Modularity and evolutionary constraints in a baculovirus gene regulatory network

Abstract\ud \ud \ud \ud Background\ud The structure of regulatory networks remains an open question in our understanding of complex biological systems. Interactions during complete viral life cycles present unique opportunities to understand how host-parasite network take shape and behave. The Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) is a large double-stranded DNA virus, whose genome may encode for 152 open reading frames (ORFs). Here we present the analysis of the ordered cascade of the AgMNPV gene expression.\ud \ud \ud \ud Results\ud We observed an earlier onset of the expression than previously reported for other baculoviruses, especially for genes involved in DNA replication. Most ORFs were expressed at higher levels in a more permissive host cell line. Genes with more than one copy in the genome had distinct expression profiles, which could indicate the acquisition of new functionalities. The transcription gene regulatory network (GRN) for 149 ORFs had a modular topology comprising five communities of highly interconnected nodes that separated key genes that are functionally related on different communities, possibly maximizing redundancy and GRN robustness by compartmentalization of important functions. Core conserved functions showed expression synchronicity, distinct GRN features and significantly less genetic diversity, consistent with evolutionary constraints imposed in key elements of biological systems. This reduced genetic diversity also had a positive correlation with the importance of the gene in our estimated GRN, supporting a relationship between phylogenetic data of baculovirus genes and network features inferred from expression data. We also observed that gene arrangement in overlapping transcripts was conserved among related baculoviruses, suggesting a principle of genome organization.\ud \ud \ud \ud Conclusions\ud Albeit with a reduced number of nodes (149), the AgMNPV GRN had a topology and key characteristics similar to those observed in complex cellular organisms, which indicates that modularity may be a general feature of biological gene regulatory networks.JVCO, CTB and AI hold FAPESP scholarships (04/12456-0, 09/16740-8 and 12/04818-5), AFB and CCMF hold CAPES-MSc and PhD scholarships and PMAZ holds a CNPq-PQ scholarship. This work was supported financially by FAPESP (Fundação de Amparo a Pesquisa do Estado de São Paulo, process: 2007/55282-0)

The pangenome of the Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV)

The alphabaculovirusAnticarsia gemmatalismultiple nucleopolyhedrovirus (AgMNPV) is the world’s most successful viral bioinsecticide. Through the 1980s and 1990s, this virus was extensively used for biological control of populations ofAnticarsia gemmatalis(Velvetbean caterpillar) in soybean crops. During this period, genetic studies identified several variable loci in the AgMNPV; however, most of them were not characterized at the sequence level. In this study we report a full genome comparison among 17 wild-type isolates of AgMNPV. We found the pangenome of this virus to contain at least 167 hypothetical genes, 151 of which are shared by all genomes. The genebro-athat might be involved in host specificity and carrying transporter is absent in some genomes, and new hypothetical genes were observed. Among these genes there is a uniquernf12-likegene, probably implicated in ubiquitination. Events of gene fission and fusion are common, as four genes have been observed as single or split open reading frames. Gains and losses of genomic fragments (from 20 to 900 bp) are observed within tandem repeats, such as in eight direct repeats and four homologous regions. Most AgMNPV genes present low nucleotide diversity, and variable genes are mainly located in a locus known to evolve through homologous recombination. The evolution of AgMNPV is mainly driven by small indels, substitutions, gain and loss of nucleotide stretches or entire coding sequences. These variations may cause relevant phenotypic alterations, which probably affect the infectivity of AgMNPV. This work provides novel information on genomic evolution of the AgMNPV in particular and of baculoviruses in general

Cell functional enviromics: Unravelling the function of environmental factors

<p>Abstract</p> <p>Background</p> <p>While functional genomics, focused on gene functions and gene-gene interactions, has become a very active field of research in molecular biology, equivalent methodologies embracing the environment and gene-environment interactions are relatively less developed. Understanding the function of environmental factors is, however, of paramount importance given the complex, interactive nature of environmental and genetic factors across multiple time scales.</p> <p>Results</p> <p>Here, we propose a systems biology framework, where the function of environmental factors is set at its core. We set forth a "reverse" functional analysis approach, whereby cellular functions are reconstructed from the analysis of dynamic envirome data. Our results show these data sets can be mapped to less than 20 core cellular functions in a typical mammalian cell culture, while explaining over 90% of flux data variance. A functional enviromics map can be created, which provides a template for manipulating the environmental factors to induce a desired phenotypic trait.</p> <p>Conclusion</p> <p>Our results support the feasibility of cellular function reconstruction guided by the analysis and manipulation of dynamic envirome data.</p

The molluscan shell secretome : unlocking calcium pathways in a changing world

How do molluscs build their shells? Despite hundreds of years of human fascination, the processes underpinning mollusc shell production are still considered a black box. We know molluscs can alter their shell thickness in response to environmental factors, but we do not have a mechanistic understanding of how the shell is produced and regulated. In this thesis I used a combination of methodologies - from traditional histology, to shell damage-repair experiments and ‘omics technologies - to better understand the molecular mechanisms which control shell secretion in two species, the Antarctic clam Laternula elliptica and the temperate blunt-gaper clam Mya truncata. The integration of different methods was particularly useful for assigning putative biomineralisation functions to genes with no previous annotation. Each chapter of this thesis found reoccurring evidence for the involvement of vesicles in biomineralisation and for the duplication and subfunctionalisation of tyrosinase paralogues. Shell damage-repair experiments revealed biomineralisation in L. elliptica was variable, transcriptionally dynamic, significantly affected by age and inherently entwined with immune processes. The high amount of transcriptional variation across 78 individual animals was captured in a single mantle regulatory gene network, which was used to predict the regulation of “classic” biomineralisation genes, and identify novel biomineralisation genes. There were some general shared patterns in the molecular control of biomineralisation between the two species investigated in this thesis, but overall, the comparative work in this thesis, coupled to the growing body of literature on the evolution of molluscan biomineralisation, suggests that biomineralisation mechanisms are surprisingly divergent

Defining the relationship between a baculoviral sulfhydryl oxidase and a potential accessory protein

Master of ScienceDivision of BiologyAna Lorena PassarelliBaculoviruses are a large, diverse, and an ecologically-important group of entomopathogens. The ac78 gene of the prototype baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV), is one of the 38 genes conserved among all baculoviruses sequenced to date. Previous studies show that Ac78 is essential for optimal production of occlusion-derived virions (ODVs) and budded virions (BVs), which are two virion types produced during baculovirus infection. However, the biochemical mechanism by which Ac78 is involved in these processes remains unknown. The AcMNPV sulfhydryl oxidase ac92 is a conserved gene, and its product, Ac92, is ODV and BV envelope-associated. Recently, the Ac78 and Ac92 homologs in Helicoverpa armigera nucleopolyhedrovirus (HearNPV) were reported to interact and co-localize to the site of BV and ODV formation. To investigate the relationship between Ac78 and Ac92, we determined their localization in the presence and absence of AcMNPV infection, performed co-immunoprecipitations to assess interaction relationships, and provided an updated report of Ac78 and Ac92 homology with other proteins. We concluded that in the absence of viral infection, Ac78 and Ac92 localized perinuclearly in the cytoplasm and that localization of Ac92 was not affected by Ac78. During AcMNPV infection, Ac78 and Ac92 co-localized within the nucleus and surrounding virus replication and assembly sites (ring zone). Co-immunoprecipitation experiments showed that at least two differentially-tagged Ac78 proteins were part of a complex in the presence of other AcMNPV proteins. Ac78 did not associate with Ac92 during AcMNPV infection. Our characterization of the relationship between Ac78 and the AcMNPV sulfhydryl oxidase is a preliminary step in a broader effort to elucidate important biochemical pathways underlying the poorly described structural changes in capsid proteins and other proteins involved in virion stability, folding, and infectivity. In a separate project, the same approach was applied in a different virus system to determine the relationship between the small accessory protein C and the measles virus (MeV) replication complex. Co-immunoprecipitation experiments showed that during MeV infection, C associated with large protein (L) and phosphoprotein (P), which comprise the MeV replication complex, and nucleoprotein (N), which encapsidates the RNA genome. Expression constructs for full-length MeV L were generated, and L was successfully expressed following transfection. Subsequent co-immunoprecipitation experiments showed that C did not precipitate with L, P, nor N when transfected in isolation from MeV infection, indicating that another factor resulting from MeV infection is necessary for the association of C with the MeV replication complex. The results of this investigation are an important step in elucidating a biochemical mechanism underlying the function of C as a quality control factor in MeV replication. MeV has been attenuated and is a highly effective vaccine against pathogenic MeV and an active subject of clinical research as an oncolytic agent for treating a number of human cancers. Taken together, the investigations of Ac78 and C and their respective relationships with the AcMNPV sulfhydryl oxidase and the MeV replication complex adds knowledge of biochemical mechanisms underlying the important functions of small accessory proteins containing less than 200 amino acids as mediators in viral replication processes of two different viral systems

On the biosynthesis of labdane-related diterpenoids by class I synthases and P450s: a

Due to their abundance in secondary metabolites, plants are a rich source of chemical diversity. Terpenoids comprise the largest class of natural products, yet many of the biosynthetic pathways leading to their production remain under explored. Using rice as a model system, the biosynthesis of labdane-related class I synthases and P450s was explored through the development of a facile, modular metabolic engineering system. Herein, details of the characterization of the rice class I labdane-related synthase family and CYP76M7 are included. Accordingly, it was found that rice possesses a stemodene synthase, bi-selective syn-labdatriene/ent-sandaracopimaradiene synthase, and a cassadiene C11 hydroxylating P450. Phytochemical analysis of rice extracts revealed the presence of the characterized diterpenes made by class I synthases. Additionally, it was found that the class I synthases exhibit a high degree of plasticity with respect to substrate selection and product outcome, including the ability of OsKSL4 T696I to produce aphidicolene. Furthermore, the first bacterial class I synthases involved in gibberellin production has been identified and characterized. Kaurene oxidase is the first cytochrome P450 involved in the biosynthesis of gibberellin growth hormones. Oxygen labeling experiments revealed this multifunctional P450 undergoes successive hydroxylation reactions with retention of intermediates through a gem-diol to form a carboxylic acid. The studies presented in this thesis detail the development of a metabolic engineering system to investigate terpenoid biosynthesis, the characterization of class I synthases and P450s, and mechanistic investigations of class I synthases and P450s involved in labdane-related biosynthesis

A structural classification of protein-protein interactions for detection of convergently evolved motifs and for prediction of protein binding sites on sequence level

BACKGROUND: A long-standing challenge in the post-genomic era of Bioinformatics is the prediction of protein-protein interactions, and ultimately the prediction of protein functions. The problem is intrinsically harder, when only amino acid sequences are available, but a solution is more universally applicable. So far, the problem of uncovering protein-protein interactions has been addressed in a variety of ways, both experimentally and computationally. MOTIVATION: The central problem is: How can protein complexes with solved threedimensional structure be utilized to identify and classify protein binding sites and how can knowledge be inferred from this classification such that protein interactions can be predicted for proteins without solved structure? The underlying hypothesis is that protein binding sites are often restricted to a small number of residues, which additionally often are well-conserved in order to maintain an interaction. Therefore, the signal-to-noise ratio in binding sites is expected to be higher than in other parts of the surface. This enables binding site detection in unknown proteins, when homology based annotation transfer fails. APPROACH: The problem is addressed by first investigating how geometrical aspects of domain-domain associations can lead to a rigorous structural classification of the multitude of protein interface types. The interface types are explored with respect to two aspects: First, how do interface types with one-sided homology reveal convergently evolved motifs? Second, how can sequential descriptors for local structural features be derived from the interface type classification? Then, the use of sequential representations for binding sites in order to predict protein interactions is investigated. The underlying algorithms are based on machine learning techniques, in particular Hidden Markov Models. RESULTS: This work includes a novel approach to a comprehensive geometrical classification of domain interfaces. Alternative structural domain associations are found for 40% of all family-family interactions. Evaluation of the classification algorithm on a hand-curated set of interfaces yielded a precision of 83% and a recall of 95%. For the first time, a systematic screen of convergently evolved motifs in 102.000 protein-protein interactions with structural information is derived. With respect to this dataset, all cases related to viral mimicry of human interface bindings are identified. Finally, a library of 740 motif descriptors for binding site recognition - encoded as Hidden Markov Models - is generated and cross-validated. Tests for the significance of motifs are provided. The usefulness of descriptors for protein-ligand binding sites is demonstrated for the case of &amp;quot;ATP-binding&amp;quot;, where a precision of 89% is achieved, thus outperforming comparable motifs from PROSITE. In particular, a novel descriptor for a P-loop variant has been used to identify ATP-binding sites in 60 protein sequences that have not been annotated before by existing motif databases