One-step generation of error-prone PCR libraries using Gateway® technology

<p>Abstract</p> <p>Background</p> <p>Error-prone PCR (epPCR) libraries are one of the tools used in directed evolution. The Gateway^®technology allows constructing epPCR libraries virtually devoid of any background (<it>i.e</it>., of insert-free plasmid), but requires two steps: the BP and the LR reactions and the associated <it>E. coli </it>cell transformations and plasmid purifications.</p> <p>Results</p> <p>We describe a method for making epPCR libraries in Gateway^®plasmids using an LR reaction without intermediate BP reaction. We also describe a BP-free and LR-free sub-cloning method for in-frame transferring the coding sequence of selected clones from the plasmid used to screen the library to another one devoid of tag used for screening (such as the green fluorescent protein). We report preliminary results of a directed evolution program using this method.</p> <p>Conclusions</p> <p>The one-step method enables producing epPCR libraries of as high complexity and quality as does the regular, two-step, protocol for half the amount of work. In addition, it contributes to preserve the original complexity of the epPCR product.</p

MeDor: a metaserver for predicting protein disorder

<p>Abstract</p> <p>Background</p> <p>We have previously shown that using multiple prediction methods improves the accuracy of disorder predictions. It is, however, a time-consuming procedure, since individual outputs of multiple predictions have to be retrieved, compared to each other and a comprehensive view of the results can only be obtained through a manual, fastidious, non-automated procedure. We herein describe a new web metaserver, MeDor, which allows fast, simultaneous analysis of a query sequence by multiple predictors and provides a graphical interface with a unified view of the outputs.</p> <p>Results</p> <p>MeDor was developed in Java and is freely available and downloadable at: <url>http://www.vazymolo.org/MeDor/index.html</url>. Presently, MeDor provides a HCA plot and runs a secondary structure prediction, a prediction of signal peptides and transmembrane regions and a set of disorder predictions. MeDor also enables the user to customize the output and to retrieve the sequence of specific regions of interest.</p> <p>Conclusion</p> <p>As MeDor outputs can be printed, saved, commented and modified further on, this offers a dynamic support for the analysis of protein sequences that is instrumental for delineating domains amenable to structural and functional studies.</p

Structural Disorder within Henipavirus Nucleoprotein and Phosphoprotein: From Predictions to Experimental Assessment

Henipaviruses are newly emerged viruses within the Paramyxoviridae family. Their negative-strand RNA genome is packaged by the nucleoprotein (N) within α-helical nucleocapsid that recruits the polymerase complex made of the L protein and the phosphoprotein (P). To date structural data on Henipaviruses are scarce, and their N and P proteins have never been characterized so far. Using both computational and experimental approaches we herein show that Henipaviruses N and P proteins possess large intrinsically disordered regions. By combining several disorder prediction methods, we show that the N-terminal domain of P (PNT) and the C-terminal domain of N (NTAIL) are both mostly disordered, although they contain short order-prone segments. We then report the cloning, the bacterial expression, purification and characterization of Henipavirus PNT and NTAIL domains. By combining gel filtration, dynamic light scattering, circular dichroism and nuclear magnetic resonance, we show that both NTAIL and PNT belong to the premolten globule sub-family within the class of intrinsically disordered proteins. This study is the first reported experimental characterization of Henipavirus P and N proteins. The evidence that their respective N-terminal and C-terminal domains are highly disordered under native conditions is expected to be invaluable for future structural studies by helping to delineate N and P protein domains amenable to crystallization. In addition, following previous hints establishing a relationship between structural disorder and protein interactivity, the present results suggest that Henipavirus PNT and NTAIL domains could be involved in manifold protein-protein interactions

A estrutura temática e o fluxo de informação na tradução da língua japonesa para o vernáculo

O presente trabalho tem por objetivo apresentar a Estruturação Temática na tradução da língua japonesa para o vernáculo tendo por base o conto Kami de Yasunari Kawabata e sua tradução para a língua portuguesa O cabelo. A pesquisa é desenvolvida na perspectiva da Lingüística Sistémico-Funcional，de Halliday (1994, 2004), que credita à organização textual a principal responsabilidade pelo desenvolvimento da informação, e no conceito de ondas de informação e de periodicidade preconizadas por Martin e Rose (2003) para a análise do fluxo de informação que organiza o discurso, tanto no conto original quanto na sua tradução.The purpose of this article is to present how the Thematic Structure occurs in the translation of Kawabata Yasunari’s short story Kami from Japanese language to Portuguese (O cabelo). Drawing upon Halliday (1994,2004)，who ascribe to textual organization the main responsibility for information development and upon the concept of waves of information and periodicity of Martin &amp; Rose (2003), the flow of information was analyzed in order to find out how it organizes discourse in the original text and in its translation

Haiku in Brazil : Assimilation or a New Poetic Accomplishment?

サンパウロ大学, 2008年10月13日-15

OS PRIMÓRDIOS DO RELACIONAMENTO DE PORTUGAL COM O JAPÃO

Na virada do séc. XV para o séc. XVI, com a descoberta do Cabo da Boa Esperança por Vasco da Gama, o caminho para o Oriente abre-se, inevitavelmente, ao Ocidente

Split-GFP Reassembly Assay: Strengths and Caveats from a Multiparametric Analysis

The split-Green Fluorescent Protein (GFP) reassembly assay is a powerful approach to study protein-protein interactions (PPIs). In this assay, two proteins, respectively, fused to the first seven and the last four β-strands of GFP are co-expressed in E. coli where they can bind to each other, which reconstitutes the full-length GFP. Thus, the fluorescence of the bacteria co-expressing the two fusion proteins accounts for the interaction of the two proteins of interest. The first split-GFP reassembly assay was devised in the early 2000s in Regan's lab. During the last ten years, we have been extensively using this assay to study the interactions of an intrinsically disordered protein (IDP) with two globular partners. Over that period, in addition to accumulating molecular information on the specific interactions under study, we progressively modified the original technique and tested various parameters. In those previous studies, however, we focused on the mechanistic insights provided by the approach, rather than on the method itself. Since methodological aspects deserve attention and the best bipartite reporter to study PPIs involving IDPs remains to be identified, we herein focus on technical aspects. To this end, we first revisit our previous modifications of the original method and then investigate the impact of a panel of additional parameters. The present study unveiled a few critical parameters that deserve consideration to avoid pitfalls and obtain reliable results

Probing Structural Transitions in the Intrinsically Disordered C-Terminal Domain of the Measles Virus Nucleoprotein by Vibrational Spectroscopy of Cyanylated Cysteines

A proof-of-concept demonstration that our methodology (infrared spectroscopy and cyanylated cysteine) is useful for monitoring structural transitions in unstructure proteins. --author-supplied descriptio

A estrutura temática de um conto na tradução do japonês para o português: um enfoque sistêmico-funcional

A estrutura temática de uma oração é composta de Tema, o ponto de partida da mensagem, que dá proeminência a alguns elementos que a compõem, estabelecendo a base de interpretação para o restante da oração, o Rema. Pesquisadores mostram que a escolha do Tema gera implicações comunicativas em traduções literárias, interferindo na interpretação da mensagem. Buscando contribuir para o estudo dessa questão, este artigo compara um conto da literatura japonesa moderna e sua tradução para o português, para verificar a diferença na realização da estrutura temática nessas línguas, e a interferência dessa diferença na interpretação do conto. As análises, com o apoio da Linguística Sistêmico-Funcional, mostram que as diferenças na estruturação temática são motivadas por diferenças devidas à tipologia linguística, levando a diferenças de intepretação do texto.Thematic structure of a clause is composed of Theme, the starting point of the message that lends prominence to some elements that compose it, thus establishing the base to interpret the remainder of the clause, the Rheme. Researchers show that the choice of the Theme generate communicational implications in literary translation, interfering in the interpretation of the message. This thesis compares one short story taken from modern Japanese literature and its translation into Portuguese with the aim of analyzing the difference in the realization of the thematic structure in these languages, and the difference in the interpretation of the short story. The article is supported by the premises of Systemic-Functional Linguistics. The analyses show that differences in structure are driven by differences due to linguistic typology, and lead to differences in the text Interpretation