The structure and composition of the nuclear matrices prepared from a mouse mammary adenocarcinoma cell (line 66) by digestion with DNase I and several proteases (PRT-matrices) were characterized by protein and DNA gel electrophoresis, flow cytometry, and scanning electron microscopy. The characteristics of these PRT-matrices were compared with the characteristics of conventionally prepared nuclear matrices that employ a high salt extraction step (HS-matrices) in order to select a preparation that can be used in biochemical and/or biophysical studies where salt extraction compromises either the analysis or the interpretation of the data. Of the characterized PRT-matrices, only those prepared with Type XIV protease (pronase) had most of the characteristics of HS-matrices. They, (i) maintained their structural integrity, (ii) had ≤5% of their nuclear DNA associated with the matrix, (iii) had no evidence of higher-order chromatin structure, and (iv) had a DNA size distribution in the range of 400-1100 bp. The major difference between the PRT-matrices and the HS-matrices was a decrease in the protein content of the PRT-matrices. Although the PRT-matrices may not be appropriate for studying the unique nuclear matrix associated proteins that are involved in functions such as replication, transcription, and differentiation, they are clearly suitable for studying the properties of the nuclear matrix associated DNA.
|Number of pages||7|
|State||Published - Oct 1991|
Bibliographical noteFunding Information:
1 Supported by Grant CA-45156 from the National of the National Institutes of Health. ’ Present address: Department of Biophysics, Mental Health and Neurosciences, Bangalore,
The flow cytometry and electron microscopy were performed in the Flow Cytometry Facility and Core Electron Microscopy Facility of the Cancer Center of Wake Forest University, respectively. These facilities are partially supported by NIH Grant CA-12197 from the National Cancer Institute. We thank R. C. Fletcher and K. Grant from these shared facilities for their help. We also thank Drs. W. H. St. Clair and R. R. Hantgan for providing some of the proteases, Dr. S. Swarts for helpful discussions, J. Anderson for technical support, E. Henson for photography, and D. Cantrell for preparation of this manuscript.
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology