Characterization of nuclear matrices prepared without salt extraction.
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 less than or equal to 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.
Dwarakanath
B S
BS
Department of Radiology, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, North Carolina 27103.
Wallen
C A
CA
St Clair
D K
DK
Wheeler
K T
KT
D000230:Adenocarcinoma; D000818:Animals; D002461:Cell Line, Transformed; D004247:DNA / Q000032:analysis; D003850:Deoxyribonuclease I / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D008321:Mammary Glands, Animal; D051379:Mice; D008855:Microscopy, Electron, Scanning; D015530:Nuclear Matrix / Q000737:chemistry*; D009687:Nuclear Proteins / Q000032:analysis; D010447:Peptide Hydrolases / Q000378:metabolism; D012965:Sodium Chloride
D009687:Nuclear Proteins; D012965:Sodium Chloride; D004247:DNA; D003850:Deoxyribonuclease I; D010447:Peptide Hydrolases