Chapter 17 Systems for the Study of Nuclear Assembly, DNA Replication, and Nuclear Breakdown in Xenopus laevis Egg Extracts

Abstract

Publisher Summary This chapter describes the conditions for preparing cell-free extracts from Xenopus eggs, which allows the study of nuclear dynamics and has proved fruitful in elucidating the structural and functional requirements for nuclear reassembly, DNA replication, and nuclear disassembly. In the amphibian Xenopus laevis , egg fertilization is followed by a period of rapid and synchronous cell division, which occurs in the complete absence of transcription or of any increase in total mass of the egg. During oogenesis, the egg must synthesize and store a large reservoir of nuclear components for use during the rapid proliferation that occurs during early development. The existence of such a reservoir of nuclear components permits the development of systems for the study of nuclear dynamics in vitro . Xenopus sperm provides an abundant source of chromatin, which after removal of plasma and nuclear membranes, may be easily purified. The chapter diagrammatically presents the preparation of the S phase extract and membrane and cytosol fractions for nuclear assembly. The inhibition of DNA replication by aphidicolin prevents the subsequent activation of maturation promoting factor (MPF) and entry into mitosis, indicating the presence of a control system that monitors the replication state of DNA and regulates the activation of MPF.