Preparation of clam oocyte extracts for cell cycle studies

Publisher Summary This chapter discusses the preparation of clam oocyte extracts for cell cycle studies. It was in clam oocytes that cyclins were first identified, cloned, and sequenced, the biological activity of cyclins was first demonstrated, the molecular role of cyclins as positive regulatory subunits of the cell cycle regulatory kinase cdc2 was first demonstrated, and components of the cyclin destruction machinery were first identified. Concentrated homogenates of clam oocytes are translationally active, reproduce faithfully at least some events of the cell cycle, and can be frozen, transported on dry ice, and stored at-80° for at least 5 years without loss of activity. Because of these advantages, clam extracts are excellent for biochemical fractionation and purification of components of the cell cycle regulatory machinery. Clam oocyte extracts are used for the fractionation of the system responsible for cell cycle stage-specific degradation of cyclin B, and for the isolation of several of its components: a cyclin-selective ubiquitin carrier protein E2-C, the cyclosome-APC complex containing a cell cycle-regulated ubiquitin ligase activity, and an okadaic acid-sensitive phosphatase that dephosphorylates and inactivates the cyclosome.