Xenopus oocytes extract preparation

In addition, we present methods for the preparation of HeLa cell extracts that can support import in vitro. In general, we find for both proteins and snRNPs that the Xenopus egg extract is the most efficient in terms of the speed of nuclear import and the final level of intranuclear accumulation. The Xenopus oocyte extract, HeLa cell extract, and reticulocyte lysates are generally less efficient in our hands. We have not been able to reconstitute nuclear import using extracts from Drosophila early embryos (C. Dingwall, unpublished). 

The Xenopus system has proven instrumental in determining the mechanism controlling exit from mitosis at the metaphase/anaphase transition. Studies in this area have relied heavily on extracts prepared from fully mature oocytes/ unfertilized eggs that are arrested at metaphase of the second meiotic division. Upon Ca2+ addition, anaphase is initiated and the extract enters the first embryonic cell cycle to replicate DNA. The activity responsible for metaphase arrest was discovered by Masui at the same time as MPF (Masui Markert 1971), and given the name cytostatic factor (CSF). CSF has never been purified... 

A variety of extract preparations have been used to support protein and snRNP import in vitro (Adam et ai, 1991 Moore and Blobel, 1992 Newmeyer and Wilson, 1991 Marshallsay and Luhrmann, 1994 Palacios et al, 1996). The most commonly used sources are Xenopus oocytes and eggs and rabbit reticulocyte lysate. The biochemical fractionation of these extracts led to the identification of the essential soluble protein factors for nuclear protein import (see Section I). Detailed protocols for the surgical removal of ovary from Xenopus and the induction of egg laying have been provided in a previous volume in this series (Kay and Peng, 1991). Here we present specific modifications of the basic protocols to give optimal nuclear protein and snRNP import in vitro. 

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