Thesis (Ph. D.)--Washington State University, August 2009. / Title from PDF title page (viewed on Aug. 24, 2009). "Department of Molecular Plant Sciences." Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 1999. / Vita. Includes bibliographical references (leaves 102-112). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D.)--Oregon State University, 2009. / Printout. Includes bibliographical references (leaves 145-156). Also available on the World Wide Web.

Orientador: Renato Vicentini dos Santos / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T20:26:13Z (GMT). No. of bitstreams: 1 Canesin_LucasEduardoCosta_M.pdf: 8460459 bytes, checksum: 3fde4864bd253faed92b7dfd9071be3c (MD5) Previous issue date: 2014 / Resumo: Recentemente, a análise de dados de genômica comparativa, buscando elucidar melhor a hipótese nula de modelos evolutivos, i.e. evolução neutra, originou uma nova teoria que eleva o tamanho populacional como principal fator evolutivo. Populações pequenas estão sujeitas a forte influência de deriva genética, o que causa o aumento da entropia do genoma. A complexidade genômica, leia-se conteúdo de sequencias informativas, como genes, é então um subproduto do aumento da entropia e a seleção teria então um papel secundário, sobretudo como moduladora do processo evolutivo. Assumindo este modelo, a emergência e degeneração de transcritos linhagem-específicos estão submetidas primariamente a evolução neutra. A transcrição pervasiva, sobretudo em linhagens germinais, é o agente causal do nascimento de genes e a fixação destes, frente ao reduzido tamanho populacional de eucariotos multicelulares, como as plantas Saccarum officinarum e Sorghum bicolor, ocorre por deriva genética. A inserção de novos genes, que são inicialmente neutros ou levemente deletérios, em redes funcionais ainda é pouco compreendida. A integração se torna gradativamente mais robusta com a evolução individual destes loci. Neste contexto, este estudo buscou identificar genes codificadores e não-codificadores de proteínas de recente emergência em cana-de-açúcar e sorgo a fim de se elucidar a hipótese de que sua arquitetura gênica e integração em redes biológicas apresentam padrões evolutivos comuns. Para isso, realizamos a identificação de lncRNAs de cana a partir de bancos de cDNA, o que permitiu a caracterização da expressão desses transcritos contrastando seis variedades distintas. Em decorrência da disponibilidade do genoma de sorgo, a identificação de genes linhagem-específicos codificadores e não codificadores pode ser resolvida com maior precisão. Pudemos determinar uma correlação entre a sua arquitetura gênica e integração nas redes biológicas e sua idade relativa. Apesar da correlação encontrada, o efeito mais forte observado em transcritos não codificadores revelam outros fatores que devem estar influenciando sua evolução. Levantamos a hipótese de que o evento de tradução possa elevar a eficiência da seleção negativa sobre o transcrito emergente, o que resultaria no turnover mais acentuado de lincRNAs e maior conservação de genes linhagem-específicos / Abstract: Recently, comparative genomics studies, aiming to better elucidate the null hypothesis of models of evolution, i. e. the neutral evolution, originate a new theory that elects the population size as the main factor acting in evolution. Small populations are subject to stronger influence of genetic drift, which raises genomic entropy. Genomic complexity, which means the information content in genome, such as genes, is a byproduct of the high entropy levels and selection would then display a secondary role, mainly as a modulator of the evolutionary process. Assuming this model, the emergence and degeneration of lineage-specific transcripts are primarily subject to neutral evolution. The pervasive transcription, especially in germinal cell lines, is the causal agent of birth of genes and their fixation, in face to the reduced population size of multicellular eukaryotes, as Saccarum officinarum and Sorghum bicolor plant species, is ruled by genetic drift. The integration of new genes, initially neutral or weakly deleterious, in functional networks is still poorly understood. The integration becomes more robust with the individual historical evolutionary path of these loci. In this context, this study aimed identify protein coding and noncoding genes of recent emergence in in sugarcane and sorghum to elucidate the hypothesis that the gene architecture and integration in biological networks display common patterns of evolution. We then identified sugarcane lncRNAs from public cDNA databases that allowed us to characterize the expression of these transcripts in six different contrasting varieties of sugarcane. As sorghum bicolor genome is available, the identification of lineage-specific coding and noncoding could be done to a higher resolution. We could then determine a correlation between gene architecture and network integration with its relative age. Despite the correlation observed, a stronger effect seen in noncoding transcripts reveals other factors that may be influencing their evolution. We propose the hypothesis that the translation event may increase negative selection efficiency over the emerging transcript, what would result in the stronger turnover of lincRNAs and higher conservation levels of coding lineage-specific genes / Mestrado / Bioinformatica / Mestre em Genética e Biologia Molecular

Chapter 3 Groundnut Bud Necrosis Virus (GBNV) is a tripartite ambisense RNA plant virus that belongs to serogroup IV of Tospovirus genus. Non-Structural protein-m (NSm), which functions as movement protein in tospoviruses, is encoded by the M RNA. In this chapter, we demonstrate that despite the absence of any putative transmembrane domain, GBNV NSm associates with membranes when expressed in E. coli as well as in N. benthamiana. Incubation of refolded NSm with liposomes ranging in size from 200-250 nm resulted in changes in the secondary and tertiary structure of NSm. A similar behaviour was observed in the presence of anionic and zwitterionic detergents. Furthermore, the morphology of the liposomes was found to be modified in the presence of NSm. Deletion of coiled coil domain resulted in the inability of in planta expressed NSm to interact with membranes. Further, when the C-terminal coiled coil domain alone was expressed, it was found to be associated with membrane. These results demonstrate that NSm associates with membranes via the C-terminal coiled coil domain and such an association may be important for movement of viral RNA from cell to cell. Further NSm was shown to be phosphorylated by N. benthamiana and tomato crude sap as observed in other movement proteins. Chapter 4 This chapter deals with localization of NSm to PD and identification of domain involved in localization. For this purpose NSm and its mutants were cloned in pEAQ:GFP vector and transiently expressed in N. benthamiana by infiltration of transformed Agrobacteria. The GFP tagged NSm was visualized by confocal microscopy. The results demonstrated that NSm forms punctate structures and localizes to PD as confirmed by colocalization of mCherry: PDLP1a, a PD marker which resides in PD, with GFP:NSm. To find out the domain involved in PD localization, sequential deletion mutants were made. It was found that C-terminal domain is involved in PD localization. On the other hand, N-terminal unfolded region was dispensable for PD localization. This is the first report of a coiled coil domain shown to be involved in PD localization. It has also been demonstrated that GBNV NSm interacts with NP. Further, membrane floatation assay carried in presence of NP suggested that interaction of NSm and NP affected membrane association of NSm. These results were further confirmed by localization studies of NSm in presence of NP. It was found that there was considerable relocalization of both NSm and NP. NSm was observed to be present in cytoplasm as well as on the membrane. At the same time, NP was observed on membrane apart from being present in the cytoplasm. When N-terminal 50 amino acids (unfolded) region of NSm was deleted and colocalization studies were carried out, it was found that NSm and NP do not colocalize, suggesting that NSm interacts with NP via the unfolded region and helps in the relocalization of NP to the membrane. Chapter 5 This chapter deals with the pathway of targeting NSm to PD. To decipher the pathway, followed by NSm, an inhibitor of endomembrane or vesicle mediated transport, Brefeldin A (BFA) was used. When GFP-NSm was expressed it was observed to form punctate structure at PD as before. Upon treatment with BFA, green islands were observed in the cytoplasm suggesting that ER was involved in targeting NSm to PD. Similarly, LatB, inhibitor of actin mediated targeting of protein to membrane, also abrogated the localization of NSm to PD. In order to further understand the role of ER in targeting NSm to PD, an ER marker, ER-GFP (GFP fused to HDEL peptide that directs it to ER) was coexpressed with GBNV NSm fused to mCherry. It was observed that NSm colocalizes with ER-GFP as yellow puncta on PD. The puncta appeared as patches and the whole ER-network was converted to vesicles. This was further confirmed by coexpressing ER-GFP with NSm without any tag. The green fluorescent vesicles were observed preferentially near cell membrane. To delineate the region of NSm involved in vesicle formation, point mutants and deletion mutants of NSm were generated without the tag and coexpressed with ER-GFP. When N-terminal 203 amino acids were deleted, NSm was able to transform ER membranes to vesicles suggesting that these residues are dispensable for vesicle formation. Interestingly, the deletion of coiled coil domain leads to cytosolic location of NSm. Furthermore, the C-terminal coiled coil domain when expressed alone was capable of inducing vesicle formation. This is the first report of involvement of such a domain in ER membrane association and vesicle formation.

In the present thesis, two positive sense single-stranded RNA viruses have been used as models to understand the structure and function of viral-encoded proteins. One of them, Pepper Vein Banding Virus (PVBV; genus Potyvirus; family Potyviridae) is a flexuous, rod-shaped virus that encodes for a polyprotein of size ~340 kDa. The polyprotein undergoes proteolytic processing by viral-encoded proteases, of which Nuclear Inclusion-a Protease (NIa-Pro) is the major protease. It is a serine-like cysteine protease which cleaves between a Q/A or Q/S, present in the context of the heptapeptide recognition sequence. The temporal regulation of intermediates and mature proteins released by NIa-Pro cleavage is crucial for a successful infection. In the present study, histidine-tagged NIa-Pro, Viral Protein genome-linked (VPg), and the cleavage site mutant (E191A) VPg-Pro were over-expressed in E. coli and purified. The protease activity of NIa-Pro was monitored using an HPLC-based protease assay developed using a peptide substrate. NIa-Pro protease activity was found to get modulated upon interaction with VPg and upon undergoing phosphorylation. Both these events have been found to involve the face of NIa-Pro which contains the solvent-exposed Trp143. Mutational studies and molecular dynamics analyses provide evidence that this residue is buried upon interaction of NIa-Pro with VPg, and any perturbation of its orientation influences the active site Cys151 via an extensive interaction network. This interaction was found to enhance the velocity of NIa-Pro protease activity, especially if the two domains were present in trans (VPg+Pro). In addition, the main-chain –NH2 group of Trp143 was found to be hydrogen-bonded to the side chain –OH group of Ser129, the residue which was identified to undergo phosphorylation by host plant kinases. Interestingly, when the two domains were present in cis (E191A VPg-Pro), no phosphorylation was observed. Mutations of Ser129 (to phosphorylation-mimic Asp or phosphorylation-deficient Ala residues) which affected this H-bond were found to disturb Trp143 and Cys151 orientation, which drastically reduced the protease activity of NIa-Pro. Within the polyprotein, VPg is present at the N-terminus of NIa-Pro and the cleavage site between them is suboptimal (E/A). In the present study, VPg-Pro was shown to be covalently linked to the genomic RNA present in the virions. Interestingly, during purification, VPg could only be purified from the soluble when it was expressed at the N-terminus of NIa-Pro. A series of bioinformatics and biophysical analysis of VPg showed that PVBV VPg, like other potyviral VPgs, exists as a molten-globule. Moreover, while VPg was shown to harbour the Walker motifs, it was found to exhibit an ATPase activity only when it was present with the NIa-Pro (especially in cis). Lys47 and Asp88:Glu89 were found crucial for optimal activity. Over all the results demonstrated that there is a reciprocal modulation of structure and function of the VPg and NIa-Pro domains. These results can explain the possible significance of an impeded cleavage rate between the two domains of VPg-Pro during PVBV infection. The precursor, VPg-Pro, could offer the advantage of evading the inhibitory phosphorylation of NIa-Pro by the host, as well as drive certain viral processes by virtue of its ATPase activity. And subsequent cleavage of the domains and their trans interaction could offer a higher turnover rate which might assist sufficient CP production required for viral morphogenesis. Another virus, Tobacco Streak Virus (TSV) that belongs to the Ilarvirus genus of the Bromoviridae family is a spherical virus which forms pleiomorphic icosahedral virus particles. It has a tripartite genome and each RNA is encapsidated individually. In the present thesis, TSV was used as a model to understand the properties of its structural protein-the coat protein (CP), with the aim of deciphering TSV assembly process. Thus, the CP gene from TSV RNA 3 was cloned and over-expressed in E. coli. The coat protein thus expressed formed virus-like particles (VLPs), which could be disassembled into dimers using high CaCl2 concentrations. Reassembly of VLPs was possible from dimers even in the absence of any nucleic acid. Mutational analysis of the N-terminal disordered domain showed that 26 amino acid residues from the amino-terminus could be crucial for capsid heterogeneity while, zinc-binding domain was essential for assembly. Overall, the present study shows that the flexible W-C loop of PVBV NIa-Pro, the disordered N-terminal region of PVBV VPg and the disordered N-terminal region of TSV CP harbour residues crucial for regulation of protein function. Such regulatory elements would ultimately allow viruses to maintain a smaller protein number, and thus a smaller genome size.