CHARACTERIZING REGULATORY REGIONS OF CYCLIN D IN STRONGYLOCENTROTUS PURPURATUS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The cyclin D gene is an important regulator of cell division and differentiation. In this project, "cis-regulatory" DNA regions that control when and where cyclin D is activated during embryonic development of the purple sea urchin Strongylocentrotus purpuratus are being analyzed to determine their specific roles. Sea urchins and vertebrates share a common ancestor, so knowledge gained from this study can be used to understand aspects of human biology. In humans, cyclin D genes are mis-regulated in certain cancers and neurodegenerative diseases, so this research has health implications. In the current project, potential cis-regulatory regions have been identified computationally based on their similarity to regions in the cyclin D gene of the sea urchin Lytechinus variegatus, as well as by various additional criteria. These candidate cis-regulatory regions were incorporated individually into a "reporter" transgene encoding green fluorescent protein (GFP), for microinjection into fertilized sea urchin eggs. As the injected embryos develop, GFP expression can be studied by microscopy to determine where in the embryo the cis-regulatory region of interest exerts its action, and quantitatively, by a technique termed Real-Time PCR. From these methods, we have isolated two regions that regulate the activation of the cyclin D gene in the purple sea urchin. These regions are being broken down further, by mutating subregions hypothesized to be required for regulatory function, then re-examining the abilities of the altered regions to induce activation of GFP.