Site-specific chromosomal integration of large synthetic constructs

We have developed an effective, easy-to-use two-step system for the site-directed insertion of large genetic constructs into arbitrary positions in the Escherichia coli chromosome. The system uses λ-Red mediated recombineering accompanied by the introduction of double-strand DNA breaks in the chromosome and a donor plasmid bearing the desired insertion fragment. Our method, in contrast to existing recombineering or phage-derived insertion methods, allows for the insertion of very large fragments into any desired location and in any orientation. We demonstrate this method by inserting a 7-kb fragment consisting of a venus-tagged lac repressor gene along with a target lacZ reporter into six unique sites distributed symmetrically about the chromosome. We also demonstrate the universality and repeatability of the method by separately inserting the lac repressor gene and the lacZ target into the chromosome at separate locations around the chromosome via repeated application of the protocol

Quantitative Characteristics of Gene Regulation by Small RNA

An increasing number of small RNAs (sRNAs) have been shown to regulate critical pathways in prokaryotes and eukaryotes. In bacteria, regulation by trans-encoded sRNAs is predominantly found in the coordination of intricate stress responses. The mechanisms by which sRNAs modulate expression of its targets are diverse. In common to most is the possibility that interference with the translation of mRNA targets may also alter the abundance of functional sRNAs. Aiming to understand the unique role played by sRNAs in gene regulation, we studied examples from two distinct classes of bacterial sRNAs in Escherichia coli using a quantitative approach combining experiment and theory. Our results demonstrate that sRNA provides a novel mode of gene regulation, with characteristics distinct from those of protein-mediated gene regulation. These include a threshold-linear response with a tunable threshold, a robust noise resistance characteristic, and a built-in capability for hierarchical cross-talk. Knowledge of these special features of sRNA-mediated regulation may be crucial toward understanding the subtle functions that sRNAs can play in coordinating various stress-relief pathways. Our results may also help guide the design of synthetic genetic circuits that have properties difficult to attain with protein regulators alone

The Future of Affirmative Action: The Legal Imperative Nationally and the Ohio Experience

This presentation of the legal future of affirmative action will be divided into five sections. The introductory material serves as a general introduction to the issues. The second section will review the origins and evolution of affirmative action. This section will also attempt to provide a definition of this complex concept. The third will provide a detailed analysis of Regents of the University of California v. Bakke. Bakke represents the Supreme Court\u27s first attempt to resolve the legal complexities of affirmative action. The Court\u27s holdings in Bakke have shaped the debate for over 20 years. The fourth segment of this chapter will review current affirmative action case law. Close attention will be paid to emerging legal distinctions essential to an accurate application of affirmative action principles in the future. Within this section, close attention will be paid to current controversies such as California Proposition 209 (the state constitutional amendment which outlaws the use of affirmative action), Hopwood v. State of Texas (nonminority students who challenged a law school\u27s affirmative action admissions program), and Taxman v. Board of Education of the Township of Piscataway (a teacher\u27s challenge to a school board\u27s affirmative action plan of retaining minority teachers over nonminority teachers in regard to layoff decisions). The last section will explain the Ohio Experience with affirmative action

Transcription by the numbers redux: experiments and calculations that surprise

The study of transcription has witnessed an explosion of quantitative effort both experimentally and theoretically. In this article we highlight some of the exciting recent experimental efforts in the study of transcription with an eye to the demands that such experiments put on theoretical models of transcription. From a modeling perspective, we focus on two broad classes of models: the so-called thermodynamic models that use statistical mechanics to reckon the level of gene expression as probabilities of promoter occupancy, and rate-equation treatments that focus on the temporal evolution of the activity of a given promoter and that make it possible to compute the distributions of messenger RNA and proteins. We consider several appealing case studies to illustrate how quantitative models have been used to dissect transcriptional regulation