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Flow cytometric studies of the nuclear matrix
W D Wright1, R Higashikubo, J L Roti Roti
1Washington University School of Medicine, Section of Cancer Biology, St. Louis, Missouri 63108.
Cytometry
|May 1, 1989
Summary
We developed a flow cytometry method to analyze nuclear matrix protein and nucleic acid content. Heat shock increases nuclear protein, which sequesters double-stranded RNA, protecting it from degradation.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- The nuclear matrix is a dynamic structure involved in DNA replication, transcription, and RNA processing.
- Understanding changes in nuclear matrix composition under stress is crucial for cell biology research.
Purpose of the Study:
- To establish a flow cytometry-based method for quantifying protein and nucleic acid content in nuclear matrices.
- To investigate alterations in nuclear matrix composition following heat shock.
Main Methods:
- Nuclear matrices were isolated from nuclei via DNase I digestion and high-salt extraction.
- Fluorescence-activated flow cytometry was used to measure protein (FITC) and double-stranded nucleic acid (PI) content.
- Matrices were subjected to enzymatic (RNase A) and chemical (heat shock) treatments.
Main Results:
- Flow cytometry successfully measured nuclear matrix protein and nucleic acid content.
- Heat-shocked cells showed increased nuclear protein content and protected double-stranded RNA after RNase A digestion.
- Excess nuclear protein in heat-shocked cells was found to sequester matrix-associated RNA.
- Polyacrylamide gel electrophoresis identified increased and novel heat-shock proteins within the nuclear matrix.
Conclusions:
- Flow cytometry is a viable method for studying nuclear matrix composition and detecting changes due to protein alterations.
- Heat shock induces significant changes in nuclear matrix protein content, leading to RNA sequestration and protection.