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Nuclear actin and histones from the myxomycete Physarum flavicomum
Abstract:
A method was established for the isolation and purification of nuclei in high yield from the microplasmodia of Physarum flavicomum. Purified nuclei was resistant to breakage by methods commonly employed for isolated plant and animal nuclei. Incubation of nuclei with 5 mM dithiothreitol at pH 9.2 was found to be the simplest and most effective method for breaking the nuclei. Several methods for the extraction of nuclear protein were compared. Incubation of nuclear lysates with either 2 M NaCl, with or without 5 M urea, or 1 M CaCl2 resulted in the extraction of nuclear actin together with histones. The histones were chemically fractionated into the five basic groups common to other eucaryotic tissue. Amino acid analyses of the total histone were also performed. Nuclear actin was found to have a molecular weight of 41,000 +/- 4,000 daltons as determined by SDS polyacrylamide gel electrophoresis. The amino acid composition of the nuclear actin was established.
Insights
Researchers developed a method to isolate and purify nuclei from Physarum flavicomum. They found dithiothreitol effectively breaks these nuclei, allowing for the extraction and analysis of nuclear proteins like histones and actin.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- The slime mold Physarum flavicomum presents unique challenges for nuclear isolation due to its robust nuclear structure.
- Understanding nuclear protein composition requires efficient methods for nuclei isolation and subsequent extraction.
Purpose of the Study:
- To establish a high-yield method for isolating and purifying nuclei from Physarum flavicomum microplasmodia.
- To determine an effective method for breaking these purified nuclei.
- To compare protein extraction methods and characterize nuclear actin and histones.
Main Methods:
- Isolation and purification of nuclei from Physarum flavicomum microplasmodia.
- Testing various methods for nuclei breakage, including dithiothreitol.
- Comparing protein extraction from nuclear lysates using different salt and urea concentrations.
- Chemical fractionation of histones and amino acid analysis.
- Determination of nuclear actin molecular weight and amino acid composition via SDS-PAGE.
Main Results:
- A high-yield nuclei isolation method was established for Physarum flavicomum.
- Dithiothreitol at pH 9.2 proved to be the most effective for breaking nuclei.
- Extraction with 2 M NaCl (with or without 5 M urea) or 1 M CaCl2 co-extracted nuclear actin and histones.
- Histones were fractionated into five basic groups, and their amino acid analyses were performed.
- Nuclear actin was identified with a molecular weight of 41,000 +/- 4,000 daltons and its amino acid composition was determined.
Conclusions:
- The developed method allows for efficient isolation and purification of Physarum flavicomum nuclei.
- Nuclear proteins, including histones and a distinct nuclear actin, can be successfully extracted and characterized.
- This study provides a foundation for further investigation into the molecular mechanisms of Physarum flavicomum nuclear proteins.