Molecular mechanisms underlying the control of antigenic variation in African trypanosomes

African trypanosomes escape the host adaptive immune response by switching their dense protective coat of Variant Surface Glycoprotein (VSG). Each cell expresses only one VSG gene at a time from a telomeric expression site (ES). The [`]pre-genomic' era saw the identification of the range of pathways involving VSG recombination in the context of mono-telomeric VSG transcription. A prominent feature of the early post-genomic era is the description of the molecular machineries involved in these processes. We describe the factors and sequences recently linked to mutually exclusive transcription and VSG recombination, and how these act in the control of the key virulence mechanism of antigenic variatio

Molecular studies on the sweet potato virus disease and its two causal agents

The studies presented in this thesis contribute to an increased understanding of the molecular aspects, variability and interaction of the two most important viral pathogens of sweet potato (Ipomoea batatas L): Sweet potato feathery mottle virus (SPFMV) and Sweet potato chlorotic stunt virus (SPCSV), which cause the severe sweet potato virus disease (SPVD) when co-infecting sweet potato plants. SPVD is the most important disease affecting sweet potato in Africa, and may be the most important virus disease of sweet potato globally. The coat protein gene sequences of several African SPFMV isolates were determined and compared by phylogenetic analyses. Results showed that East African SPFMV isolates were genetically distinct. They could furthermore be divided into two serotypes which differed in their ability to systemically infect the sweet potato cultivar Tanzania. The aetiology of SPVD was studied in sweet potato plants co-infected with SPFMV and SPCSV using nucleic acid hybridisation, bioassays, tissue printing and thin section immunohistochemistry. Resistance to SPFMV in East African sweet potato cultivars was found to be due to inhibition of virus replication rather than movement and resistance was suppressed by infection with SPCSV, resulting in a ca. 600-fold increase in titres of SPFMV. Furthermore, in SPVD affected plants SPFMV is detected outside of the phloem, whereas SPCSV is detected only inside the phloem, which suggests novel as yet unknown mechanisms how SPCSV synergises SPFMV. The genomic sequence of SPCSV was determined. It was composed of two RNA molecules (9407 and 8223 nucleotides), representing the second largest (+)ssRNA genome of plant viruses. The genomic organization of SPCSV revealed novel features for the genus Crinivirus, such as i) the presence of a gene putatively encoding an ribonuclease III-like protein, ii) near-identical, 208 nucleotides long 3’-sequences on both viral RNAs, and iii) the placement of the SHP gene at a new position on the genome of SPCSV relative to other closteroviridae. Northern analyses showed the presence of several sub-genomic RNAs, of which the accumulation was temporally regulated in infected tissues. The 5’-ends of seven sub-genomic RNAs were determined using a PCR based method, which indicated that the sgRNAs were capped

Antigenic variation in the African trypanosome: molecular mechanisms and phenotypic complexity

Antigenic variation is an immune evasion strategy that has evolved in viral, bacterial and protistan pathogens. In the African trypanosome this involves stochastic switches in the composition of a variant surface glycoprotein (VSG) coat, using a massive archive of silent VSG genes to change the identity of the single VSG expressed at a time. VSG switching is driven primarily by recombination reactions that move silent VSGs into specialized expression sites, though transcription-based switching can also occur. Here we discuss what is being revealed about the machinery that underlies these switching mechanisms, including what parallels can be drawn with other pathogens. In addition, we discuss how such switching reactions act in a hierarchy and contribute to pathogen survival in the face of immune attack, including the establishment and maintenance of chronic infections, leading to host-host transmission

Callose homeostasis at plasmodesmata: molecular regulators and developmental relevance

Plasmodesmata are membrane-lined channels that are located in the plant cell wall and that physically interconnect the cytoplasm and the endoplasmic reticulum (ER) of adjacent cells. Operating as controllable gates, plasmodesmata regulate the symplastic trafficking of micro- and macromolecules, such as endogenous proteins [transcription factors (TFs)] and RNA-based signals (mRNA, siRNA, etc.), hence mediating direct cell-to-cell communication and long distance signaling. Besides this physiological role, plasmodesmata also form gateways through which viral genomes can pass, largely facilitating the pernicious spread of viral infections. Plasmodesmatal trafficking is either passive (e.g., diffusion) or active and responses both to developmental and environmental stimuli. In general, plasmodesmatal conductivity is regulated by the controlled build-up of callose at the plasmodesmatal neck, largely mediated by the antagonistic action of callose synthases (CalSs) and beta-1,3-glucanases. Here, in this theory and hypothesis paper, we outline the importance of callose metabolism in PD SEL control, and highlight the main molecular factors involved. In addition, we also review other proteins that regulate symplastic PD transport, both in a developmental and stress-responsive framework, and discuss on their putative role in the modulation of PD callose turn-over. Finally, we hypothesize on the role of structural sterols in the regulation of (PD) callose deposition and outline putative mechanisms by which this regulation may occur

Sequence elements controlling expression of Barley stripe mosaic virus subgenomic RNAs in vivo.

Barley stripe mosaic virus (BSMV) contains three positive-sense, single-stranded genomic RNAs, designated alpha, beta, and gamma, that encode seven major proteins and one minor translational readthrough protein. Three proteins (alphaa, betaa, and gammaa) are translated directly from the genomic RNAs and the remaining proteins encoded on RNAbeta and RNAgamma are expressed via three subgenomic messenger RNAs (sgRNAs). sgRNAbeta1 directs synthesis of the triple gene block 1 (TGB1) protein. The TGB2 protein, the TGB2' minor translational readthrough protein, and the TGB3 protein are expressed from sgRNAbeta2, which is present in considerably lower abundance than sgRNAbeta1. A third sgRNA, sgRNAgamma, is required for expression of the gammab protein. We have used deletion analyses and site-specific mutations to define the boundaries of promoter regions that are critical for expression of the BSMV sgRNAs in infected protoplasts. The results reveal that the sgRNAbeta1 promoter encompasses positions -29 to -2 relative to its transcription start site and is adjacent to a cis-acting element required for RNAbeta replication that maps from -107 to -74 relative to the sgRNAbeta1 start site. The core sgRNAbeta2 promoter includes residues -32 to -17 relative to the sgRNAbeta2 transcriptional start site, although maximal activity requires an upstream hexanucleotide sequence residing from positions -64 to -59. The sgRNAgamma promoter maps from -21 to +2 relative to its transcription start site and therefore partially overlaps the gammaa gene. The sgRNAbeta1, beta2, and gamma promoters also differ substantially in sequence, but have similarities to the putative homologous promoters of other Hordeiviruses. These differences are postulated to affect competition for the viral polymerase, coordination of the temporal expression and abundance of the TGB proteins, and constitutive expression of the gammab protein

Physicochemical analysis of rotavirus segment 11 supports a 'modified panhandle' structure and not the predicted alternative tRNA-like structure (TRLS)

.Rotaviruses are a major cause of acute gastroenteritis, which is often fatal in infants. The viral genome consists of 11 double-stranded RNA segments, but little is known about their cis-acting sequences and structural elements. Covariation studies and phylogenetic analysis exploring the potential structure of RNA11 of rotaviruses suggested that, besides the previously predicted "modified panhandle" structure, the 5' and 3' termini of one of the isoforms of the bovine rotavirus UKtc strain may interact to form a tRNA-like structure (TRLS). Such TRLSs have been identified in RNAs of plant viruses, where they are important for enhancing replication and packaging. However, using tRNA mimicry assays (in vitro aminoacylation and 3'- adenylation), we found no biochemical evidence for tRNA-like functions of RNA11. Capping, synthetic 3' adenylation and manipulation of divalent cation concentrations did not change this finding. NMR studies on a 5'- and 3'-deletion construct of RNA11 containing the putative intra-strand complementary sequences supported a predominant panhandle structure and did not conform to a cloverleaf fold despite the strong evidence for a predicted structure in this conserved region of the viral RNA. Additional viral or cellular factors may be needed to stabilise it into a form with tRNA-like properties

Computing and data processing

The applications of computers and data processing to astronomy are discussed. Among the topics covered are the emerging national information infrastructure, workstations and supercomputers, supertelescopes, digital astronomy, astrophysics in a numerical laboratory, community software, archiving of ground-based observations, dynamical simulations of complex systems, plasma astrophysics, and the remote control of fourth dimension supercomputers

Proteomics of Cytochrome c Oxidase-Negative versus -Positive Muscle Fiber Sections in Mitochondrial Myopathy

The mosaic distribution of cytochrome c oxidase(+) (COX+) and COX - muscle fibers in mitochondrial disorders allows the sampling of fibers with compensated and decompensated mitochondrial function from the same individual. We apply laser capture microdissection to excise individual COX+ and COX- fibers from the biopsies of mitochondrial myopathy patients. Using mass spectrometry-based proteomics, we quantify >4,000 proteins per patient. While COX+ fibers show a higher expression of respiratory chain components, COX- fibers display protean adaptive responses, including upregulation of mitochondrial ribosomes, translation proteins, and chaperones. Upregulated proteins include C1QBP, required for mitoribosome formation and protein synthesis, and STOML2, which organizes cardiolipin-enriched microdomains and the assembly of respiratory supercomplexes. Factoring in fast/slow fiber type, COX (-) slow fibers show a compensatory upregulation of beta-oxidation, the AAA(+) protease AFG3L1, and the OPA1-dependent cristae remodeling program. These findings reveal compensatory mechanisms in muscle fibers struggling with energy shortage and metabolic stress

Geminivirus C2 protein represses genes involved in sulphur assimilation and this effect can be counteracted by jasmonate treatment

Geminiviruses are plant viruses that infect a broad range of crops and cause extensive losses worldwide, having an important economic impact. C2, a multifunctional pathogenicity factor encoded by geminiviruses, has been recently shown to suppress the responses to jasmonates in the host plant, which might at least partially explain its well-established role in pathogenicity. Sulphur is one of the essential macro-elements for plant life, and is considered to have a role in plant defence, in a phenomenon named sulphur-induced resistance (SIR) or sulphur-enhanced defence (SED). In this work, we show that geminivirus C2 protein represses the expression of genes involved in the sulphur assimilation pathway in Arabidopsis, but, interestingly, this effect can be neutralized by exogenous jasmonate treatment. These preliminary results may raise the idea that geminiviruses might be affecting sulphur metabolism, and maybe counteracting SIR/SED, through the manipulation of the jasmonate signalling pathway, which would define a novel strategy in plant-virus interactions and may unveil SIR/SED as an important player in the plant defence against viruses.Ministerio de Ciencia y Innovación/FEDER AGL2007-66062-C02-02/AGR AGL2010-22287-CO2European Regional Development Fund (ERDF) BIO2010-15201Junta de Andalucía BIO­27