Ultrastructural and biochemical comparisons of nuclear matrices prepared by high salt or LIS extraction.
We have directly compared two independently published methods for isolating operationally defined nuclear matrices by studying EM ultrastructure, protein composition and distribution of replicating DNA. Nuclear matrices prepared by extraction with 2 M NaCl consisted of fibrous pore complex lamina, residual fibrillar and granular components of nucleoli and interchromatin granules, and an extensive anastomosing internal fibrous network. These matrices were enriched in high molecular weight nonhistone proteins but were virtually devoid of histones. Consistent with previously published data, newly-replicated DNA was resistant to this high salt extraction. Nuclear matrices prepared by extraction of nuclei with 25 mM lithium 3,5-diiodosalicylate, LIS, also contained fibrous pore complex lamina, but lacked morphologically distinct residual nucleoli and were markedly depleted in internal structure. The reduced amounts and complexity of proteins associated with the LIS matrix were consistent with the ultrastructural data. Moreover, much less newly-replicated DNA was recovered in LIS matrices. The data show that LIS dissociates nuclear ultrastructure and extracts both protein and DNA in proportion to the concentration used, regardless of whether nuclei or high salt nuclear matrices are used as starting material. While the data suggest that LIS may not necessarily be an optimal reagent for preparing nuclear matrices containing internal structural elements from all tissue sources, it may be useful for selectively solubilizing and analyzing components of the nuclear matrix.
Department of Medicine, Baylor College of Medicine, Houston, TX 77030.
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Smith
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Ochs
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Lin
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Chinault
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism / Q000648:ultrastructure*; D006224:Cricetinae; D004247:DNA / Q000302:isolation & purification; D004261:DNA Replication; D006801:Humans; D006822:Hybrid Cells / Q000378:metabolism / Q000648:ultrastructure; D008854:Microscopy, Electron; D009687:Nuclear Proteins / Q000302:isolation & purification
D009687:Nuclear Proteins; D004247:DNA