Skip to main content
NCBI home page
Plant Physiology logoLink to Plant Physiology
. 1982 Sep;70(3):704–708. doi: 10.1104/pp.70.3.704

Nuclear Buoyant Density Determination and the Purification and Characterization of Wild-Type Neurospora Nuclei Using Percoll Density Gradients 1

Katrin J Talbot 1, Peter J Russell 1
PMCID: PMC1065756  PMID: 16662561

Abstract

A procedure has been developed using Percoll density gradients for the isolation and purification of nuclei from germinated conidia of wild-type Neurospora crassa St. Lawrence strain 74A. Crude nuclei were purified isopycnically in gradients of Percoll, which is silica coated with polyvinylpyrrolidone. A DNA:RNA:protein ratio of 1:3.5:6.5 was found in purified nuclei. Cytoplasmic contamination was found to be negligible in the nuclear preparations, as determined by electron microscopy and by following a radioactively-labeled ribosome tag during the isolation procedure. A small amount of endogenous ribonuclease activity was detected in the crude nuclear preparations, but not in suspensions of nuclei purified in the Percoll gradients. Ribosomal RNA was extracted from the nuclei in good yields, and electrophoretic analysis indicated the presence of precursor rRNA molecules, as well as the mature 17S and 25S rRNA species. Using the Percoll gradient system, the buoyant density of purified Neurospora nuclei was determined to be 1.08 grams per milliliter based on refractive index measurements.

Full text

PDF
704

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  2. Hautala J. A., Conner B. H., Jacobson J. W., Patel G. L., Giles N. H. Isolation and characterization of nuclei from Neurospora crassa. J Bacteriol. 1977 May;130(2):704–713. doi: 10.1128/jb.130.2.704-713.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Johri M. M., Varner J. E. Enhancement of RNA synthesis in isolated pea nuclei by gibberellic acid. Proc Natl Acad Sci U S A. 1968 Jan;59(1):269–276. doi: 10.1073/pnas.59.1.269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Luthe D. S., Quatrano R. S. Transcription in Isolated Wheat Nuclei: I. ISOLATION OF NUCLEI AND ELIMINATION OF ENDOGENOUS RIBONUCLEASE ACTIVITY. Plant Physiol. 1980 Feb;65(2):305–308. doi: 10.1104/pp.65.2.305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. MASTER R. W. POSSIBLE SYNTHESIS OF POLYRIBONUCLEOTIDES OF KNOWN BASE-TRIPLET SEQUENCES. Nature. 1965 Apr 3;206:93–93. doi: 10.1038/206093b0. [DOI] [PubMed] [Google Scholar]
  6. Pertoft H., Laurent T. C., Lås T., Kågedal L. Density gradients prepared from colloidal silica particles coated by polyvinylpyrrolidone (Percoll). Anal Biochem. 1978 Jul 15;88(1):271–282. doi: 10.1016/0003-2697(78)90419-0. [DOI] [PubMed] [Google Scholar]
  7. Russell P. J., Hammett J. R., Selker E. U. Neurospora crassa cytoplasmic ribosomes; ribosomal ribonucleic acid synthesis in the wild type. J Bacteriol. 1976 Aug;127(2):785–793. doi: 10.1128/jb.127.2.785-793.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Schlitt S. C., Russell P. J. Neurospora crassa cytoplasmic ribosomes: isolation and characterization of a cold-sensitive mutant defective in ribosome biosynthesis. J Bacteriol. 1974 Nov;120(2):666–671. doi: 10.1128/jb.120.2.666-671.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Sturani E., Magnani F., Alberghina F. A. Inhibition of ribosomal RNA synthesis during a shift-down transition of growth in Neurospora crassa. Biochim Biophys Acta. 1973 Aug 24;319(2):153–164. doi: 10.1016/0005-2787(73)90006-3. [DOI] [PubMed] [Google Scholar]

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES