| Autor: |
Strand, Roger, Bøe, Roald, Flatmark, Torgeir |
| Zdroj: |
Molecular & Cellular Biochemistry; Oct1994, Vol. 139 Issue 2, p149-157, 9p |
| Abstrakt: |
Standard protocols for in vitro transcription assay (nuclear run-off) include 10-40% (v/v) glycerol (of various ionic strength) in the medium used for resuspension/storage of the isolated nuclei. In the present work the morphological and functional properties of nuclei isolated from rat liver have been studied as a function of the content of glycerol, sucrose and inorganic ions (K and Mg) in the resuspension medium. In contrast to earlier reports, glycerol was found not to be essential to maintain morphological integrity and RNA polymerase activity in frozen/stored nuclei. Nuclear pellets, resuspended and stored in isoosmotic sucrose media, were found to give morphologically intact and transcriptionally active nuclei. Furthermore, these nuclei displayed a higher specific hybridization signal for the differentially expressed genes encoding peroxisomal β-oxidation enzymes, relative to the total RNA synthesis, than nuclei resuspended and stored in a hyperosmotic glycerol-containing medium. The concentrations of inorganic ions were also found to affect nuclear morphology. Flow cytometry indicated DNA leakage from nuclei at insufficient concentrations of K and Mg, and high ionic strength favoured aggregation and disintegration of nuclei. Our findings indicate that quantitative results from nuclear run-off experiments should be interpreted with caution until the process of transcription in isolated nuclei is better understood. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
| Externí odkaz: |
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