Isolation of large quantities of functional, cytoplasm-free Xenopus laevis oocyte nuclei.
A procedure is described that facilitates the isolation of large quantities of nuclei (germinal vesicles or GVs) from late stage Xenopus laevis oocytes. The method was developed by modifying techniques described in two published reports (F. Scalenghe et al. (1978) Chromosoma 66, 299-308; I. Ruberti et al. (1989) Anal. Biochem. 180, 177-180). Methods were chosen which optimize yields of GVs and homologous recombination activity. This procedure yields up to 90% of the total GVs present in 70 ml of oocytes. Proteins in the extract made from these GVs are predominantly of nuclear origin; little cytoplasmic contamination is detected as measured by alpha-tubulin content. The extract catalyzes the complete recombination of linear, terminally homologous DNA, as observed in injected oocytes. The extract also performs other nuclear processes including transcription, repair-type DNA synthesis, chromatin assembly, NDP kinase reactions, and dsRNA deamination. Because this procedure greatly increases the yield of GVs over manual isolation protocols, and the resulting extract is capable of carrying out a variety of typical nuclear processes, it should expedite the purification of components found in oocyte GVs, including proteins required for homologous recombination.
Lehman
C W
CW
Department of Biochemistry, University of Utah School of Medicine, Salt Lake City 84132.
Carroll
D
D
D000818:Animals; D002457:Cell Extracts / Q000235:genetics / Q000302:isolation & purification; D002467:Cell Nucleus / Q000502:physiology*; D002843:Chromatin / Q000378:metabolism / Q000502:physiology; D003593:Cytoplasm / Q000737:chemistry; D004247:DNA / Q000096:biosynthesis; D003641:Deamination; D005260:Female; D009687:Nuclear Proteins / Q000302:isolation & purification; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism; D009865:Oocytes / Q000648:ultrastructure*; D012330:RNA, Double-Stranded / Q000378:metabolism; D011995:Recombination, Genetic / Q000502:physiology; D014158:Transcription, Genetic / Q000502:physiology; D014982:Xenopus laevis
D002457:Cell Extracts; D002843:Chromatin; D009687:Nuclear Proteins; D012330:RNA, Double-Stranded; D004247:DNA; D009701:Nucleoside-Diphosphate Kinase