Intergenic and external transcribed spacers of ribosomal RNA genes in lizard-infecting Leishmania: molecular structure and phylogenetic relationship to mammal-infecting Leishmania in the subgenus Leishmania (Leishmania)

To establish the relationships of the lizard- and mammal-infecting Leishmania, we characterized the intergenic spacer region of ribosomal RNA genes from L. tarentolae and L. hoogstraali. The organization of these regions is similar to those of other eukaryotes. The intergenic spacer region was approximately 4 kb in L. tarentolae and 5.5 kb in L. hoogstraali. The size difference was due to a greater number of 63-bp repetitive elements in the latter species. This region also contained another element, repeated twice, that had an inverted octanucleotide with the potential to form a stem-loop structure that could be involved in transcription termination or processing events. The ribosomal RNA gene localization showed a distinct pattern with one chromosomal band (2.2 Mb) for L. tarentolae and two (1.5 and 1.3 Mb) for L. hoogstraali. The study also showed sequence differences in the external transcribed region that could be used to distinguish lizard Leishmania from the mammalian Leishmania. The intergenic spacer region structure features found among Leishmania species indicated that lizard and mammalian Leishmania are closely related and support the inclusion of lizard-infecting species into the subgenus Sauroleishmania proposed by Saf'janova in 1982.

Detection and molecular characterization of group A rotavirus from hospitalized children in Rio de Janeiro, Brazil, 2004

Rotavirus is a major cause of infantile acute diarrhea, causing about 440,000 deaths per year, mainly in developing countries. The World Health Organization has been recommending the assessment of rotavirus burden and strain characterization as part of the strategies of immunization programs against this pathogen. In this context, a prospective study was made on a sample of 134 children with acute diarrhea and severe dehydration admitted to venous fluid therapy in two state hospitals in Rio de Janeiro, Brazil, from February to September 2004. Rotavirus where detected by polyacrylamide gel electrophoresis (PAGE) and by an enzyme-linked immunoassay to rotavirus and adenovirus (EIARA) in 48% of the children. Positive samples for group A rotavirus (n = 65) were analyzed by reverse transcription/heminested multiplex polymerase chain reaction to determine the frequency of G and [P] genotypes and, from these, 64 samples could be typed. The most frequent G genotype was G1 (58%) followed by G9 (40%). One mixed infection (G1/G9) was detected. The only [P] genotype identified was [8]. In order to estimate the rotavirus infection frequency in children who acquired diarrhea as hospital infection in those hospitals, we studied 24 patients, detecting the pathogen in 41% of them. This data suggest that genotype G9 is an important genotype in Rio de Janeiro, with implications to the future strategies of vaccination against rotavirus, reinforcing the need of continuous monitoring of circulating strains of the pathogen, in a surveillance context.

Molecular approaches for the detection of Schistosoma mansoni: possible applications in the detection of snail infection, monitoring of transmission sites, and diagnosis of human infection

The detection of specific DNA sequences by polymerase chain reaction (PCR) has proved extremely valuable for the analysis of genetic disorders and the diagnosis of a variety of infectious disease pathogens. However, the application to the detection of Schistosoma mansoni is rare, despite a recommendation of the World Health Organization that a major focus of research on schistosomiasis should be on the development and evaluation of new strategies and tools for control of the disease. In this context, a few studies were published for the detection of the parasite in snails, monitoring of cercariae in water bodies, and diagnosis of human infection. The present minireview describes sensitive and specific PCR based systems to detect S. mansoni, indicating possible applications in the detection of snail infection, monitoring of transmission sites, and diagnosis of human infection.

Genomic organization, fine-mapping, and expression of the human islet-brain 1 (IB1)/c-Jun-amino-terminal kinase interacting protein-1 (JIP-1) gene.

Islet-brain 1 (IB1), a regulator of the pancreatic beta-cell function in the rat, is homologous to JIP-1, a murine inhibitor of c-Jun amino-terminal kinase (JNK). Whether IB1 and JIP-1 are present in humans was not known. We report the sequence of the 2133-bp human IB1 cDNA, the expression, structure, and fine-mapping of the human IB1 gene, and the characterization of an IB1 pseudogene. Human IB1 is 94% identical to rat IB1. The tissue-specific expression of IB1 in human is similar to that observed in rodent. The IB1 gene contains 12 exons and maps to chromosome 11 (11p11.2-p12), a region that is deleted in DEFECT-11 syndrome. Apart from an IB1 pseudogene on chromosome 17 (17q21), no additional IB1-related gene was found in the human genome. Our data indicate that the sequence and expression pattern of IB1 are highly conserved between rodent and human and provide the necessary tools to investigate whether IB1 is involved in human diseases.

Molecular findings from influenza A(H1N1)pdm09 detected in patients from a Brazilian equatorial region during the pandemic period

After the World Health Organization officially declared the end of the first pandemic of the XXI century in August 2010, the influenza A(H1N1)pdm09 virus has been disseminated in the human population. In spite of its sustained circulation, very little on phylogenetic data or oseltamivir (OST) resistance is available for the virus in equatorial regions of South America. In order to shed more light on this topic, we analysed the haemagglutinin (HA) and neuraminidase (NA) genes of influenza A(H1N1)pdm09 positive samples collected during the pandemic period in the Pernambuco (PE), a northeastern Brazilian state. Complete HA sequences were compared and amino acid changes were related to clinical outcome. In addition, the H275Y substitution in NA, associated with OST resistance, was investigated by pyrosequencing. Samples from PE were grouped in phylogenetic clades 6 and 7, being clustered together with sequences from South and Southeast Brazil. The D222N/G HA gene mutation, associated with severity, was found in one deceased patient that was pregnant. Additionally, the HA mutation K308E, which appeared in Brazil in 2010 and was only detected worldwide the following year, was identified in samples from hospitalised cases. The resistance marker H275Y was not identified in samples tested. However, broader studies are needed to establish the real frequency of resistance in this Brazilian region.

Clinical implications of molecular neuropathology and biomarkers for malignant glioma.

Malignant gliomas are currently diagnosed based on morphological criteria and graded according to the World Health Organization classification of primary brain tumors. This algorithm of diagnosis and classification provides clinicians with an estimated prognosis of the natural course of the disease. It does not reflect the expected response to specific treatments beyond surgery (eg, radiotherapy or alkylating chemotherapy). Clinical experience has revealed that gliomas sharing similar histomorphological criteria might indeed have different clinical courses and exhibit highly heterogenous responses to treatments. This was very impressively demonstrated first for oligodendrogliomas. The presence or lack of combined deletions of the chromosomal segments 1p/19q was associated with different benefit from radiotherapy and chemotherapy. We review current molecular markers for malignant gliomas and discuss their current and future impact on clinical neuro-oncology.

Structural determinants for membrane association and dynamic organization of the hepatitis C virus NS3-4A complex.

Hepatitis C virus (HCV) NS3-4A is a membrane-associated multifunctional protein harboring serine protease and RNA helicase activities. It is an essential component of the HCV replication complex and a prime target for antiviral intervention. Here, we show that membrane association and structural organization of HCV NS3-4A are ensured in a cooperative manner by two membrane-binding determinants. We demonstrate that the N-terminal 21 amino acids of NS4A form a transmembrane alpha-helix that may be involved in intramembrane protein-protein interactions important for the assembly of a functional replication complex. In addition, we demonstrate that amphipathic helix alpha(0), formed by NS3 residues 12-23, serves as a second essential determinant for membrane association of NS3-4A, allowing proper positioning of the serine protease active site on the membrane. These results allowed us to propose a dynamic model for the membrane association, processing, and structural organization of NS3-4A on the membrane. This model has implications for the functional architecture of the HCV replication complex, proteolytic targeting of host factors, and drug design.

Organization of lin genes and IS6100 among different strains of hexachlorocyclohexane-degrading Sphingomonas paucimobilis: evidence for horizontal gene transfer.

The organization of lin genes and IS6100 was studied in three strains of Sphingomonas paucimobilis (B90A, Sp+, and UT26) which degraded hexachlorocyclohexane (HCH) isomers but which had been isolated at different geographical locations. DNA-DNA hybridization data revealed that most of the lin genes in these strains were associated with IS6100, an insertion sequence classified in the IS6 family and initially found in Mycobacterium fortuitum. Eleven, six, and five copies of IS6100 were detected in B90A, Sp+, and UT26, respectively. IS6100 elements in B90A were sequenced from five, one, and one regions of the genomes of B90A, Sp+, and UT26, respectively, and were found to be identical. DNA-DNA hybridization and DNA sequencing of cosmid clones also revealed that S. paucimobilis B90A contains three and two copies of linX and linA, respectively, compared to only one copy of these genes in strains Sp+ and UT26. Although the copy number and the sequence of the remaining genes of the HCH degradative pathway (linB, linC, linD, and linE) were nearly the same in all strains, there were striking differences in the organization of the linA genes as a result of replacement of portions of DNA sequences by IS6100, which gave them a strange mosaic configuration. Spontaneous deletion of linD and linE from B90A and of linA from Sp+ occurred and was associated either with deletion of a copy of IS6100 or changes in IS6100 profiles. The evidence gathered in this study, coupled with the observation that the G+C contents of the linA genes are lower than that of the remaining DNA sequence of S. paucimobilis, strongly suggests that all these strains acquired the linA gene through horizontal gene transfer mediated by IS6100. The association of IS6100 with the rest of the lin genes further suggests that IS6100 played a role in shaping the current lin gene organization.

The molecular architecture of the TNF superfamily.

Ligands of the TNF (tumour necrosis factor) superfamily have pivotal roles in the organization and function of the immune system, and are implicated in the aetiology of several acquired and genetic diseases. TNF ligands share a common structural motif, the TNF homology domain (THD), which binds to cysteine-rich domains (CRDs) of TNF receptors. CRDs are composed of structural modules, whose variation in number and type confers heterogeneity upon the family. Protein folds reminiscent of the THD and CRD are also found in other protein families, raising the possibility that the mode of interaction between TNF and TNF receptors might be conserved in other contexts.

Complex organization of CTF/NF-I, C/EBP, and HNF3 binding sites within the promoter of the liver-specific vitellogenin gene.

Vitellogenin genes are expressed specifically in the liver of female oviparous vertebrates under the strict control of estrogen. To explain this tissue-specific expression, we performed a detailed analysis of the Xenopus laevis vitellogenin gene B1 promoter by DNase I footprinting and gel mobility-shift assays. We characterized five binding sites for the ubiquitous factor CTF/NF-I. Two of these sites are close to the TATA-box, whereas the others are located on both sides of the estrogen responsive unit formed by two imperfect estrogen response elements. Moreover two liver-enriched factors, C/EBP and HNF3, were found to interact with multiple closely spaced proximal promoter elements in the first 100 base pairs upstream of the TATA-box. To confirm the physiological significance of this in vitro analysis, in vivo DNase I footprinting experiments were carried out using the ligation-mediated polymerase chain reaction technique. The various cis-elements characterized in vitro as binding sites for known transcription factors and more particularly for liver-enriched transcription factors are efficiently recognized in vivo as well, suggesting that they play an important role in the control of the liver-specific vitellogenin gene B1 expression.

Genomic organization and gene expression in a chromosomal region of Leishmania major.

Little is known about the relation between the genome organization and gene expression in Leishmania. Bioinformatic analysis can be used to predict genes and find homologies with known proteins. A model was proposed, in which genes are organized into large clusters and transcribed from only one strand, in the form of large polycistronic primary transcripts. To verify the validity of this model, we studied gene expression at the transcriptional, post-transcriptional and translational levels in a unique locus of 34kb located on chr27 and represented by cosmid L979. Sequence analysis revealed 115 ORFs on either DNA strand. Using computer programs developed for Leishmania genes, only nine of these ORFs, localized on the same strand, were predicted to code for proteins, some of which show homologies with known proteins. Additionally, one pseudogene, was identified. We verified the biological relevance of these predictions. mRNAs from nine predicted genes and proteins from seven were detected. Nuclear run-on analyses confirmed that the top strand is transcribed by RNA polymerase II and suggested that there is no polymerase entry site. Low levels of transcription were detected in regions of the bottom strand and stable transcripts were identified for four ORFs on this strand not predicted to be protein-coding. In conclusion, the transcriptional organization of the Leishmania genome is complex, raising the possibility that computer predictions may not be comprehensive.

Gene organization of the mating type regions in the ectomycorrhizal fungus Laccaria bicolor reveals distinct evolution between the two mating type loci

In natural conditions, basidiomycete ectomycorrhizal fungi such as Laccaria bicolor are typically in the dikaryotic state when forming symbioses with trees, meaning that two genetically different individuals have to fuse or 'mate'. Nevertheless, nothing is known about the molecular mechanisms of mating in these ecologically important fungi. Here, advantage was taken of the first sequenced genome of the ectomycorrhizal fungus, Laccaria bicolor, to determine the genes that govern the establishment of cell-type identity and orchestrate mating. The L. bicolor mating type loci were identified through genomic screening. The evolutionary history of the genomic regions that contained them was determined by genome-wide comparison of L. bicolor sequences with those of known tetrapolar and bipolar basidiomycete species, and by phylogenetic reconstruction of gene family history. It is shown that the genes of the two mating type loci, A and B, are conserved across the Agaricales, but they are contained in regions of the genome with different evolutionary histories. The A locus is in a region where the gene order is under strong selection across the Agaricales. By contrast, the B locus is in a region where the gene order is likely under a low selection pressure but where gene duplication, translocation and transposon insertion are frequent.

Role of myosin V in actin cable organization in fission yeast

Functional specialization is tightly linked to the ability of eukaryotic cells to acquire a particular shape. Cell morphogenesis, in turn, relies on the capacity to establish and maintain cell "polarity", which is achieved by orienting the trafficking of signaling molecules and organelles towards specific cellular locations and/or membrane domains. The "oriented" transport is based upon cytoskeletal polymers, microtubules and actin filaments, which serve as tracks for molecular motors. These latter generate motion that is translated either into pulling forces or directed transport. Fission yeast, a rod-like unicellular eukaryote, shapes itself by restricting growth at cell tips through the concerted activity of microtubules and actin cables. Microtubules, which assemble into 2-6 bundles and run parallel to the long axis of the cell, serve to orient growth to the tips. Growth is supported by the actin cytoskeleton, which provides tracks, the cables, for motor-based transport of secretory vesicles. The molecular motors, which bind cargos and deliver them to the tips along cables, are also known as type V myosins (hereafter indicated as myosin V). How the bundles of parallel actin filaments, i.e. the cables, extend from the tips through the cell and whether they serve any other purpose, besides providing tracks, is poorly understood. It is also unclear how the crosstalk between the two cytoskeletal systems is achieved. These are the basic questions I addressed during my PhD. The first part of the thesis work (Chapter two) suggests that the sole function of actin cables in polarized growth is to serve as tracks for motors. The data indicate that cells may have evolved two cytoskeletal systems to provide robustness to the polarization process but in principle a unique cytoskeleton might have been able to direct and support polarized growth. How actin cables are organized within the cell to optimize cargo transport is addressed later on (Chapter three). The major finding, based on the actin cable defect of cells lacking myosin Vs, is that actin filaments self-organize through the activity of the transport motors. In fact, by delivering cargos to cell tips and exerting physical pulling forces on actin filaments, Myosin Vs contribute not only to polarize cargo transport but also actin tracks. Among the cargos transported by Myosin V, which may be relevant to its function in organizing cables, there is likely the endoplasmic reticulum (ER). Actin cables, which run parallel to cortical ER, may serve as tracks for Myosin V. Myosin V-driven displacement, in turn, may account for the dynamic expansion and organization of ER during polarized growth as suggested in Chapter four. The last part of the work (Chapter five) highlights the existence of a crosstalk between actin and microtubules. In absence of myosin V, indeed, microtubules contribute to actin cable organization, likely playing a scaffolding/tethering function. Whether or not the kinesin 1, Klp3, plays any role in such process has to be demonstrated. In conclusion the work proposes a novel role for myosin Vs in actin organization, besides its transport function, and provides molecular tools to further dissect the role of this type of myosin in fission yeast. - La spécialisation fonctionnelle est étroitement connectée à la capacité des cellules eucaryotes d'acquérir une forme particulière. La morphogenèse cellulaire à son tour, est basée sur la capacité d'établir et de maintenir la polarité cellulaire, polarité réalisée en orientant le trafic des molécules signales et des organelles vers des zones cellulaires spécifiques. Ce transport directionnel dépend des polymères du cytosquelette, microtubules et microfilaments, qui servent comme des voies pour les moteurs moléculaires. Ces derniers engendrent du mouvement, traduit soit en force de traction soit en transport directionnel. La levure fissipare, un eucaryote unicellulaire en forme de bâtonnet, acquière sa forme en limitant sa croissance aux extrémités par l'action concertée des microtubules et de l'actine. Les microtubules, qui s'assemblent de façon antiparallèle et parcourent la cellule parallèlement à l'axe longitudinal, servent à orienter la croissance aux extrémités. Cette croissance est permise par le cytosquelette d'actine, fournissant des voies, les câbles, pour le transport actif des vésicules de sécrétion. Les moteurs moléculaires, responsables de ce transport actif sont aussi appelés myosines de type V (par la suite appelés myosines V). La manière dont ces câbles s'étendent depuis l'extrémité jusqu'à l'intérieur de la cellule est peu connue. De plus, on ignore également si ces câbles présentent une fonction autre que le transport. L'interaction entre les deux cytosquelettes est également obscure. Ce sont ces questions de base auxquelles j'ai tenté de répondre lors de ma thèse. La première partie de cette thèse (chapitre II) suggère que les câbles d'actine, pendant la croissance polarisée, fonctionnent uniquement comme des voies pour les moteurs moléculaires. Les données indiqueraient que les cellules ont fait évoluer deux systèmes de cytosquelette pour assurer plus de robustesse au processus de polarisation, bien que, comme nous le verrons, un système unique est suffisant. Au chapitre III, nous verrons comment les câbles d'actine sont organisés à l'intérieur de la cellule afin d'optimiser le transport des cargo. La découverte majeure, réalisée en observant des cellules dont la myosine V fait défaut, est que ces filaments d'actine s'auto organisent grâce au passage des moteurs moléculaires le long de ces voies. En réalité, en délivrant les cargos aux extrémités de la cellule et en exerçant des forces de traction sur les câbles, les myosines V contribuent non seulement à polariser le transport mais également à polariser les voies elles mêmes. Nous verrons également au chapitre IV, que parmi les cargos importants pour l'organisation des câbles, il y aurait le réticulum endoplasmique (RE). En effet, les câbles d'actine, qui s'étalent parallèlement au RE cortical, pourraient servir comme voie pour la myosine V. Cette dernière en retour pourrait être responsable de l'expansion dynamique et de l'organisation du RE pendant la croissance polarisée.

Expression of foraging and Gp-9 are associated with social organization in the fire ant Solenopsis invicta.

The aim of this study was to investigate levels of expression of two major genes, the odorant binding protein Gp-9 (general protein-9) and foraging, that have been shown to be associated with behavioural polymorphisms in ants. We analysed workers and young nonreproductive queens collected from nests of the monogyne (single reproductive queen per nest) and polygyne (multiple reproductive queens) social forms of Solenopsis invicta. In workers but not young queens, the level of foraging expression was significantly associated with social form and the task performed (ie localization in the nest or foraging area). The level of expression of Gp-9 was also associated with social form and worker localization. In addition there was a higher level of expression of the Gp-9(b) allele compared with the Gp-9(B) allele in the heterozygote workers and the young nonreproductive queens. Finally, in the polygyne colonies the level of expression of foraging was not significantly associated with the Gp-9 genotype for either workers or young nonreproductive queens, suggesting that both genes have independent non-epistatic effects on behaviour in S. invicta.