Using competition assays to quantitatively model cooperative binding by transcription factors and other ligands.

BACKGROUND: The affinities of DNA binding proteins for target sites can be used to model the regulation of gene expression. These proteins can bind to DNA cooperatively, strongly impacting their affinity and specificity. However, current methods for measuring cooperativity do not provide the means to accurately predict binding behavior over a wide range of concentrations. METHODS: We use standard computational and mathematical methods, and develop novel methods as described in Results. RESULTS: We explore some complexities of cooperative binding, and develop an improved method for relating in vitro measurements to in vivo function, based on ternary complex formation. We derive expressions for the equilibria among the various complexes, and explore the limitations of binding experiments that model the system using a single parameter. We describe how to use single-ligand binding and ternary complex formation in tandem to determine parameters that have thermodynamic relevance. We develop an improved method for finding both single-ligand dissociation constants and concentrations simultaneously. We show how the cooperativity factor can be found when only one of the single-ligand dissociation constants can be measured. CONCLUSIONS: The methods that we develop constitute an optimized approach to accurately model cooperative binding. GENERAL SIGNIFICANCE: The expressions and methods we develop for modeling and analyzing DNA binding and cooperativity are applicable to most cases where multiple ligands bind to distinct sites on a common substrate. The parameters determined using these methods can be fed into models of higher-order cooperativity to increase their predictive power

Kinetic control of eukaryotic chromatin structure by recursive topological restraints

Chromatin structure undergoes many changes during the cell cycle and in response to regulatory events. A basic unit of chromatin organization is the nucleosome core particle. However, very little is known about how nucleosomes are arranged into higher-order structures in vivo, even though the efficiency and precision of cell division imply high levels of structural organization. We propose abandoning the current paradigm of chromatin organization based on thermodynamics of the lowest energy state and replace it with the idea of a topologically restrained, high-energy structure. We propose that DNA is subject to a recursive topological restraint, and is anchored by hemicatenates that are part of the chromosomal scaffold. Long-distance _cis_-regulation of transcription is a natural consequence of recursive topological restraint. This new theory of chromatin structure has a multitude of consequences for key aspects of cellular biology

Capturing the ‘ome’ : the expanding molecular toolbox for RNA and DNA library construction

All sequencing experiments and most functional genomics screens rely on the generation of libraries to comprehensively capture pools of targeted sequences. In the past decade especially, driven by the progress in the field of massively parallel sequencing, numerous studies have comprehensively assessed the impact of particular manipulations on library complexity and quality, and characterized the activities and specificities of several key enzymes used in library construction. Fortunately, careful protocol design and reagent choice can substantially mitigate many of these biases, and enable reliable representation of sequences in libraries. This review aims to guide the reader through the vast expanse of literature on the subject to promote informed library generation, independent of the application

Spartan Daily, April 1, 1986

Volume 86, Issue 39https://scholarworks.sjsu.edu/spartandaily/7428/thumbnail.jp

Spartan Daily, November 4, 1986

Volume 87, Issue 48https://scholarworks.sjsu.edu/spartandaily/7505/thumbnail.jp

Error Detection and Recovery in Software Development

Software rarely works as intended when it is first written. Software engineering research has long been concerned with assessing why software fails and who is to blame, or why a piece of software is flawed and how to prevent such faults in the future. Errors are examined in the context of bugs, elements of source code that produce undesirable, unexpected and unintended deviations in behaviour. Though error is a prevalent, mature topic within software engineering, error detection and recovery are less well understood. This research uses rich qualitative methods to study error detection and recovery in professional software development practice. It has considered conceptual representations of error in software engineering research and trade literature. Using ethnographic principles, it has gathered accounts given by professional developers in interviews and in video-recorded paired interaction. Developers performing a range of tasks were observed, and findings were compared to theories of human error formed in psychology and safety science. Three empirical studies investigated error from the perspective of developers, recon- structing the view they hold when errors arise, to build a catalogue of active encounters with error in conceptual design, at the desk and after the fact. Analyses were structured to consider development holistically over time, rather than in terms of discrete tasks. By placing emphasis on “local rationality”, analytical focus was redirected from outcomes toward factors that influence performance. The resultant observations are assembled in an account of error handling in software development as personal and situated (in time and the developer’s environment), with implications for the changing nature of expertise

Spartan Daily, October 14, 1986

Volume 87, Issue 33https://scholarworks.sjsu.edu/spartandaily/7490/thumbnail.jp