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Research Introduction
We are studying the molecular mechanisms that control cell division during development, using the genetically tractable model organism, Drosophila (fruit fly). Our long-standing approach has been to identify putative new cell cycle regulatory proteins using yeast two-hybrid technologies, then to study the functions of the new proteins and their genes using a variety of molecular and genetic approaches. Initially we identified new cell cycle regulators by elaborating a network of interacting proteins centered on Cyclin-dependent kinases (Cdks). These highly conserved S/T protein kinases are well-known regulators of critical transitions in the cell division cycle of all eukaryotes. More recently, we have taken advantage of the data emerging from another project in our lab aimed at mapping protein interactions on a proteome-wide scale; the ultimate goal of that project is to construct a protein interaction map (PIM) depicting the binary interactions among most of the ~14,000 proteins encoded by the Drosophila genome.
One set of data is shown in the adjacent figure. This is a PIM generated by using ~100 known or suspected cell cycle regulators, including Cdk1 and Cdk2, in high throughput screens to detect possible interactions with ~14,000 Drosophila proteins. This type of interaction map can provide insights about regulatory pathways, which often consist of networks of interacting proteins. All of the gene and protein interaciton data can be accessed from the Drosophila Interactions Database.
Some members of our laboratory have chosen to study one or a few proteins in the interaction networks in more detail. One example is cyclin J. As can be seen in the PIM, we have found that cyclin J interacts with Cdk2 in yeast two-hybrid assays. We became particularly interested in cyclin J after finding that the protein is expressed only during the early embryonic divisions that lack defined gap phases between DNA synthesis and mitosis. We hypothesized that cyclin J may be a unique regulator that contributes to these rapid unusual divisions. We have found that cyclin J associates with active Cdk2 in early embryos and that disruption of this activity with antibodies or with peptide aptamers (see below), results in delay of the nuclear cycles. The figure shows an example of an embryo that has been injected (at the left) with a peptide aptamers that inhibits cyclin J-associated kinase activity. Notice that the nuclei near the site of injection have paused in the division cycle, whereas the nuclei in the right two thirds of the embryo have progressed through the cycle and divided - there are more of them. We are currently using genetic methods to further understand cyclin J function. Other projects in the laboratory are using similar approaches to study other proteins in the cell cycle PIM.
Protein networks provide a good starting point to study the functions of individual proteins and regulatory pathways. However, additional approaches are needed to verify interactions and to test the actual functions of particular interactions in vivo. We are addressing this need by using yeast two-hybrid methods to identify peptides that bind specifically to target proteins and disrupt their activity or interactions with other proteins. We then express these so-called peptide 'aptamers' in vivo and examine the resulting phenotypes. The figure (below) shows an example in which we expressed a peptide aptamer that targets a cell cycle regulator (Cdk2) needed for proper cell divisions in the Drosophila eye. The misshapen eye on the right resulted from expression of the aptamer, while the normal looking eye on the left expressed a control peptide.
Last modified: Friday, February 3, 2006 10:35:07 AM
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