de le quantifier dans une expérience de déplacement. Cette méthodologie a notamment permis de déterminer l'affinité d'un ligand de référence pour le récepteur V1A à la vasopressine. / Since the advent of soft ionization techniques such as MALDI or ESI, mass spectrometry has become an indispensable tool for the study of biomolecules, and particularly proteins. The specific ionization mechanism of MALDI leads to a spectral discrimination phenomenon generally regarded as a restriction. The α-cyano 4-hydroxy cinnamic acid (HCCA) pattern grafting on a peptide can take advantage by introducing a spectral discrimination in favor of the labeled compound. Two applications have been developed during this thesis, based on HCCA tagging.A limitation of phosphoproteomic studies is the low ionization of phosphorylated peptides. Many purification methods have been reported to circumvent this problem. We studied an alternative approach which consists in specifically grafting a HCCA moiety on a phosphorylated position in order to amplify the signal of interest peptide.At the same time, we have developed a methodology for the study of ligand peptidic binding to its receptor which does not require radioactivity. The covalent HCCA tagged ligand has allowed us to detect and quantify it, in a binding displacement assay. More particularly, this methodology allowed us to determine the affinity of a reference ligand for the V1A vasopressin receptor.

Les virus Nipah (NiV) et Hendra (HeV) appartiennent au genre Henipavirus au sein de la famille des Paramyxoviridae. Cette famille comporte de nombreux pathogènes tel que le virus de la rougeole (MeV). Les paramyxovirus possèdent un génome de type ARN simple brin encapsidé par la nucléoprotéine (N) au sein d'une nucléocapside hélicoïdale. N interagit avec la phosphoprotéine (P) et cette dernière recrute la polymérase (L) qui assure la transcription et la réplication du génome viral. L'objectif de mon projet de thèse était de caractériser les protéines N et P ainsi que les interactions qui existent entre elles chez les trois virus, NiV, HeV et MeV. A la différence du MeV, qui a été intensivement étudié au cours des dernières années, les données moléculaires et structurales sur les Henipavirus étaient très limitées. A l'aide d'analyses computationnelles, nous avons pu déchiffrer l'organisation modulaire de N et de P, et nous avons montré que les régions, C-terminale de N (NTAIL) et N-terminale de P (PNT), sont prédites comme intrinsèquement désordonnées (RIDs). Les RIDs sont des régions fonctionnelles dépourvues de structures secondaires et tertiaires stables dans des conditions physiologiques. En utilisant des approches biochimiques et biophysiques, nous avons confirmé que NTAIL et PNT sont désordonnées. Elles conservent toutefois des structures secondaires transitoires qui pourraient correspondre à des éléments de reconnaissance moléculaire (ou MoREs) impliqués dans de transitions structurales en présence d'un partenaire. / The Paramyxoviridae family includes many important human and animal pathogens, such as measles virus (MeV), a morbillivirus, and the emerging Nipah (NiV) and Hendra (HeV) viruses, members of the Henipavirus genus. Paramyxoviruses possess a negative-strand RNA genome that is encapsidated by the nucleoprotein (N) into a helical nucleocapsid. N interacts with the phosphoprotein (P), and this latter recruits the polymerase that ensures genome replication and transcription. My PhD project has mainly focused on the characterization of the N and P proteins and on the interactions between these two proteins from the three cognate viruses, namely NiV, HeV and MeV. While MeV has been extensively studied through the past years, structural and molecular information on Henipavirus N and P proteins were rather scarce. Using computational analyses, we deciphered the modular organization of Henipavirus N and P. Intrinsically disordered regions (IDRs) were predicted within these proteins, notably at the C-terminus of N (referred to as NTAIL), and at the N-terminus of P (referred to as PNT). IDRs are functional despite they lack of a well-defined 3-D structure under physiological conditions. Biochemical and biophysical approaches pointed out a mostly disordered state for both NTAIL and PNT, although they were shown to contain short-order prone segments (i.e. molecular recognition elements, MoREs). These latter are involved in partner recognition and in disorder-to-order transitions. The C-terminal domains of the P proteins (referred to as PXD) were found to bind to NTAIL and to induce an α-helical transition thereof.

A raiva é uma doença aguda, progressiva e infecciosa do sistema nervoso central de mamíferos, causada pelo vírus da raiva (RABV). Embora possa ser prevenida por vacina, continua sendo um grave problema de saúde pública, além de ser responsável pela morte de seres humanos e muitos outros animais, incluindo os de interesse econômico. Este estudo teve como objetivo avaliar a relação entre polimorfismos dos genes que codificam as proteínas P e L de amostras de RABV pertencentes a variantes antigênicas 2 e 3 e períodos de incubação e títulos em camundongos. Para isso, foram selecionadas amostras isoladas de diferentes reservatórios de raiva de mamíferos das Ordens Carnivora e Chiroptera e amostras de bovinos, de áreas endêmicas para o vírus da raiva. As sequências obtidas foram utilizadas para a construção de árvores filogenéticas para procurar os padrões de segregação de linhagens. Os resultados mostraram que não houve marcadores ou polimorfismos que explicam as variações nos períodos de incubação e de letalidade entre cepas pertencentes a variantes antigênicas 2 e 3. Esta informação pode ser usada para discussões sobre a importância de reservatórios de raiva, a dinâmica do vírus da manutenção e evolução das diferentes formas desta zoonose entre os animais infectados, contribuindo para um estudo mais aprofundado sobre a busca de marcadores moleculares para patogênese. / Rabies is an acute, progressive and infectious disease of the central nervous system of mammals, caused by Rabies virus (RABV). Although preventable by vaccine, it remains a serious public health problem, and is responsible for the death of humans and many other animals, including those of economic interest. This study aimed to assess the relationship between polymorphisms in genes encoding the P and L proteins of RABV samples belonging to antigenic variants 2 and 3 and incubation periods and titers in mice. For this, samples isolated from different mammalian rabies reservoirs of the Orders Carnivora and Chiroptera and samples of cattle from endemic areas for rabies virus were selected. The sequences obtained were used to construct phylogenetic trees to search for the segregation patterns of strains. The results showed that there were no markers or polymorphisms that explain variations in incubation periods and lethality amongst strains belonging to antigenic variants 2 and 3. This information might be used for discussions about the importance of rabies reservoirs, the dynamics of the virus maintenance and evolution of the different forms of this zoonotic disease among infected animals, contributing to further study about the search for molecular markers for pathogenesis.

O Vírus Respiratório Sincicial Humano (HRSV) causa doença respiratória principalmente em recém-nascidos e bebês. A infecção por HRSV exerce forte interferência sobre a localização de actina, fenômeno que propomos estar relacionado à interação de TPM com M e P, levando ao rearranjo dos microtúbulos. Os objetivos gerais do projeto são de analisar interações in vitro, em bactéria e em célula entre TPM com M e P. Os dados obtidos indicam que ocorre a interação in vitro entre M e TPM 3 mas não ocorre entre P e TPM 3. Os estudos realizados em célula indicam a interação de M e P com TPM 3. Com siRNA para TPM 3 os dados não foram conclusivos e para a super expressão de TPM 3 verificamos que ocorre inibição da replicação viral. Testamos uma droga que desacopla TPM 3 dos filamentos de actina e os resultados indicam inibição da replicação viral. Concluímos com esses dados que TPM 3 tem papel fundamental no ciclo replicativo do vírus e que sua interação com M tem potencial para ser explorada como alvo terapêutico no desenvolvimento de antivirais contra HRSV. / Human Respiratory Syncytial Virus (HRSV) causes respiratory disease in newborns and babies. The HRSV infection exerts strong interference on intracellular location of actin, a phenomenon that we propose to be connected to the interaction of TPM with M and P, leading to the rearrangement of microtubules. In this project we propose to analyze interactions in vitro, in bacteria and in cells between TPM and P or M. The obtained data indicate that an in vitro interaction between M and TPM occurs but does not occur between P and TPM. Cell studies indicate an interaction of M and P with TPM. With siRNA fot TPM 3 the data were not conclusive, and for overexpression of TPM 3 an inhibition of virus replication was shown. Also, in cell, we obtained results indicating that the use of a cytoskeletal destabilizing drug is affecting viral replication. These data indicate that Tropomyosin plays a key role in the virus cycle and therefore has the potential to be exploited as a therapeutic target in the development of antiviral drugs against HRSV.

Gene expression profiles obtained by means of semi-quantitative RT-PCR showed that both PPP1R1B and PPP1R3C were frequently silenced in multiple carcinomas. Bisulfite treated tumor DNA was subjected to Methylation-specific PCR (MSP) using primers flanking across the ∼130bp CpG island of the promoter of the particular gene of interest. It was revealed that PPP1R1B and PPP1R3C gene silencing in the carcinoma cell lines were due to promoter CpG island hypermethylation. Such claim was further confirmed by bisulfite genomic sequencing (BGS). Treatment with 5' azacytidine and TSA restored PPP1R1B and PPP1R3C expression in carcinoma cells through demethylating the hypermethylated promoter. In terms of cancer growth inhibition, ectopic expression of PPP1R1B and PPP1R3C could significantly inhibit the proliferation of carcinoma cell lines by 40--50% and 50--60%, respectively, according to the result of anchorage-dependent colony formation assay. / Overall, we believed that PPP1R1B and PPP1R3C are the putative tumor suppressor genes in which their expression silencing through promoter CpG island hypermethylation may be strongly linked to the development of cancer. / Protein Phosphatase 1 regulatory subunits are a family of small molecules which define the substrate specificity and subcellular localization of protein phosphatase-1 upon their interactions. Downregulation of Protein Phosphatase 1 regulatory subunits were often associated with tumor initiation and progression, for example, ASPP family (PPP1R13A and PPP1R13B). In the present study, PPP1R1B and PPP1R3C were identified in which their tumor suppressor functions had been investigated. / Reduction in the level of p-ser473 Akt and p-ser552 beta-catenin could be observed when PPP1R1B expression was restored in respective carcinoma cells. In addition, the transcription activity of AP-1 decreased in the presence of full-length PPP1R1B expression as determined by Dual-Luciferase reporter assay system. Ectopic expression of PPP1R3C increased the amount of inactive pSer9-GSK-3beta as shown in the western blot analysis and a concomitant increased in p53 level was observed in colorectal carcinoma HCT116 cells. Transcription activity of NF-kappaB in HCT116 cells was increased but decreased in KYSE150 cells (ESCC) in the presence of PPP1R3C expression. Subcellular localization study using the GFP-fusion protein revealed that PPP1R1B protein was distributed throughout the cytoplasm while PPP1R3C protein was mainly localized around the nuclear membrane. / Leung, Ching Hei. / Adviser: Tak Cheung Chan. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 160-183). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

A raiva é uma doença aguda, progressiva e infecciosa do sistema nervoso central de mamíferos, causada pelo vírus da raiva (RABV). Embora possa ser prevenida por vacina, continua sendo um grave problema de saúde pública, além de ser responsável pela morte de seres humanos e muitos outros animais, incluindo os de interesse econômico. Este estudo teve como objetivo avaliar a relação entre polimorfismos dos genes que codificam as proteínas P e L de amostras de RABV pertencentes a variantes antigênicas 2 e 3 e períodos de incubação e títulos em camundongos. Para isso, foram selecionadas amostras isoladas de diferentes reservatórios de raiva de mamíferos das Ordens Carnivora e Chiroptera e amostras de bovinos, de áreas endêmicas para o vírus da raiva. As sequências obtidas foram utilizadas para a construção de árvores filogenéticas para procurar os padrões de segregação de linhagens. Os resultados mostraram que não houve marcadores ou polimorfismos que explicam as variações nos períodos de incubação e de letalidade entre cepas pertencentes a variantes antigênicas 2 e 3. Esta informação pode ser usada para discussões sobre a importância de reservatórios de raiva, a dinâmica do vírus da manutenção e evolução das diferentes formas desta zoonose entre os animais infectados, contribuindo para um estudo mais aprofundado sobre a busca de marcadores moleculares para patogênese. / Rabies is an acute, progressive and infectious disease of the central nervous system of mammals, caused by Rabies virus (RABV). Although preventable by vaccine, it remains a serious public health problem, and is responsible for the death of humans and many other animals, including those of economic interest. This study aimed to assess the relationship between polymorphisms in genes encoding the P and L proteins of RABV samples belonging to antigenic variants 2 and 3 and incubation periods and titers in mice. For this, samples isolated from different mammalian rabies reservoirs of the Orders Carnivora and Chiroptera and samples of cattle from endemic areas for rabies virus were selected. The sequences obtained were used to construct phylogenetic trees to search for the segregation patterns of strains. The results showed that there were no markers or polymorphisms that explain variations in incubation periods and lethality amongst strains belonging to antigenic variants 2 and 3. This information might be used for discussions about the importance of rabies reservoirs, the dynamics of the virus maintenance and evolution of the different forms of this zoonotic disease among infected animals, contributing to further study about the search for molecular markers for pathogenesis.

Establishment of the biochemical and molecular nature of cardiac development is essential for us to understand the relationship between genetic and morphological aspects of heart formation. The molecular mechanisms that underly heart development are still not clearly defined. To address this issue we have used two approaches to identify genes involved in early chick cardiac development. Differential display previously conducted in our laboratory led to the identification of two gene fragments differentially expressed in the heart that are further described in this thesis. The full-length cDNA sequence of both eukaryotic translation initiation factor-2b (eIF-2b) and NADH cytochrome b5 reductase (b5R) were isolated using library screening. The upreglation of these genes during heart development is expected given the heart is the first functional organ to form in vertebrates and protein synthesis and cell metabolism at this stage of development is maximal. Limitations in the differential display approach led to the development and optimisation of a subtractive hybridisation approach for use with small amounts of cells or tissue. To focus on cardiac gene expression during the initial phases of heart development, subtractive hybridization was performed between the cardiogenic lateral plate mesoderm of Hamburger and Hamilton stage 4 embryos and the heart primordia of stage 9 embryos. Of the 87 independent clones identified by this procedure, 59 matched known sequences with high homology, 25 matched unknown expressed sequence tag (EST) sequences with high homology, and 3 did not match any known sequence on the database. Known genes isolated included those involved in transcription, translation, cell signalling, RNA processing, and energy production. Two of these genes, high mobility group phosphoprotein A2 (HMGA2) and C1-20C, an unknown gene, were chosen for further characterisation. The role of each gene in early chick heart development and indeed development in general, was addressed using techniques such as in situ hybridisation, transfection analysis, in ovo electroporation and RNAi. HMGA2 is a nuclear phosphoprotein commonly referred to as an architectural transcription factor due to its ability to modulate DNA conformation. In keeping with this function, HMGA2/GFP fusion protein was shown to localise to the nucleus and in particular, the nucleolus. In situ hybridisation analysis suggested a role for HMGA2 in heart and somite development. HMGA2 expression was first detected at HH stage 5 in the lateral plate mesoderm, a region synonymous with cells specified to the cardiac fate. HMGA2 was also strongly expressed in the presomitic segmental plate mesoderm and as somites developed from the segmental plate mesoderm, the expression of HMGA2 showed an increasingly more restricted domain corresponding to the level of maturation of the somite. Restriction of HMGA2 expression was first detected in the dorsal region of the epithelial somite, then the dorsomedial lip of the dermomyotome, and finally the migrating epaxial myotome cells. The novel intronless gene, C1-20C, predicts a protein of 148 amino acids containing a putative zinc finger binding domain and prenyl binding motif. Zinc binding assays showed that the zinc finger domain of C1-20C/MBP fusion protein bound over six times the quantity of zinc compared to MBP alone, although not in a 1:1 stoichiometric molar ratio. C1-20C/GFP fusion protein was shown to localise to as yet unidentified intracellular cytoplasmic vesicular compartments. These compartments did not colocalise with the endosome/lysosome pathway, aparently ruling out a role for C1-20C in protein trafficking, recycling or degradation. Expression of C1-20C in the chick embryo suggests a possible role in heart and notochord development and preliminary results using siRNA suggest that C1-20C is involved in normal heart looping.