Purification of Xenopus egg nucleoplasmin and its use in chromatin assembly in vitro

Sealy, L., Burgess, R. R., Cotten, M. and Chalkley, R. (1989) Purification of Xenopus egg nucleoplasmin and its use in chromatin assembly in vitro. Methods in Enzymology, 170, pp. 612-630. (doi: 10.1016/0076-6879(89)70068-9) (PMID:2549340)

Abstract

The purification of trans-acting regulatory proteins has been studied extensively. These agents modulate the transcriptional activity of genes, though their mechanism of action is not understood. As a consequence, there has been renewed interest in reassembling chromatin containing these factors to assay the interrelationship between structure and function in vitro. However, the Xenopus laevis frog oocyte stores large quantities of histone for assembly after fertilization. These histones are associated with two karyophilic proteins, NI/N2 and nucleoplasmin. Although whole oocyte homogenates are capable of directing chromatin assembly, the many uncontrolled variables so introduced have encouraged the purification of individual factors such as nucleoplasmin. Nucleoplasmin obtained from unfertilized eggs, rather than oocytes, is far superior in promoting the formation of nucleosomes onto DNA in vitro, owing to massive additional phosphorylation of the protein. Details of the purification of nucleoplasmin from Xenopus laevis frog eggs and the use of this protein as an assembly agent are presented in this chapter.

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Cotten, Professor Matthew
• Authors: Sealy, L., Burgess, R. R., Cotten, M., and Chalkley, R.
• College/School: College of Medical Veterinary and Life Sciences > School of Infection & Immunity; College of Medical Veterinary and Life Sciences > School of Infection & Immunity > Centre for Virus Research
• Journal Name: Methods in Enzymology
• Publisher: Academic Press
• ISSN: 0076-6879
• ISSN (Online): 1557-7988