created: 10th September 1997, last updated: 10th September 1997,© 1997 ABRF
Elizabeth Fowler AutoImmune, Inc.
Dissecting Genetic Networks was the topic of the ABRF sponsored symposium held July 12 in Boston in connection with the 1997 Protein Society meeting. This symposium, organized by Paul Matsudaira (Whitehead Institute), focused on emerging techniques for investigating the functions of proteins discovered as genome sequencing progresses. Much of the required information can be produced, systematized, automated, and performed by core facilities.
Paul Matsudaira characterized ABRF members as the "Go To" people, the people approached when sophisticated analysis of biomolecules is needed. One purpose of this symposium was to point to a future direction for core facilities where they generate information needed to understand the interactions of proteins in functional networks. Matusdaira compared the assembly of information resulting from the genome project to the development of the periodic table of chemical elements. The periodic table organized the elements, predicted new elements, enabled chemical syntheses, and fostered development of new technologies and disciplines. A table of expressed genes that includes information not only on the primary structure, but also on higher order structure, control of expression, interactions with other cell constituents, and biological functions would lead to new ways of dealing with pathologies, new techniques in bio-engineering, and unforeseen new technologies. Obtaining this information will drive the development of new methods, including high-throughput means of identifying interacting proteins and techniques for functionally analyzing genetic networks. These topics were discussed by Roger Brent and Adam Arkin.
Roger Brent (Harvard Medical School) emphasized that predicting how a complex biological system will act under a particular circumstance involves careful modeling based on precise knowledge. Obtaining the information is an iterative process, which involves cycles of data production, analysis, and formulation of testable hypotheses. Producing certain kinds of information can be systematized and conducted by facilities. Becoming involved in these efforts is a potential direction for ABRF members. The approach that Brent discussed under the topic "Charting and Exploiting Genetic Networks" is one that could be implemented in a core facility.
The objective of Brent's work is to identify components interacting in a pathway. He uses a general transcription-based selection for protein-protein interactions termed the "interaction trap". His system (1) uses three components: (a) a transcriptionally inert fusion protein consisting of a lexA DNA binding domain and the test protein for which interacting proteins are sought, (b) two reporter genes under the lexA operator, and (c) an expression library in which the library proteins are fused to an activation domain. If a library protein interacts with the test protein, the activation domain is brought close enough to the lexA DNA binding domain to enable transcription of the reporter genes. In one example (2), the test protein was the cyclin-dependent kinase Cdk2. The protein was fused to the lexA DNA binding domain. The construct was expressed in the selection strain, a yeast strain containing the Leu2 and b-galactosidase genes both under control of the lexA operator. A combinatorial library encoding constrained 20-residue peptides (aptamers) was placed in the active site loop of E. coli thioredoxin fused to the lexA activation peptide. This library was introduced into the selection strain. If the expressed peptide aptamer interacted with Cdk2, the yeast grew without leucine and contained b-galactosidase activity. From 6 x 106 transformants, 14 plasmids were isolated that contained peptide aptamers that interacted with Cdk2 but not with control proteins. Binding studies with purified peptide aptamers and Cdk2 showed that these interactions were specific with dissociation constants of between 30 and 120 nM. Furthermore, these aptamers inhibited phosphorylation of histone H1 by Cdk2/cyclin E kinase. Thus, this selection system offers an approach to dissecting protein interaction networks.
Adam Arkin (Stanford University) described the modeling of cellular regulatory networks, including stochastic or random events. In networks where the protein product of one gene regulates the expression of another gene, there is a time delay between the activation of the first promoter and the second. During this time, the first protein accumulates to a level sufficient to activate the second gene, and this delay provides information about the expression level of the first protein. The experimental data describing the induction of the lacZ gene in E. coli or the lysogeny of phage lambda can be modeled by a stochastic process in which events in a single cell are influenced by random short bursts of variable numbers of protein molecules. This can result in large differences in the time between successive events in regulatory cascades across a cell population. The model shows that the random pattern of expression of competitive effectors can probabilistically produce different outcomes for individual cells that, in turn, can partition the cell population into different phenotypes. This type of model can explain, for example, the observation that lysogeny in phage lambda is never 100%.
The importance of understanding genetic regulatory networks is underscored by the fact that the Office of Naval Research (ONR) has recently started a new program in this area. Eric Eisenstadt, from the ONR, described this program briefly. The philosophy underlying the genetic network program is that understanding the fundamental structure and function of regulatory networks can lead to the development of new technologies such as neural networks, biologically inspired algorithms, and biologically manufactured devices.
As evidenced by all the talks during this interesting afternoon, sequencing the genome is a first step towards a number of exciting possibilities. Understanding the interactions among gene products is an even more ambitious task. The resulting information explosion will lead us in directions we have yet to envision. Core facilities, with their expertise in sophisticated methods for analyzing biological molecules, can play important roles in obtaining this information.
References
1. Gyuris, J., Golemis, E., Chertkov, H., and Brent, R. Cdi1, a Human G1 and S Phase Protein Phosphatase that Associates with Cdk2. Cell 75, 791-803, 1993.
2. Colas, P, Cohen, B., Jessen, T., Grishina, I., McCoy, J., and Brent, R., Genetic Selection of Peptide Aptimers that Recognize and Inhibit Cyclin-dependent Kinase 2. Nature 380, 548-550, 1996.
Elizabeth Fowler may be contacted at AutoImmune, Inc.,128 Spring St., Lexington, MA 02173, Tel: (617) 860-0710Fax: (617) 860-0705, E-mail: efowler@ultranet.com
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