Antigen Receptor Signaling to NF-kappa B via CARMA1, BCL10, and MALT1

The signaling pathway controlling antigen receptor-induced regulation of the transcription factor NF-kappa B plays a key role in lymphocyte activation and development and the generation of lymphomas. Work of the past decade has led to dramatic progress in the identification and characterization of new players in the pathway. Moreover, novel enzymatic activities relevant for this pathway have been discovered, which represent interesting drug targets for immuno-suppression or lymphoma treatment. Here, we summarize these findings and give an outlook on interesting open issues that need to be addressed in the future.

Biochemical characterization of recombinant human telomerase

Résumé Les télomères sont les structures ADN-protéines des extrémités des chromosomes des eucaryotes. L'ADN télomérique est constitué de courtes séquences répétitives. L'intégrité des télomères est essentielle pour protéger les extrémités des chromosomes contre les systèmes de dégradations et pour les distinguer des cassures de l'ADN double brin. Parce que la machinerie de la réplication de l'ADN n'est pas capable de répliquer l'extrémité des chromosomes, les télomères raccourcissent au fur et à mesure des cycles de réplication. Dès que les télomères atteignent une longueur critique, leur structure protectrice est perdue. Cela induit un signal de dommage de l'ADN et l'arrêt du cycle cellulaire. Pour contrebalancer le raccourcissement des télomères, les cellules qui s'auto régénèrent, dont les cellules de la moelle osseuse, les lymphocytes activés et 80-90% des cellules cancéreuses, expriment la télomérase. C'est une ribonucléoprotéine qui a la capacité de synthétiser des séquences télomériques par transcription inverse d'une courte séquence contenue dans sa propre sous-unité ARN avec laquelle elle est associée. La télomérase humaine est une enzyme processive au niveau de l'addition des nucléotides et aussi des répétitions télomériques. La télomérase de levure et la télomérase humaine sont toutes deux dimériques et il a été montré que la télomérase humaine recombinante contient deux ARN qui coopèrent pour fonctionner ainsi que deux sous-unités catalytiques. Cependant, il n'a pas encore été montré quel est le rôle de la dimérisation dans l'activité de la télomérase. Afin d'élucider ce rôle, nous avons exprimé, reconstitué et purifié la télomérase humaine dimérique recombinante. Et pour étudier l'effet d'ARN mutants sur l'activité de la télomérase, nous avons développé une méthode pour reconstituer et enrichir en hétérodimères de télomérase. Les hétérodimères contiennent une sous-unité ARN sauvage et une sous-unité ARN mutée au niveau de la séquence de la matrice. Sur l'ARN muté nous avons introduit une étiquette aptamer ARN-S1 puis nous avons purifié la télomérase via l'etiquette Si. Nous avons montré que la dimérisation est essentielle pour l'activité de la télomérase. Nos données indiquent que chaque télomérase du dimère allonge leur substrat, l'ADN télomérique, indépendamment l'une de l'autre à chaque cycle d'élongation mais que l'addition itérative de répétitions télomériques nécessite une coopération entre les deux télomérases du dimère. Nous proposons donc un modèle dans lequel les deux télomérases du dimères se lient et allongent deux substrats télomères et que pendant l'élongation processive les deux enzymes subissent un changement de conformation de manière coordonnée, ce changement va permettre le repositionnement des substrats pour d'autres cycles d'additions de répétitions télomériques. Dyskeratosis congenita est une maladie mortelle due majoritairement au disfonctionnement de la moelle osseuse. Dans la forme autosomale de la maladie, l'ARN de la télomérase contient des mutations. En utilisant notre système de reconstitution, nous avons montré que ces ARN mutés, qui ont perdu leur activité enzymatique dans le cas d'un homodimère de mutants, sont dominant négatifs quand ils sont présents dans les hétérodimères sauvage/mutant. Cet effet trans-dominant négatif pourrait contribuer à la progression de la maladie. Abstract Telomeres are protein-DNA structures at the ends of linear eukaryotic chromosomes. The telomeric DNA consists of tandemly repeated sequences. Telomeric integrity is essential to protect chromosomal ends from nucleolytic degradation and to prevent their recognition as DNA double strand breaks. Due to the inability of the conventional DNA replication machinery to replicate terminal DNA stretches, telomeres shorten with continuous rounds of DNA replication. As soon as telomeres reach a critical length, their protective structure is lost and the deprotected telomeres will induce a DNA damage response leading to cell cycle arrest. To counteract telomere shortening, self-renewing cells, including bone marrow cells, activated lymphocytes and 80-90% of cancer cells express the cellular reverse transcriptase telomerase, which has the capacity to synthesize telomeric repeats by reverse transcription of a short template sequence encoded by its stably associated RNA subunit. Human telomerase is a processive enzyme for nucleotide as well as repeat addition. Both yeast and human telomerase are dimeric enzymes and recombinant human telomerase has been shown to contain two functionally cooperating RNAs and most probably also two protein subunits. However, it has remained unclear how dimerization may contribute to telomerase activity. To study the role of dimerization, we expressed, reconstituted and purified recombinant human telomerase. We also developed a new method to reconstitute and enrich for telomerase heterodimers containing wild-type (wt) and mutant telomerase RNA subunits. To this end we introduced an S1-RNA-aptamer tag into telomerase RNA and purified telomerase reconstituted with a mixture of untagged and tagged RNA via the S1-tag. Using this experimental system, we introduced template mutations in the tagged RNA subunit and examined the effect of mutant RNAs on wt telomerase activity in wt/mutant heterodimers. We obtained evidence that dimerization is essential for telomerase activity. Our data indicate that the two subunits elongate telomere substrates independently of each other during single rounds of elongation, but that iterative addition of telomeric repeats requires cooperation between the two subunits. We suggest a model, in which dimeric telomerases bind and elongate two telomere substrates and that the two subunits undergo coordinated conformational changes during processive elongation that enable repositioning the substrates for subsequent rounds of repeat addition. Dyskeratosis congenita is a multisystemic disease with bone marrow failure as the major cause of death. The autosomal form of this disease was found to harbor mutations in the telomerase RNA. Using our reconstitution system, we tested whether mutant dyskeratosis telomerase RNAs behaved in a dominant negative manner. We observed that dyskeratosis telomerase RNA mutants, which lacked enzymatic activity were dominant negative, when present in wt/ mutant heterodimers. The transdominant negative effect of these mutants may contribute to disease progression.

Significant genetic and phenotypic changes arising from clonal growth of a single spore of an arbuscular mycorrhizal fungus over multiple generations.

Arbuscular mycorrhizal fungi (AMF) are highly successful plant symbionts. They reproduce clonally producing multinucleate spores. It has been suggested that some AMF harbor genetically different nuclei. However, recent advances in sequencing the Glomus irregulare genome have indicated very low within-fungus polymorphism. We tested the null hypothesis that, with no genetic differences among nuclei, no significant genetic or phenotypic variation would occur among clonal single spore lines generated from one initial AMF spore. Furthermore, no additional variation would be expected in the following generations of single spore lines. Genetic diversity contained in one initial spore repeatedly gave rise to genetically different variants of the fungus with novel phenotypes. The genetic changes represented quantitative changes in allele frequencies, most probably as a result of changes in the frequency of genetic variation partitioned on different nuclei. The genetic and phenotypic variation is remarkable, given that it arose repeatedly from one clonal individual. Our results highlight the dynamic nature of AMF genetics. Even though within-fungus genetic variation is low, some is probably partitioned among nuclei and potentially causes changes in the phenotype. Our results are important for understanding AMF genetics, as well as for researchers and biotechnologists hoping to use AMF genetic diversity for the improvement of AMF inoculum.

Interactions between feather-degrading bacteria and an avian host : zebra finches (Taeniopygia guttata)

Abstract: Birds harbor a variety of bacteria on their plumage, some of which can degrade feathers in vitro. Whether these keratinolytic bacteria are active on live birds and can effect feather degradation on birds is debatable. The effect of such bacteria on the body condition and behavior of birds, is unknown. Using a community of feather-degrading bacteria (EB), we investigate the interaction between the activity and load of such bacteria, on the morphology, body condition, and behavior of zebra finches (Taeniopygia guttata). In Chapter 2, we find that the elevated loads of such microbes lead to a reduction in the expression of morphological traits, such as male bill color (a sexually selected trait) and uropygial gland volume, without reducing body mass, or evoking a cellular immune response. We also suggest the presence of a carotenoid based defense response in hosts, to such elevated loads of microbes and document a sex-based difference in the source of carotenoids used for such a response. In Chapter 3, we investigated the effect of EB loads on male mate choice of zebra finches, wherein male choice of females with elevated and un-altered bacterial loads, varied with male size. We found that larger males preferred females with higher bacterial load and smaller males preferred females with lower bacterial load. Chapter 4 demonstrates that the presence of melanin in feathers reduces the growth and activity of the community of feather-degrading bacteria (EB) and that the EB community can effect feather degradation in humid conditions, without broth. Additional results also demonstrate that the EB community consists of bacteria that can attach themselves to feathers on live birds and those that can live freely on avian plumage. Finally, chapter 5 demonstrates that the self-maintenance, social and sexual behaviors of birds are implicated in the infection and horizontal transmission of bacteria. It also suggests a linked oral - faecal - genital mode of transmission of pathogens in birds. These results demonstrate that differential loads of normal flora of vertebrate hosts can effect changes in their morphology and behavior. They also shed light on the role of feather-degrading bacteria in the evolution of melanin polymorphism in birds and suggest that bacteria can be active on live birds. This thesis also highlights the importance of social and, sexual behaviors of birds, in epidemiology. Résumé: Les Oiseaux ont dans leur plumage diverses bactéries dont certaines dégradent les plumes in vitro, néanmoins. Il n'est pas clair, au vu de précédentes études, si ces bactéries kératinolytiques sont actives sur des oiseaux vivants, et si celles-ci dégradent effectivement le plumage de leur hôte, L'effet de ces bactéries sur la condition corporelle ainsi que le comportement des oiseaux n'est pas connu. A l'aide d'une communauté de bactéries dégradant les plumes (EB), non pathogènes, nous examinons les interactions entre l'activité et la charge bactérienne sur la morphologie, la condition corporelle et le comportement du diamant mandarins (Taeniopygia guttata). Dans le chapitre 2, nous montrons qu'une charge élevée de ces microbes mène à une réduction de l'expression de certains traits morphologiques, tels que la couleur du bec chez le mâle (un trait soumis à sélection sexuelle), ainsi que le volume de la glande uropygienne, sans qu'il y ait une réduction de la masse corporelle, ni déclenchement d'une réponse immune cellulaire. Nos données suggèrent la présence d'une défense chez l'hôte à des charges élevées de bactéries basée sur la présence de caroténoïdes. Nous montrons, de plus une différence liée au sexe dans la source des caroténoïdes utilisé pour cette réponse. Dans le chapitre 3 nous examinons l'influence de la charge bactérienne EB sur le choix des mâles chez le diamant mandarins. Des femelles avec une charge bactérienne normale et augmentée sont choisies par les mâles et ce choix varie avec la taille des mâles. Nous avons mis en évidence que les grands mâles préfèrent les femelles avec une charge bactérienne plus élevée. Les petits mâles préfèrent les femelles avec une charge bactérienne réduite. Le chapitre 4 démontre que la présence de mélanine dans les plumes réduit la croissance et l'activité de la communauté de bactéries dégradant le plumage (EB), et que cette communauté EB peut dégrader les plumes dans des conditions humides, sans milieu de culture liquide. De plus nous montrons que cette communauté consiste en des bactéries qui peuvent s'attacher sur les plumes d'oiseaux vivants ainsi que des bactéries libres. Pour finir nous montrons dans le chapitre 5 que la maintenance corporelle, l'interaction sociale et le comportement sexuel de ces oiseaux sont impliqués dans l'infection et la transmission horizontale de ces bactéries. Nos données suggèrent une transmission orale-fécale-génitale des pathogènes chez les oiseaux. Ces résultats montrent que des charges différentes de la flore bactérienne habituelle et non pathogène de vertébrés peuvent affecter leur morphologie et leur comportement. Ils éclaircissent également le rôle des bactéries dégradant les plumes dans l'évolution du polymorphisme mélanique chez les oiseaux et suggèrent que ces bactéries peuvent être actives sur des oiseaux vivants. Cette thèse souligne également l'importance du comportement social et sexuel des oiseaux dans l'épidémiologie.

Multifocal epithelial tumors and field cancerization: stroma as a primary determinant.

It is increasingly evident that cancer results from altered organ homeostasis rather than from deregulated control of single cells or groups of cells. This applies especially to epithelial cancer, the most common form of human solid tumors and a major cause of cancer lethality. In the vast majority of cases, in situ epithelial cancer lesions do not progress into malignancy, even if they harbor many of the genetic changes found in invasive and metastatic tumors. While changes in tumor stroma are frequently viewed as secondary to changes in the epithelium, recent evidence indicates that they can play a primary role in both cancer progression and initiation. These processes may explain the phenomenon of field cancerization, i.e., the occurrence of multifocal and recurrent epithelial tumors that are preceded by and associated with widespread changes of surrounding tissue or organ "fields."

On the cellular site of two-pore channel TPC1 action in the Poaceae.

The slow vacuolar (SV) channel has been characterized in different dicots by patch-clamp recordings. This channel represents the major cation conductance of the largest organelle in most plant cells. Studies with the tpc1-2 mutant of the model dicot plant Arabidopsis thaliana identified the SV channel as the product of the TPC1 gene. By contrast, research on rice and wheat TPC1 suggested that the monocot gene encodes a plasma membrane calcium-permeable channel. To explore the site of action of grass TPC1 channels, we expressed OsTPC1 from rice (Oryza sativa) and TaTPC1 from wheat (Triticum aestivum) in the background of the Arabidopsis tpc1-2 mutant. Cross-species tpc1 complementation and patch-clamping of vacuoles using Arabidopsis and rice tpc1 null mutants documented that both monocot TPC1 genes were capable of rescuing the SV channel deficit. Vacuoles from wild-type rice but not the tpc1 loss-of-function mutant harbor SV channels exhibiting the hallmark properties of dicot TPC1/SV channels. When expressed in human embryonic kidney (HEK293) cells OsTPC1 was targeted to Lysotracker-Red-positive organelles. The finding that the rice TPC1, just like those from the model plant Arabidopsis and even animal cells, is localized and active in lyso-vacuolar membranes associates this cation channel species with endomembrane function.

Carotid Dose Spring in Definitive Irradiation of T1N0 Squamous Cell Laryngeal Carcinoma Using Helical Tomotherapy

Purpose/Objective(s): To implement a carotid dose sparing protocol using helical Tomotherapy in T1N0 squamous cell laryngeal carcinoma.Materials/Methods: Between July and August 2010, 7 men with stage T1N0 laryngeal carcinoma were included in this study. Age ranged from 47 - 74 years. Staging included endoscopic examination, CT-scan and MRI when indicated. Planned irradiation dose was 70 Gy in 35 fractions over 7 weeks. A simple treatment planning algorithm for carotid sparing was used: maximum point dose to the carotids 35 Gy, to the spinal cord 30 Gy, and 100% PTV volume to be covered with 95% of the prescribed dose. Carotid volume of interest extended to 1 cm above and below of the PTV. Doses to the carotid arteries, to the critical organs, and to the planned target volume (PTV) with our standard laryngeal irradiation protocol was compared. Daily megavoltage scans were obtained before each fraction. When necessary, the Planned Adaptive software (TomoTherapy Inc., Madison, WI) was used to evaluatethe need for a re-planning, which has never been indicated. Dose data were extracted using the VelocityAI software (Atlanta, GA), and data normalization and dose-volume histogram (DVH) interpolation were realized using the Igor Pro software (Portland, OR).Results:A significant (p\0.05) carotid dose sparing compared to our standard protocol with an average maximum point dose of 38.3 Gy (standard deviation [SD] 4.05 Gy), average mean dose of 18.59 Gy (SD 0.83 Gy) was achieved. In all patients, 95% of the carotid volume received less than 28.4 Gy (SD 0.98 Gy). The average maximum point dose to the spinal cord was 25.8 Gy (SD 3.24 Gy). PTV was fully covered with more than 95% of the prescribed dose for all patients with an average maximum point dose of 74.1 Gy and the absolute maximum dose in a single patient of 75.2 Gy. To date, the clinical outcomes have been excellent. Three patients (42%) developed stage 1 mucositis that was conservatively managed, and all the patients presented a mild to moderate dysphonia. All adverse effects resolved spontaneously in the month following the end of treatment. Early local control rate is 100% considering a 4 - 5 months post treatment follow-up.Conclusions: Helical Tomotherapy allows a clinically significant decrease of carotid irradiation dose compared to standard irradiation protocols with an acceptable spinal cord dose tradeoff. Moreover, this technique allows the PTV to be homogenously covered with a curative irradiation dose. Daily control imaging brings added security margins especially when working with high dose gradients. Further investigations and follow-up are underway to better evaluate the late clinical outcomes especially the local control rate, late laryngeal and vascular toxicity, and expected potential impact on cerebrovascular events.

Structure and expression profile of the Arabidopsis PHO1 gene family indicates a broad role in inorganic phosphate homeostasis.

PHO1 has been recently identified as a protein involved in the loading of inorganic phosphate into the xylem of roots in Arabidopsis. The genome of Arabidopsis contains 11 members of the PHO1 gene family. The cDNAs of all PHO1 homologs have been cloned and sequenced. All proteins have the same topology and harbor a SPX tripartite domain in the N-terminal hydrophilic portion and an EXS domain in the C-terminal hydrophobic portion. The SPX and EXS domains have been identified in yeast (Saccharomyces cerevisiae) proteins involved in either phosphate transport or sensing or in sorting proteins to endomembranes. The Arabidopsis genome contains additional proteins of unknown function containing either a SPX or an EXS domain. Phylogenetic analysis indicated that the PHO1 family is subdivided into at least three clusters. Reverse transcription-PCR revealed a broad pattern of expression in leaves, roots, stems, and flowers for most genes, although two genes are expressed exclusively in flowers. Analysis of the activity of the promoter of all PHO1 homologs using promoter-beta-glucuronidase fusions revealed a predominant expression in the vascular tissues of roots, leaves, stems, or flowers. beta-Glucuronidase expression is also detected for several promoters in nonvascular tissue, including hydathodes, trichomes, root tip, root cortical/epidermal cells, and pollen grains. The expression pattern of PHO1 homologs indicates a likely role of the PHO1 proteins not only in the transfer of phosphate to the vascular cylinder of various tissues but also in the acquisition of phosphate into cells, such as pollen or root epidermal/cortical cells.

Exome Sequencing Identifies INPPL1 Mutations as a Cause of Opsismodysplasia.

Opsismodysplasia (OPS) is a severe autosomal-recessive chondrodysplasia characterized by pre- and postnatal micromelia with extremely short hands and feet. The main radiological features are severe platyspondyly, squared metacarpals, delayed skeletal ossification, and metaphyseal cupping. In order to identify mutations causing OPS, a total of 16 cases (7 terminated pregnancies and 9 postnatal cases) from 10 unrelated families were included in this study. We performed exome sequencing in three cases from three unrelated families and only one gene was found to harbor mutations in all three cases: inositol polyphosphate phosphatase-like 1 (INPPL1). Screening INPPL1 in the remaining cases identified a total of 12 distinct INPPL1 mutations in the 10 families, present at the homozygote state in 7 consanguinous families and at the compound heterozygote state in the 3 remaining families. Most mutations (6/12) resulted in premature stop codons, 2/12 were splice site, and 4/12 were missense mutations located in the catalytic domain, 5-phosphatase. INPPL1 belongs to the inositol-1,4,5-trisphosphate 5-phosphatase family, a family of signal-modulating enzymes that govern a plethora of cellular functions by regulating the levels of specific phosphoinositides. Our finding of INPPL1 mutations in OPS, a severe spondylodysplastic dysplasia with major growth plate disorganization, supports a key and specific role of this enzyme in endochondral ossification.

Evolutionary forces affect multigenomic arbuscular mycorhizal fungi : an experimental approach using Glomus intraradices

Abstract Arbuscular Mycorhizal Fungi (AMF) are important plant symbionts that can improve floristic diversity and ecosystem productivity. These important fungi are obligate biotrophs and form symbioses with roots of the majority of plant species, improving plant nutrient acquisition in exchange of photosynthates. AM fungi are successful both ecologically as they occupy a very large spectrum of environments as well as host range and evolutionarily, as this symbiosis is over 400 million years old. These fungi grow and reproduce clonally by hyphae and multinucleate spores. AMF are coenocytic and recent work has shown that they harbor genetically different nuclei and that AMF populations are genetically diverse. How AMF species diversity is maintained has been addressed theoretically and experimentally at the community level. Much less attention has been drawn to understand how genetic diversity is maintained within populations although closely related individuals are more likely to compete for the same resources and occupy similar niches. How infra-individual genetic diversity is shaped and maintained has received even less attention. In Chapter 2, we show that individuals from a field population may differ in their symbiotic efficiency under reduced phosphate availability: We show there is genetic variation in an AMF field population for fitness-related growth traits in response to different phosphate availability acid host species. Furthermore, AFLP fingerprints of the same individuals growing in contrasting environments diverged suggesting that the composition in nuclei of AMF is dynamical and affected by environmental factors. Thus environmental heterogeneity is likely to play an important role for the maintenance of genetic diversity at the population level. In Chapter 3 we show that single spores do not inherit necessarily the same genetic material. We have found genetic divergences using two different types of molecular marker, as well as phenotypic divergences among single spore lines. Our results stress the importance of considering these organisms as a multilevel hierarchical system and of better knowing their life cycle. They have important consequences for the understanding of AMF genetics, ecology and the development of commercial AMF inocculum. Résumé Les champignons endomycorhiziens arbusculaires (CEA) sont d'importants symbiontes pour les plantes, car ils augmentent la diversité et la productivité des écosystèmes. Ces importants symbiontes sont des biotrophes obligatoires et forment une symbiose avec la plupart des plantes terrestres. Ils améliorent l'acquisition de substances nutritives de leurs hôtes en échange de sucres obtenus par photosynthèse. Ces champignons ont un grand succès écologique, ils colonisent une grande rangée d'environnements ainsi que d'hôtes. Ils ont aussi un succès évolutif certain de part le fait que cette symbiose existe depuis plus de 400 millions d'années. Les CEA sont asexués et croissent clonalement en formant des hyphes et des spores multinuclées. Les CEA sont des coenocytes et des travaux de recherche récents ont montré qu'ils possèdent des noyaux génétiquement différents. D'autres travaux ont aussi révélé que les populations de CEA sont génétiquement diversifiées. Comment la diversité des CEA est maintenue a seulement été adressée par des études théoriques et expérimentalement au niveau des communautés. Très peu d'attention a été portée sur le maintien de la diversité génétique infra et inter populationnelle, or ce sont les individus les plus proches génétiquement qui vont entrer en compétition pour des ressources et niches similaires. La formation et le maintien de la diversité intra-individu des CEA a reçu très peu d'attention. Dans le chapitre 2, nous montrons que des individus CEA d'un même champ différent dans leur efficacité symbiotique lorsque la concentration en phosphoré est réduite. Nous montrons qu'il existe de la variance génétique dans une population de CEA provenant d'un même champ en réponse à différentes concentrations de phosphore, ainsi qu'en réponse à différentes espèces d'hôtes, et ceci pour des traits de croissance vraisemblablement liés au succès reproducteur. De plus grâce à des AFLP nous avons pu montrer que le génome de ces individus subissent des changements lorsqu'ils croissent dans des environnements contrastés. Ceci suggère que les noyaux génétiquement différents des CEA sont des entités dynamiques. Il est fort probable que l'hétérogénéité environnementale joue un rôle dans le maintien de la diversité génétique des populations de CEA. Dans le chapitre 3, nous montrons que toutes les spores d'un même mycélium parental de CEA ne reçoivent pas exactement le même contenu génétique. Nous avons mis en évidence des divergences entre des Lignées monosporales en utilisant deux types de marqueur moléculaires, ainsi que des différences phénotypiques. Nos résutats soulignent l'importance de considézer ces organismes comme dés systëmes hiérarchiques mufti-niveaux, ainsi que de mieux connaître leur cycle de vie. Nos résultats ont d'importantes conséquences pour la compréhension du système génétique des CEA, ainsi que de leur évolution, leur écológie, mais également des conséquences pour la production d' inoccultim commercial.

Complete Nucleotide Sequence of Mouse Mammary Tumor Virus from JYG Chinese Wild Mice: Absence of Bacterial Insertion Sequences in the Cloned Viral gag Gene.

Mammary tumors of a newly isolated strain of Chinese wild mouse (JYG mouse) harbor exogenous mouse mammary tumor virus (MMTV). The complete nucleotide sequence of exogenous JYG-MMTV was determined on the proviral 5' long terminal repeat (LTR)(partial)-gag-pol-env-3' LTR (partial) fragment cloned into a plasmid vector and the cDNA sequence from JYG-MMTV producing cells. Similarly to the other MMTV species the LTR of JYG-MMTV contains an open reading frame (ORF). The amino acid sequence of the JYG-MMTV ORF resembles that of SW-MMTV (92% identity) and endogenous Mtv-7 (93% identity) especially at the C-terminal region. Thus, a functional similarity in T-cell receptor V beta recognition as a superantigen is implicated among these MMTV species. Analysis of the viral gag nucleotide sequence revealed that this gene is not disrupted by the bacterial insertion sequence IS1 or IS2, which have been reported to be present in the majority of the plasmids containing the gag region. Comparison of amino acid sequences of JYG-MMTV with those of BR6-MMTV showed that over 96% of the amino acids of gag, pol, protease and env products are identical. These results suggest the intact nature of the nucleotide sequence of the near full-length MMTV genome cloned in the plasmid.

Homologous sex chromosomes in three deeply divergent anuran species.

Comparative genomic studies are revealing that, in sharp contrast with the strong stability found in birds and mammals, sex determination mechanisms are surprisingly labile in cold-blooded vertebrates, with frequent transitions between different pairs of sex chromosomes. It was recently suggested that, in context of this high turnover, some chromosome pairs might be more likely than others to be co-opted as sex chromosomes. Empirical support, however, is still very limited. Here we show that sex-linked markers from three highly divergent groups of anurans map to Xenopus tropicalis scaffold 1, a large part of which is homologous to the avian sex chromosome. Accordingly, the bird sex determination gene DMRT1, known to play a key role in sex differentiation across many animal lineages, is sex linked in all three groups. Our data provide strong support for the idea that some chromosome pairs are more likely than others to be co-opted as sex chromosomes because they harbor key genes from the sex determination pathway.

Mutations in FGF17, IL17RD, DUSP6, SPRY4, and FLRT3 Are Identified in Individuals with Congenital Hypogonadotropic Hypogonadism.

Congenital hypogonadotropic hypogonadism (CHH) and its anosmia-associated form (Kallmann syndrome [KS]) are genetically heterogeneous. Among the >15 genes implicated in these conditions, mutations in FGF8 and FGFR1 account for ∼12% of cases; notably, KAL1 and HS6ST1 are also involved in FGFR1 signaling and can be mutated in CHH. We therefore hypothesized that mutations in genes encoding a broader range of modulators of the FGFR1 pathway might contribute to the genetics of CHH as causal or modifier mutations. Thus, we aimed to (1) investigate whether CHH individuals harbor mutations in members of the so-called "FGF8 synexpression" group and (2) validate the ability of a bioinformatics algorithm on the basis of protein-protein interactome data (interactome-based affiliation scoring [IBAS]) to identify high-quality candidate genes. On the basis of sequence homology, expression, and structural and functional data, seven genes were selected and sequenced in 386 unrelated CHH individuals and 155 controls. Except for FGF18 and SPRY2, all other genes were found to be mutated in CHH individuals: FGF17 (n = 3 individuals), IL17RD (n = 8), DUSP6 (n = 5), SPRY4 (n = 14), and FLRT3 (n = 3). Independently, IBAS predicted FGF17 and IL17RD as the two top candidates in the entire proteome on the basis of a statistical test of their protein-protein interaction patterns to proteins known to be altered in CHH. Most of the FGF17 and IL17RD mutations altered protein function in vitro. IL17RD mutations were found only in KS individuals and were strongly linked to hearing loss (6/8 individuals). Mutations in genes encoding components of the FGF pathway are associated with complex modes of CHH inheritance and act primarily as contributors to an oligogenic genetic architecture underlying CHH.

High-level molecular diversity of copper-zinc superoxide dismutase genes among and within species of arbuscular Mycorrhizal fungi.

In the ecologically important arbuscular mycorrhizal fungi (AMF), Sod1 encodes a functional polypeptide that confers increased tolerance to oxidative stress and that is upregulated inside the roots during early steps of the symbiosis with host plants. It is still unclear whether its expression is directed at scavenging reactive oxygen species (ROS) produced by the host, if it plays a role in the fungus-host dialogue, or if it is a consequence of oxidative stress from the surrounding environment. All these possibilities are equally likely, and molecular variation at the Sod1 locus can possibly have adaptive implications for one or all of the three mentioned functions. In this paper, we analyzed the diversity of the Sod1 gene in six AMF species, as well as 14 Glomus intraradices isolates from a single natural population. By sequencing this locus, we identified a large amount of nucleotide and amino acid molecular diversity both among AMF species and individuals, suggesting a rapid divergence of its codons. The Sod1 gene was monomorphic within each isolate we analyzed, and quantitative PCR strongly suggest this locus is present as a single copy in G. intraradices. Maximum-likelihood analyses performed using a variety of models for codon evolution indicated that a number of amino acid sites most likely evolved under the regime of positive selection among AMF species. In addition, we found that some isolates of G. intraradices from a natural population harbor very divergent orthologous Sod1 sequences, and our analysis suggested that diversifying selection, rather than recombination, was responsible for the persistence of this molecular diversity within the AMF population.

PH01 gene family in Arabidopsis thaliana and Physcomitrella patens

Summary Inorganic phosphate (Pi) is a main limiting nutrient to the growth and production yield of plants in many agro-ecosystems. Plants have evolved a series of metabolic and developmental adaptations to cope with low Pi availability. PH01 has been identified as a protein involved in the loading of Pi into the xylem of roots in Arabidopsis. In this study, the PHO1 gene family in both higher plant Arabidopsis and lower plant Physcomitrella was characterized. Additional ten PHO1 homologues in Arabidopsis and three homologues in Physcomitrella were identified. All proteins harbor a SPX tripartite domain in the N-terminal hydrophilic portion and an EXS domain in the highly conserved C-terminal hydrophobic portion. RT-PCR analysis of the Arabidopsis PHO1 genes revealed a broad pattern of expression in leaves, roots, stems, and flowers for most genes, although two genes are expressed exclusively in flowers, indicating their potential roles not only in Pi transport but also in Pi homeostasis within the Arabidopsis plant. The regulation of gene expression by different nutrient-starvations showed that some genes are strongly up-regulated by elements other than Pi, e.g. by NO3, Mg, and Zn starvation. Northern blot and RT-PCR analysis showed distinct expression patterns of the three Physcomitrella PHO1 genes. The investigation of Pi starvation effects on some Pi-deprivation responsive genes demonstrates that Physcomitrella has evolved a similar mechanism as higher plants to respond to Pi deficiency. Promoter activity analysis for the Physcomitrella PHO1 family genes using promoter-GUS fusions revealed their expression in protonemata and gametophores but at different levels and with different patterns, suggesting these genes may play distinct roles in Pi transport and/or Pi homeostasis in the moss plant. Single knockout mutants of the three genes were generated by gene targeting and one of them displayed a reduced Pi content in the protonemata under Pi starvation. The evolution of the PHO1 family in land plants was also discussed. Together, these findings indicate that the PHO1 family genes, present in a broad range of plant species from lower plants to flowering plants, play important roles in Pi transport and homeostasis. Résumé Le phosphate inorganique (Pi) est un nutriment essentiel à la croissance des plantes et au rendement de la production végétale. Dans beaucoup d'agro-écosystèmes, ce nutriment est limitant. Les plantes ont développé des adaptations métaboliques et développementales pour palier à la faible disponibilité du Pi. Il a été démontré que la protéine PHOI est indispensable au transfert du Pi dans le xylème des racines d' Arabidopsis. Cette étude porte sur la famille de gènes définie par PHO1 ; ceci, dans deux organismes modèles : la plante Arabidopsis pour les végétaux supérieurs, et la mousse Physcomitrella pour les végétaux inférieurs. Dix homologues à PHOI dans Arabidopsis et trois homologues dans Physcomitrella ont été identifiés. Toutes les protéines encodées présentent un domaine tripartite SPX dans leur partie N terminale hydrophile et un domaine EXS dans la partie C terminale hydrophobe hautement conservée d'entre eux. L'analyse par RT-PCR de l'expression des gènes PHO1 dans Arabidopsis révèle une expression ectopique pour la plupart, à l'exception de deux gènes dont l'expression est uniquement florale ; ceci suggère l'implication de cette famille non seulement dans le transport mais aussi dans l'homéostasie du Pi dans Arabidopsis. L'observation de l'expression de ces gènes en fonction de l'absence de différents nutriments montre que certains gènes sont fortement régulés lors de carences en NO3, Mg et Zn. L'analyse par northern blot et RT-PCR met en évidence des profils d'expression distincts pour les trois gènes de Physcomitrella. Les effets de la carence en Pi sur Physcomitrella ont été étudiés par le biais de gènes dépendants connus pour Arabidopsis, les résultats suggèrent un mode de réponse à cette carence conservé entre les végétaux inférieurs et supérieurs. La localisation tissulaire de l'expression de la famille PHO1 dans la mousse a été étudiée au moyen du gène rapporteur GUS fusionné aux différents promoteurs. Ceci a révélé leur expression dans les protonemata et les gametophores, mais à des intensités et avec des profils différents, ce qui suggère des implications distinctes dans le transport et/ou l'homéostasie du Pi dans la mousse. Des simples mutants knockout ont été générés pour chaque gène de mousse ; l'un d'eux présente une diminution du contenu protonemal en Pi lorsque soumis à une carence en Pi. L'évolution de la famille PHO1 dans les plantes terrestres est également discutée. Ensemble, ces résultats indiquent que les gènes de la famille PHO1 sont présents dans une large gamme de plantes allant des végétaux inférieurs aux supérieurs, et cette étude démontre que leur conservation se justifie potentiellement par le fait qu'ils sont probablement impliqués dans des mécanismes conservés de transport et d'homéostasie du Pi.