Introduction of asymmetry in the naturally symmetric restriction endonuclease EcoRV to investigate intersubunit communication in the homodimeric protein.

Type II restriction endonucleases are dimers of two identical subunits that together form one binding site for the double-stranded DNA substrate. Cleavage within the palindromic recognition site occurs in the two strands of the duplex in a concerted manner, due to the action of two catalytic centers, one per subunit. To investigate how the two identical subunits of the restriction endonuclease EcoRV cooperate in binding and cleaving their substrate, heterodimeric versions of EcoRV with different amino acid substitutions in the two subunits were constructed. For this purpose, the ecorV gene was fused to the coding region for the glutathione-binding domain of the glutathione S-transferase and a His6-tag, respectively. Upon cotransformation of Escherichia coli cells with both gene fusions stable homo- and heterodimers of the EcoRV variants are produced, which can be separated and purified to homogeneity by affinity chromatography over Ni-nitrilotriacetic acid and glutathione columns. A steady-state kinetic analysis shows that the activity of a heterodimeric variant with one inactive catalytic center is decreased by 2-fold, demonstrating that the two catalytic centers operate independently from each other. In contrast, heterodimeric variants with a defect in one DNA-binding site have a 30- to 50-fold lower activity, indicating that the two subunits of EcoRV cooperate in the recognition of the palindromic DNA sequence. By combining a subunit with an inactive catalytic center with a subunit with a defect in the DNA-binding site, EcoRV heterodimers were produced that only nick DNA specifically within the EcoRV recognition sequence.

Communication in Cyberspace

This Article examines a problem in cybercrime law that is both persistent and pervasive. What counts as “communication” on the Internet? Defining the term is particularly important for crimes such as cyberstalking, cyberharassment, and cyberbullying, where most statutes require a showing that the alleged perpetrator “communicated” with the victim or impose a similar requirement through slightly different language. This Article takes up the important task of defining communication. As a foundation to our discussion, we provide the first comprehensive survey of state statutes and case law relating to cyberstalking, cyberharassment, and cyberbullying. We then examine the realities of the way people use the Internet to develop a definition of “communication” that reflects those realities. That is, we aim to provide effective tools by which prosecutors can address wrongful conduct without punishing innocuous behavior or chilling speech. We conclude by proposing a model statute that appropriately defines “communication.” We recommend that state legislatures adopt the statute or modify existing laws to match it in pertinent part and demonstrate how the statute would apply in a range of situations.

The Annual of scientific discovery: or, Year-book of facts in science and art.

1869

Drivers of populism - A four-country comparison of party communication in the run-up to the 2014 European Parliament elections

The aim of this paper is to analyze what different types of populist communication styles emerged during the 2014 EP election campaign and under which conditions political parties selected specific populist communication styles. To do this, a comparative quantitative content analysis of press releases in the run-up of the latest EP elections has been conducted for parties in France, Germany, Austria and Greece. The paper presents a definition of populism based on the contemporary academic discourse, which focuses on the transnational nature of the European political field. It is shown that populist party communication is more pronounced on the fringes of the political spectrum and in countries struggling with severe macroeconomic difficulties. Contrary to intuitive expectations, the perceived populist rhetoric of exclusivity in the context of the European sovereign debt crisis, which is identified as a central feature of right-wing populism, barely takes place within populist party communication.

Careers in science.

Mode of access: Internet.

A syllabus for Communication in business management /

Mimeographed.