Identification of the dominant translation start site in the attB1 sequence of the pET-DEST42 Gateway vector

Gateway technology is a powerful system for converting a single entry vector into a wide variety of expression vectors. We expressed recombinant influenza matrix protein M1 (FMP), a potent antigen for cytotoxic T cells, using the Gateway vector pET-DEST42 containing the FMP cDNA, and purified the expressed FMP as a single 32 kDa recombinant protein. N-terminal and internal protein sequencing, however, showed that the recombinant FMP contained an extra 10 amino acids fused to the N-terminal of native FMP. Further investigation of the DNA sequence adjacent to the 5'-FMP cDNA indicated that the TTG in the attB1 site (30bp upstream of the ATG in the 5'-FMP cDNA) behaved as a dominant translation start site, resulting in a 10 amino acid extension of the recombinant FMP. Thus, it is possible that recombinant proteins produced by this Gateway vector contain unexpected vector-derived peptides, which may affect experimental outcomes. (c) 2006 Elsevier Inc. All rights reserved.

Translation of the flavivirus Kunjin NS3 gene in cis but not its RNA sequence or secondary structure is essential for efficient RNA packaging

Our previous studies using trans-complementation analysis of Kunjin virus (KUN) full-length cDNA clones harboring in-frame deletions in the NS3 gene demonstrated the inability of these defective complemented RNAs to be packaged into virus particles (W. J. Liu, P. L. Sedlak, N. Kondratieva, and A. A. Khromykh, J. Virol. 76:10766-10775). In this study we aimed to establish whether this requirement for NS3 in RNA packaging is determined by the secondary RNA structure of the NS3 gene or by the essential role of the translated NS3 gene product. Multiple silent mutations of three computer-predicted stable RNA structures in the NS3 coding region of KUN replicon RNA aimed at disrupting RNA secondary structure without affecting amino acid sequence did not affect RNA replication and packaging into virus-like particles in the packaging cell line, thus demonstrating that the predicted conserved RNA structures in the NS3 gene do not play a role in RNA replication and/or packaging. In contrast, double frameshift mutations in the NS3 coding region of full-length KUN RNA, producing scrambled NS3 protein but retaining secondary RNA structure, resulted in the loss of ability of these defective RNAs to be packaged into virus particles in complementation experiments in KUN replicon-expressing cells. Furthermore, the more robust complementation-packaging system based on established stable cell lines producing large amounts of complemented replicating NS3-deficient replicon RNAs and infection with KUN virus to provide structural proteins also failed to detect any secreted virus-like particles containing packaged NS3-deficient replicon RNAs. These results have now firmly established the requirement of KUN NS3 protein translated in cis for genome packaging into virus particles.

Maternal transmission efficiency of Wolbachia superinfections in Aedes albopictus populations in Thailand

We examined the transmission efficiency of 2 strains of Wolbachia bacteria that cause cytoplasmic incompatibility in field populations of Aedes albopictus by polymerase chain reaction assay. We found mainland and island populations throughout Thailand to be superinfected with group A and B bacteria. Of 320 Wolbachia-positive adult mosquitoes, 97.5% were infected with both groups. Single infected individuals of each Wolbachia group were encountered in nearly equal numbers. We screened 550 offspring from 80 field-collected mothers and found the transmission efficiency of group A Wolbachia to be 96.7% and that of group B Wolbachia to be 99.6%. Mothers that did not transmit both Wolbachia infections to all of their offspring were significantly larger in size than those with perfect transmission fidelity. We discuss our findings in relation to the prospects of the use of Wolbachia as a gene-driving mechanism.

Review of 'The Thai Village Economy in the Past'

This charmingly old-fashioned little book was first published in Thai in 1984, and now appears in an elegant English translation. The two major intellectual influences that gave it birth are rather older, dating from the 1950s, 1960s, and early 1970s. The first developed from the intersection of the academic pre-eminence of varieties of Marxist thinking about the Third World and the struggles of anticolonial peasant-based revolutionaries and produced a high age of romanticism about the Southeast Asian village and the unfortunate victims who inhabited them. The origins of the second are more uncertain, but probably represent, paradoxically, a Thai appropriation of those Western social-science constructions of Thai cultural uniqueness which were especially popular in the 1950s and 1960s.

Bringing the state to England: Andrew Tooke's translation of Samuel Pufendorf's 'De officio hominis et civis'

Andrew Tooke's 1691 English translation of Samuel Pufendorf's De officio hominis et civis, published as The Whole Duty of Man According to the Law of Nature, brought Pufendorf's manual fo statist natural law into English politics at a moment of temporary equilibrium in the unfinished contest between Crown and Parliament for the rights and powers of sovereignty. Drawing on the authors' re-edition of The Whole Duty of Man, this article describes and analyses a telling instance of how--by translation--the core political terms and concepts of the German natural jurist's 'absolutist' formulary were reshaped for reception in the different political culture of late seventeenth-century England.