Dissection of the Cell Cycle Using Cell-Free Extracts From Xenopus Laevis

Ford, C. C. and Lindsay, H. (1995) Dissection of the Cell Cycle Using Cell-Free Extracts From Xenopus Laevis. Advances in Molecular and Cell Biology, 13 (C). pp. 181-217. ISSN 1569-2558

Abstract

In the ten years since Lohka and Masui developed extracts from amphibian eggs capable of nuclear assembly, DNA replication, and mitotic chromosome condensation, the use of these and derivative extracts has expanded prodigiously. The relative ease with which these extracts are made from naturally synchronous starting material has provided an exceptional approach to many aspects of the cell cycle. The methods used to generate these extracts are considered first, together with the features that contribute to nuclear construction. A correctly assembled nucleus is essential for S phase, which is the dominant interphase activity of early embryonic cell cycles. Having initiated S phase, mitosis can be delayed under particular circumstances, and it seems to us that the source of the signal delaying mitosis is the replication complexes themselves. Extracts for studying M phase can be prepared from unfertilized eggs or from interphase cells approaching mitosis, but always in the presence of a calcium-chelating agent. However, free calcium in extracts is present at physiological levels. A calcium-mediated event is required, not only in exit from meiotic metaphase, but also for inactivation of cdc2 kinase during mitosis.