The role of the vacuolar H+ - ATPase in membrane fusion

RésuméLa H+-ATPase vacuolaire (V-ATPase) est un complexe enzymatique composé de deux secteurs multimériques (VQ et Vi) dont l'association dans la cellule est réversible. Le secteur intramembranaire de la V-ATPase (V0) interagit physiquement avec des protéines SNARE et stimule la fusion homotypique des vacuoles de la levure (lysosomes), la sécrétion de neurotransmetteurs et d'insuline, la fusion entre phagosome et lysosome ainsi que la sécrétion des corps multivésiculaires par un mécanisme inconnu. Dans cette étude j'ai identifié des résidues d'acides amines situés dans des sous-unités de V0 impliqués dans le mécanisme de fusion des vacuoles mais non essentiels pour l'acidification vacuolaire par la V-ATPase. j'ai utilisé un protocole de mutagenèse aléatoire pour produire des libraries de mutants des sous unités de V0. Ces libraries ont été analysées in vivo afin d'identifier des alleles qui permettent la translocation des protons mais produisent une vacuole fragmentée, phénotype indiquant un défaut dans la fusion membranaire. Les vacuoles des mutants ont été isolées et caractéisées en utilisant une grande variété d'outils biochimiques pour déterminer précisément l'impact des différentes mutations sur l'accomplissement d'événements clés du processus de fusion.J'ai identifié des mutations associées à des défauts spécifiques de la fusion dans plusieurs sous-unités de V0. Dans les protéolipides c, c' et c" ces mutations se concentrent dans la partie cytosolique des domaines transmembranaires. Elles renforcent les associations entre les secteurs de la V-ATPase et entre V0 et les SNAREs. Dans la fusion vacuolaire ces mutations permettent la formation de complexes SNAREs en trans mais inhibent l'induction de la fusion. Par contre, la deletion de la sous- unité d influence les étapes de la fusion qui précèdent la formation des complexes trans-SNAREs. Mes résultats démontrent que V0 joue des rôles différents dans plusieurs étapes de la fusion et que ces fonctions sont liées au système des SNAREs. Ils différencient génétiquement les activités de V0 dans la translocation des protons et dans la fusion et identifient de nombreux résidus importants pour la fusion vacuolaire. De plus, compte tenu de la grande conservation de sequence des protéolipides chez les eukaryotes les mutations identifiées dans cette l'étude apportent de nouvelles informations pour analyser la fonction de V0 dans des organismes multicellulaires pour lesquels la function catalytique de la V-ATPase est essentielle à la survie.Résumé pour le large publicLe transport de protéines et de membranes est important pour maintenir la fonction des organelles dans la cellule. Il s'excerce au niveau des vesicules. La fusion membranaire est un processus élémentaire de ce transport. Pour fusionner deux membranes, il faut la coordination de deux activités: le rapprochement et la déstabiiization des deux membranes. La collaboration d'un ensemble de proteins conservés chez les eukaryotes, est nécessaire pour catalyser ces activités. Les proteins SNAREs sont les protagonistes principaux dans la fusion membranaire. Néanmoins, d'autres protéines, comme des Rab-GTPases et des chaperonnes, sont nécessaires pour permettre ce phénomène de fusion. Toutes ces protéines sont temporairement associées avec les SNAREs et leur fonction dans la fusion membranaire est souvent directement liée à leur activité dans cette association. Le secteur transmembranaire V0 de la V-ATPase rnteragit avec des SNAREs et est essentiel pour la fusion dans une variété de systèmes modèles comme la mouche, la souris et la levure. Le secteur V0 est composé de six protéines différentes. Avec te secteur Va, qui réside dans le cytosol, il forme la V-ATPase dont la fonction principale est l'acidification des organelles par translocation des protons à travers la membrane par un mécanisme ressemblant à celui d'une pompe. V0joue un role dans la fusion membranaire, indépendamment de son activité catalytique liée au pompage des protons, et ce rôle est encore largement méconnu à ce jour. Le but de ma thèse était de mieux comprendre l'implication de V0 dans ce contexte.Pour étudier des activités liées à la V-ATPase, la levure est un excellent modèle d'étude car elle survie à une inactivation de l'enzyme alors que le meme traitement serait léthal pour des organismes multicellulaires. Dans ma thèse j'ai utilisé la fusion homotypique de la vacuole de levure comme système modèle pour étudier le rôle de V0 dans la fusion. J'ai muté des gènes qui encodent des sous- unités de V0 et les ai introduit dans des souches privées des gènes respectifs. Dans les librairies de souches portant différentes versions de ces gènes j'ai cherché des clones exprimant une V-ATPase intacte et fonctionnelle mais qui possèdent une vacuole fragmentée. Le plus souvent, une vacuole fragmentée indique un défaut dans la fusion vacuolaire. Dans les trois types de protéolipides qui composent un cylindre dans le secteur V0, j'ai trouvé des clones avec une vacuole fragmentée. Après avoir isolé les mutations responsable de ce type de morphologie vacuolaire, j'ai isolé les vacuoles de ces clones pour étudier leur activités dans différentes étapes de la fusion vacuolaire. Les résultats de ces analyses mettent en évidence une implication de V0 dans plusieurs étapes de la fusion vacuolaire. Certaines mutations sélectionnées dans mon étude inhibent une étape précoce de la fusion qui inclue la dissociation des complexes SNARE, tandis que d'autres mutations inhibent une étape tardive du processus de fusion qui inclue la transmission d'une force disruptive dans la membrane.AbstractThe membrane-integral V0 sector of the vacuolar H+-ATPase (V-ATPase) interacts with SNARE proteins. V0 stimulates fusion between yeast vacuoles (lysosomes) (Peters et al., 2001b), secretion of neurotransmitters and insulin (Hiesinger et al., 2005a, Sun-Wada et al., 2006a), phagosome-lysosome fusion (Peri and Nusslein-Volhard, 2008) and secretion of multivesicular bodies (Liegeois et al., 2006b) by a yet unknown mechanism. In my thesis, I identified sites in V0 subunits that are involved in yeast vacuole fusion but dispensable for the proton pumping by the V-ATPase. I randomly mutagenized V0 subunits and screened in vivo for mutant alleles that support proton pumping but cause fragmented vacuoles, a phenotype indicative of a fusion defect. Mutant vacuoles were isolated and analyzed in a cell-free system, allowing assay of key events in fusion, such as trans-SNARE pairing, lipid transition and fusion pore opening (Reese et al., 2005b).Mutants with selective fusion defects were found in several V0 subunits. In the proteolipids c, c' and c", critical mutations are concentated in the cytosolic half of the transmembrane domains. These mutations rendered the V-ATPase holoenzyme more stable and modulated V0-SNARE associations. In vacuole fusion critical proteolipid mutations permitted trans-SNARE pairing but impeded the induction of lipid flow between the membranes. Deletion of subunit d, by contrast, influenced early stages of fusion that precede trans-SNARE pairing. My results show that V0 acts in several steps of the fusion process and that its function is intimately connected to the SNARE system. They genetically separate the proton pump and fusion activities of V0 and identify numerous critical residues. Given the high sequence conservation of proteolipids in eukaryotic life, the identified mutations may be helpful in analyzing the fusion function of V0 also in mammalian cells, where V- ATPase pump function is essential for survival.

Simultaneous Mendelian and clonal genome transmission in a sexually reproducing, all-triploid vertebrate.

Meiosis in triploids faces the seemingly insuperable difficulty of dividing an odd number of chromosome sets by two. Triploid vertebrates usually circumvent this problem through either asexuality or some forms of hybridogenesis, including meiotic hybridogenesis that involve a reproductive community of different ploidy levels and genome composition. Batura toads (Bufo baturae; 3n = 33 chromosomes), however, present an all-triploid sexual reproduction. This hybrid species has two genome copies carrying a nucleolus-organizing region (NOR+) on chromosome 6, and a third copy without it (NOR-). Males only produce haploid NOR+ sperm, while ova are diploid, containing one NOR+ and one NOR- set. Here, we conduct sibship analyses with co-dominant microsatellite markers so as (i) to confirm the purely clonal and maternal transmission of the NOR- set, and (ii) to demonstrate Mendelian segregation and recombination of the NOR+ sets in both sexes. This new reproductive mode in vertebrates ('pre-equalizing hybrid meiosis') offers an ideal opportunity to study the evolution of non-recombining genomes. Elucidating the mechanisms that allow simultaneous transmission of two genomes, one of Mendelian, the other of clonal inheritance, might shed light on the general processes that regulate meiosis in vertebrates.

Organisation and role of actin microfilaments during cellular growth and morphogenesis in the moss Physcomitrella patens

ABSTRACT The network of actin cytoskeleton is composed of actin filaments (F-actin) that are made by polymerisation of actin monomers and actin binding proteins. It is required for growth and morphogenesis of eukaryotic cells. The labelling of F-actin with constitutively expressed GFP-Talin (Kost et al., 1998) reveals the organisation of cellular actin networks in plants. Due to the lack of information on actin cytoskeleton through gametophytic development of the model moss plant Physcornitrella patens, stable transgenic lines overexpressing GFP-Talin were generated to detect F-actin structures. It is shown that the 35S promoter driven expression is not suitable for F-actin labelling in all cells. When it is replaced by the inducible heat-shock promoter Gmhsp17.3 from soybean, one hour mild heat stress at 37°C followed by recovery at 25°C is enough to induce efficient and transient labelling in all tissues without altering cellular morphology. The optimal observations of F-actin structures at different stages of moss development can be done between 12-18 hours after the induction. By using confocal microscopy, we demonstrate that stellated actin arrays were densely accumulated at the growing tip in regenerating protoplasts, apical protonemal cells and rhizoids and connected with a fine dispersed F-actin mesh. Following three-dimensional growth, the cortical star-like structures are widespread in the meristematic cells of developing bud and young gametophores. On the contrary, undulating networks of actin cables are found at the final stage of cell differentiation. During redifferentiation of mature leaf cells into protonemal filaments the rather stagnant web of actin cables is replaced by diffuse actin meshwork. In eukaryotes, nucleation of the actin monomers prior to their polymerization is driven by the seven-subunit ARP2/3 complex and formins. We cloned the gene encoding the ARP3 subunit of P. patens and generated arp3 mutants of the moss through gene disruption. The knockout of ARP3 affects the elongation of chloronemal cells and blocks further differentiation of caulonemal cells and rhizoids, and the gametophores are slightly stunted compared to wild-type. The arp mutants were created in the heat-shock inducible GFP-Talin strains allowing us to visualise a disorganised actin network and a lack of star-like actin cytoskeleton arrays. We conclude that ARP2/3 dependent nucleation of actin filaments is critical for the growth of filamentous cells, which in turn influences moss colonization. In complementation assays, the overexpression of Physcomitrella and Arab idopsis ARP3 genes in the moss arp3 mutant results in full recovery of wild type phenotype. In contrast the ARP3 subunit of fission yeast is not able to complement the moss arp3 mutant of moss indicating that regulation of the ARP2/3 dependent actin nucleation diverged in different kingdoms. RESUME Le réseau d'actine est composé de filaments de F-actine et d'un ensemble de protéines s'y attachant (Actin binding proteins). Le réseau d'actine est nécessaire à la croissance et à la morphogenèse de toutes les cellules eucaryotes. Chez les plantes, le marquage ainsi que l'étude de l'organisation du réseau d'actine ont été réalisés en utilisant une fusion GFP-Talin (Kost et al., 1998) exprimée sous le control d'un promoteur constitutif. Afin d'étudier les structures F-actine dans les cellules de Physcomitrella Patens et pour combler le manque d'information sur le développement des gamétophores, des lignées transgéniques stables surexprimant GFP-Talin ont été crées. Nous avons démontré que l'utilisation du promoteur 35S est inadéquate pour le marquage complet et homogène des filaments d'actine dans toutes les cellules de P. patens. Par contre, l'utilisation du promoteur inductible Gmhsp17.3 nous a permis de réaliser un marquage transitoire et général dans tous les tissus de la mousse. Une heure de choc thermique à 37°C suivis d'un temps de récupération de 12-18h à 25°C sont les conditions optimales (sans dommages cellulaires) pour l'observation des structures F-actine à différentes étapes de développement de la mousse. En utilisant la microscopie confocale, nous avons observé l'existence de structures F-actine accumulées en forme d'étoiles. Ces structures, qui sont liées au réseau de microfilaments d'actine, ont été observées dans les protoplastes en régénération, les cellules des protonema apicales ainsi que dans les rhizoïdes. En suivant la croissance tridimensionnelle, ces structures en étoiles ont été observées dans les cellules meristématiques des bourgeons et des jeunes gamétophores. Par contre, dans les cellules différentiées ces structures laissent place à des réseaux de câbles épais. Nous avons également remarqué que durant la redifferentiation des cellules foliaires le réseau de câbles de F-actine est remplacé par un réseau de F-actine diffus. Dans les cellules eucaryotes, la nucléation des filaments d'actirie précédant leur polymérisation est contrôlé par sept sous unités du complexe ARP2/3 et par des formines. Nous avons isolé le gène codant pour la sous unité ARP3 de P. patens et nous avons crée des mutants arp3 par intégration ciblée (Knockout). L'élongation des cellules chloronema est clairement affectée dans les mutants arp3. La différentiation des caulonemata et des rhizoïdes est bloquée et les gametophores sont légèrement plus courts comparé au type sauvage. A fin d'étudier l'organisation des filaments d'actines dans les mutants arp3, nous avons aussi réalisé un arp3-knockout dans la lignée Hsp-GFP-Talin. La nouvelle lignée générée nous a permis de visualiser une désorganisation du réseau d'actine et une absence complète de structures de F-actine accumulée en forme d'étoiles. Les résultats obtenus nous amènent à conclure que la nucléation (ARP2/3 dépendante) des filaments d'actine est indispensable à la croissance des cellules filamenteuses. Par conséquent, les filaments d'actine semblent avoir un rôle dans la colonisation des milieux par les mousses. Nous avons également procédé à des essais de complémentation du mutant arp3. La surexpression des gènes ARP3 de Physcomitrella et d'Arabidopsis dans les cellules du mutant arp3 rétabli complètement le phénotype WT. Par contre, le gène ARP3 des levures n'est pas suffisant pour complémenter la même mutation dans les cellules de mousses. Ce résultat démontre que les mécanismes de régulation de la nucléation des filaments d'actine (ARP2/3 dépendante) sont différents entre les différents groupes d'eucaryotes.

A dual functional origin of transfer in the ICEclc genomic island of Pseudomonas knackmussii B13.

Genomic islands (GEIs) are large DNA segments, present in most bacterial genomes, that are most likely acquired via horizontal gene transfer. Here, we study the self-transfer system of the integrative and conjugative element ICEclc of Pseudomonas knackmussii B13, which stands model for a larger group of ICE/GEI with syntenic core gene organization. Functional screening revealed that unlike conjugative plasmids and other ICEs ICEclc carries two separate origins of transfer, with different sequence context but containing a similar repeat motif. Conjugation experiments with GFP-labelled ICEclc variants showed that both oriTs are used for transfer and with indistinguishable efficiencies, but that having two oriTs results in an estimated fourfold increase of ICEclc transfer rates in a population compared with having a single oriT. A gene for a relaxase essential for ICEclc transfer was also identified, but in vivo strand exchange assays suggested that the relaxase processes both oriTs in a different manner. This unique dual origin of transfer system might have provided an evolutionary advantage for distribution of ICE, a hypothesis that is supported by the fact that both oriT regions are conserved in several GEIs related to ICEclc.

Atomic force microscopy of nucleoprotein complexes.

Recent data on the AFM studies of nucleoprotein complexes of different types are reviewed in this paper. The first section describes the progress in the sample preparation methods for AFM studies of nucleic acids and nucleoprotein complexes. The second part of this paper reviews AFM data on studies of complexes of DNA with regulatory proteins. These studies include two different types of DNA distortion induced by proteins binding: local bending of DNA at sites of protein binding and formation of large loops due to protein-protein interactions between molecules bound to distant sites along the DNA molecules (DNA looping). The prospects for use of AFM for physical mapping of genomes are discussed in this section as well. The third part of the paper reviews data on studies of complexes of DNA with non-sequence specific binding proteins. Special emphasis is given to studies of chromatin which have resulted in progress in the understanding of structure of native chromatin fiber. In this section, novel data on AFM studies of RecA-DNA filaments and complexes of dsRNA with the dsRNA-specific protein p25 are also presented. Discussion of the substrate preparation procedures in relation to the AFM studies of nucleoprotein complexes is given in the final section.

Synergistic transcriptional activation by CTF/NF-I and the estrogen receptor involves stabilized interactions with a limiting target factor.

Transcription initiation at eukaryotic protein-coding gene promoters is regulated by a complex interplay of site-specific DNA-binding proteins acting synergistically or antagonistically. Here, we have analyzed the mechanisms of synergistic transcriptional activation between members of the CCAAT-binding transcription factor/nuclear factor I (CTF/NF-I) family and the estrogen receptor. By using cotransfection experiments with HeLa cells, we show that the proline-rich transcriptional activation domain of CTF-1, when fused to the GAL4 DNA-binding domain, synergizes with each of the two estrogen receptor-activating regions. Cooperative DNA binding between the GAL4-CTF-1 fusion and the estrogen receptor does not occur in vitro, and in vivo competition experiments demonstrate that both activators can be specifically inhibited by the overexpression of a proline-rich competitor, indicating that a common limiting factor is mediating their transcriptional activation functions. Furthermore, the two activators functioning synergistically are much more resistant to competition than either factor alone, suggesting that synergism between CTF-1 and the estrogen receptor is the result of a stronger tethering of the limiting target factor(s) to the two promoter-bound activators.

trome, trEST and trGEN: databases of predicted protein sequences.

We previously introduced two new protein databases (trEST and trGEN) of hypothetical protein sequences predicted from EST and HTG sequences, respectively. Here, we present the updates made on these two databases plus a new database (trome), which uses alignments of EST data to HTG or full genomes to generate virtual transcripts and coding sequences. This new database is of higher quality and since it contains the information in a much denser format it is of much smaller size. These new databases are in a Swiss-Prot-like format and are updated on a weekly basis (trEST and trGEN) or every 3 months (trome). They can be downloaded by anonymous ftp from ftp://ftp.isrec.isb-sib.ch/pub/databases.

Evolution of gene expression in fire ants: the effects of developmental stage, caste, and species.

Ants provide remarkable examples of equivalent genotypes developing into divergent and discrete phenotypes. Diploid eggs can develop either into queens, which specialize in reproduction, or workers, which participate in cooperative tasks such as building the nest, collecting food, and rearing the young. In contrast, the differentiation between males and females generally depends upon whether eggs are fertilized, with fertilized (diploid) eggs giving rise to females and unfertilized (haploid) eggs giving rise to males. To obtain a comprehensive picture of the relative contributions of gender (sex), caste, developmental stage, and species divergence to gene expression evolution, we investigated gene expression patterns in pupal and adult queens, workers, and males of two species of fire ants, Solenopsis invicta and S. richteri. Microarray hybridizations revealed that variation in gene expression profiles is influenced more by developmental stage than by caste membership, sex, or species identity. The second major contributor to variation in gene expression was the combination of sex and caste. Although workers and queens share equivalent diploid nuclear genomes, they have highly distinctive patterns of gene expression in both the pupal and the adult stages, as might be expected given their extraordinary level of phenotypic differentiation. Overall, the difference in the proportion of differentially expressed genes was greater between workers and males than between workers and queens or queens and males, consistent with the fact that workers and males share neither gender nor reproductive capability. Moreover, between-species comparisons revealed that the greatest difference in gene expression patterns occurred in adult workers, a finding consistent with the fact that adult workers most directly experience the distinct external environments characterizing the different habitats occupied by the two species. Thus, much of the evolution of gene expression in ants may occur in the worker caste, despite the fact that these individuals are largely or completely sterile. Analyses of gene expression evolution revealed a combination of positive selection and relaxation of stabilizing selection as important factors driving the evolution of such genes.

Soil tillage affects the community structure of mycorrhizal fungi in maize roots

In this study we tested whether communities of arbuscular mycorrhizal fungi (AMF) colonizing the roots of maize (Zea mays L.) were affected by soil tillage practices (plowing, chiseling, and no-till) in a long-term field experiment carried out in Tanikon (Switzerland). AMF were identified in the roots using specific polymerase chain reaction (PCR) markers that had been developed for the AMF previously isolated from the soils of the studied site. A nested PCR procedure with primers of increased specificity (eukaryotic, then, fungal, then AMF species or. species-grouop specific) was used. Sequencing of amplified DNA confirmed that the DNA obtained from the maize roots was of AMF origin. Presence of particular AMF species or species-group was scored as a presence of a DNA product after PCR with specific primers. We also used single-strand conformation polymorphism analysis (SSCP), of amplified DNA samples to-check if the amplification of the DNA from maize roots matched the expected profile for a particular AMF isolate with a given specific primer pair. Presence of the genus Scutellospora, in maize roots was strongly reduced in plowed and chiseled soils. Fungi from the suborder Glomineae were more prevalent colonizers of maize roots growing in plowed soils, but were also present in the roots from other tillage treatments. These changes in community of AMF colonizing maize roots might be due to (1), the differences in tolerance to the tillage-induced disruption of the hyphae among the different AMF species, (2) changes in nutrient content of the soil, (3) changes in microbial activity, or (4) changes in weed populations in response to soil tillage. This is the first report on community composition of AMF in the roots of a field-grown crop plant (maize) as affected by soil tillage.

Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution.

We present here a draft genome sequence of the red jungle fowl, Gallus gallus. Because the chicken is a modern descendant of the dinosaurs and the first non-mammalian amniote to have its genome sequenced, the draft sequence of its genome--composed of approximately one billion base pairs of sequence and an estimated 20,000-23,000 genes--provides a new perspective on vertebrate genome evolution, while also improving the annotation of mammalian genomes. For example, the evolutionary distance between chicken and human provides high specificity in detecting functional elements, both non-coding and coding. Notably, many conserved non-coding sequences are far from genes and cannot be assigned to defined functional classes. In coding regions the evolutionary dynamics of protein domains and orthologous groups illustrate processes that distinguish the lineages leading to birds and mammals. The distinctive properties of avian microchromosomes, together with the inferred patterns of conserved synteny, provide additional insights into vertebrate chromosome architecture.

Farnesol-induced apoptosis in Candida albicans is mediated by Cdr1-p extrusion and depletion of intracellular glutathione.

Farnesol is a key derivative in the sterol biosynthesis pathway in eukaryotic cells previously identified as a quorum sensing molecule in the human fungal pathogen Candida albicans. Recently, we demonstrated that above threshold concentrations, farnesol is capable of triggering apoptosis in C. albicans. However, the exact mechanism of farnesol cytotoxicity is not fully elucidated. Lipophilic compounds such as farnesol are known to conjugate with glutathione, an antioxidant crucial for cellular detoxification against damaging compounds. Glutathione conjugates act as substrates for ATP-dependent ABC transporters and are extruded from the cell. To that end, this current study was undertaken to validate the hypothesis that farnesol conjugation with intracellular glutathione coupled with Cdr1p-mediated extrusion of glutathione conjugates, results in total glutathione depletion, oxidative stress and ultimately fungal cell death. The combined findings demonstrated a significant decrease in intracellular glutathione levels concomitant with up-regulation of CDR1 and decreased cell viability. However, addition of exogenous reduced glutathione maintained intracellular glutathione levels and enhanced viability. In contrast, farnesol toxicity was decreased in a mutant lacking CDR1, whereas it was increased in a CDR1-overexpressing strain. Further, gene expression studies demonstrated significant up-regulation of the SOD genes, primary enzymes responsible for defense against oxidative stress, with no changes in expression in CDR1. This is the first study describing the involvement of Cdr1p-mediated glutathione efflux as a mechanism preceding the farnesol-induced apoptotic process in C. albicans. Understanding of the mechanisms underlying farnesol-cytotoxicity in C. albicans may lead to the development of this redox-cycling agent as an alternative antifungal agent.

Role of the V-ATPase in regulation of the vacuolar fission-fusion equilibrium.

Like numerous other eukaryotic organelles, the vacuole of the yeast Saccharomyces cerevisiae undergoes coordinated cycles of membrane fission and fusion in the course of the cell cycle and in adaptation to environmental conditions. Organelle fission and fusion processes must be balanced to ensure organelle integrity. Coordination of vacuole fission and fusion depends on the interactions of vacuolar SNARE proteins and the dynamin-like GTPase Vps1p. Here, we identify a novel factor that impinges on the fusion-fission equilibrium: the vacuolar H(+)-ATPase (V-ATPase) performs two distinct roles in vacuole fission and fusion. Fusion requires the physical presence of the membrane sector of the vacuolar H(+)-ATPase sector, but not its pump activity. Vacuole fission, in contrast, depends on proton translocation by the V-ATPase. Eliminating proton pumping by the V-ATPase either pharmacologically or by conditional or constitutive V-ATPase mutations blocked salt-induced vacuole fragmentation in vivo. In living cells, fission defects are epistatic to fusion defects. Therefore, mutants lacking the V-ATPase display large single vacuoles instead of multiple smaller vacuoles, the phenotype that is generally seen in mutants having defects only in vacuolar fusion. Its dual involvement in vacuole fission and fusion suggests the V-ATPase as a potential regulator of vacuolar morphology and membrane dynamics.

Understanding mutational and selective processes that govern gene duplication in mammals

Abstract : Gene duplication is an essential source of material for the origin of genetic novelties. The reverse transcription of source gene mRNA followed by the genomic insertion of the resulting cDNA - retroposition - has provided the human genome with at least ~3600 detectable retrocopies. We find that ~30% of these retrocopies are transcribed, generally in testes. Their transcription often relies on preexisting regulatory elements (or open chromatin) close to their insertion site, which is illustrated by mRNA molecules containing retrocopies fused to their neighboring genes. Retrocopies appear to have been profoundly shaped by selection. Consistently, human retrocopies with an intact open reading (ORF) are more often transcribed than retropseudogenes, which leads to a minimal estimate of 120 functional retrogenes present in our genome. We also performed an analysis of Ka/Ks for human retrocopies. This analysis demonstrates that several intact retrocopies evolved under purifying selection and yields an estimated formation rate of ~1 retrogene per million year in the primate lineage. Using DNA sequencing and evolutionary simulations, we have identified 7 such primate-specific retrogenes that emerged on the lineage leading to humans In therian genomes, we found an excess of retrogenes with X-linked parents. Expression analyses support the idea that this "out of X" movement was driven by natural selection to produce autosomal functional counterparts for X-linked genes, which are silenced during male meiosis. Phylogenetic dating of this "out of X" movement suggests that our sex chromosomes arose about 180 MYA ago and are thus much younger than previously thought. Finally, we have also analyzed young gene duplications (and deletions) that arose by non allelic-homologous recombination and are not fixed in species. Using wild-caught and laboratory animals, we detected thousands of DNA segments that are polymorphic in copy number in mice. These copy number variants were found to profoundly alter the transcriptome of several mouse tissues. Strikingly, their influence on gene expression is not limited to the gene they contain but seems to extend to genes located up to 1.5 million bases away.

Molecular biology of baculovirus and its use in biological control in Brazil

Baculoviruses are insect viruses found mainly in Lepidoptera. The family Baculoviridae is taxonomically divided in two genera, Nucleopolyhedrovirus and Granulovirus, which differ by occlusion body morphology. NPVs (Nucleopolyhedroviruses) have polyhedrical inclusion bodies (PIBs) containing multiple viral particles, while GVs (Granuloviruses) appear to be generally single particles occluded in oval shaped occlusion bodies. During the life cycle, two different viral progenies are produced: BV (Budded Virus) and PDV (Polyhedra Derived Virus), which are essential for the infectious process and virus propagation in host cells. Baculoviruses are being used for pest control and they are especially safe due to their specificity and invertebrate-restricted host range. Baculoviruses have been used as vectors for high level protein expression ofheterologous genes from prokaryotic and eukaryotic organisms. Also, recombinant DNA techniques have allowed the production of genetically modified viral insecticides. This study is a review on the taxonomy, structure, replication and molecular biology of baculoviruses, as well as their use as bioinsecticides in Brazil.

Pseudogenes in the ENCODE regions: consensus annotation, analysis of transcription, and evolution.

Arising from either retrotransposition or genomic duplication of functional genes, pseudogenes are "genomic fossils" valuable for exploring the dynamics and evolution of genes and genomes. Pseudogene identification is an important problem in computational genomics, and is also critical for obtaining an accurate picture of a genome's structure and function. However, no consensus computational scheme for defining and detecting pseudogenes has been developed thus far. As part of the ENCyclopedia Of DNA Elements (ENCODE) project, we have compared several distinct pseudogene annotation strategies and found that different approaches and parameters often resulted in rather distinct sets of pseudogenes. We subsequently developed a consensus approach for annotating pseudogenes (derived from protein coding genes) in the ENCODE regions, resulting in 201 pseudogenes, two-thirds of which originated from retrotransposition. A survey of orthologs for these pseudogenes in 28 vertebrate genomes showed that a significant fraction ( approximately 80%) of the processed pseudogenes are primate-specific sequences, highlighting the increasing retrotransposition activity in primates. Analysis of sequence conservation and variation also demonstrated that most pseudogenes evolve neutrally, and processed pseudogenes appear to have lost their coding potential immediately or soon after their emergence. In order to explore the functional implication of pseudogene prevalence, we have extensively examined the transcriptional activity of the ENCODE pseudogenes. We performed systematic series of pseudogene-specific RACE analyses. These, together with complementary evidence derived from tiling microarrays and high throughput sequencing, demonstrated that at least a fifth of the 201 pseudogenes are transcribed in one or more cell lines or tissues.