Molecular Clock Mechanisms/Gene-Protein Networks

The Molecular Clock Mechanisms/Gene-Protein Networks Cluster draws heavily upon the techniques of molecular biology and biochemistry to study the molecular building blocks of biological clocks. Such gene-based research has increasingly permitted the dissection of key components of the biological clock and provide a means for comparison of features of the biological clock across species lines. Current research in this cluster falls principally in three categories: (i) Comparative molecular mechanisms, (ii) Cell signaling/clock control of transcription, and (iii) gene networks.

Comparative molecular mechanisms

Investigators Golden, Kay, Evans, and Brody are exploring the basic architecture of oscillators through the exploitation of genetic techniques as a way of dissecting the complex biological phenomena of the biological clock. Using single gene mutants and genomics, CCB investigators endeavor to advance the description of the genes and proteins which are involved directly or indirectly in the core oscillator mechanism of the biological clock. These studies build upon knowledge about existing mutants by analyzing genetic interactions between mutants in multiple genes relevant to the biological clock. By comparing the findings from these investigations of key mutations in key biological clock genes across many different organisms, these studies should allow us to understand the basic architecture of oscillators across species lines.

Cell signaling/clock control of transcription

The functioning and importance of the clock mechanism can be explored at the molecular level through the paradigm of input functions and output functions. Analysis of input functions for biological clocks depends on an elucidation of certain receptors for cell signaling which prompts a process influencing the regulation of genes essential to the functioning of the clock. Such cell signaling of interest to chronobiological systems are both internal and external in nature. Meanwhile, the output functions in this paradigm are the so called clock-controlled genes whose control by the clock is driven in part by receptor-mediated cell signaling that is dependent upon these internal and external signals. The regulation of the clock-controlled genes provides a window on the complexity of the clockworks in that the clock-controlled genes are subject to regulation by the clock at all levels: transcriptional, post-transcriptional, translational, and post-translational. The CCB is actively pursuing all aspects of inquiry into clock-controlled genes, and comparison of such molecular level outputs of clock function across various species is yielding important new insights into the fundamental mechanisms of clock function. For example, the analysis of the roles of the cAMP and CREB signaling in the oscillator mechanism in addition to downstream (output) signaling is expected to break new ground in our understanding clock control of cellular physiology.

Gene Networks

The circadian clock can be understood at the single cell level to be essentially an oscillating gene network with the rhythmic expression of multiple clock-related genes collectively manifesting the oscillator function. One approach to studying a complex network such as the oscillating gene network of a biological clock is to perturb the function of one of the genes experimentally, and to test the effects on the network’s oscillatory function. Breakthroughs in the development of new tools which permit the use of luciferase and fluorescent reporters of clock gene expression have caused explosive growth in the monitoring of clock function in cells from a variety of different organisms such as the bacterium Synechococcus (Golden), the fungus Neurospora (Brody), the fruit fly Drosophila (Kay), plants (Kay, Chory), and mice (Welsh). For example, cells from mutant mice in which specific clock genes have been altered or deleted (Welsh, Kay) have revealed cellular phenotypes of clock function that are qualitatively different from the behavioral phenotypes determined previously at the whole animal level. Thus, these studies of the influence of the function of a single gene in the gene network permits a finer level of dissection of the functioning of the biological clock and a deeper understanding of the contributions of its components. In some efforts at CCB, new cell-level data are serving as the basis for revised computational models of the intracellular circadian clock, which notably display similarities with properties of other oscillatory gene networks such as delayed negative feedback loops and/or positive feedback (Hasty). The CCB is driving new collaborations between experimentalists and modelers at UCSD to improve our understanding of cellular circadian clock mechanisms.

Increasingly, it is becoming possible to explore the oscillatory gene network of the biological clock through the synthetic construction of a network from defined, understood components. Applying synthetic approach, simple oscillators with periods of a few hours have been constructed in bacteria (Hasty). Using these simplified model systems, investigators at CCB are able to test specific hypotheses about the importance of various network features for the oscillatory function of the network. These reduced model systems then lend further insight into the oscillatory networks comprising the biological clock. For example, synthetic studies in oscillatory gene networks have already demonstrated that the time delay in a negative feedback loop is a critical design feature which can be expected to be an important part of the clockworks for many biological clocks. Similarly, exploration in model systems has yielded the insight that the addition of positive feedback improves precision and tunability in an oscillatory gene network (Hasty). The CCB is further driving investigation of the biological clock through construction of synthetic oscillatory networks with collaborations between circadian biologists, biophysicists (Hwa), and synthetic biologists.