THE CHOICE OF RESUSPENSION MEDIUM FOR ISOLATED RAT-LIVER NUCLEI - EFFECTS ON NUCLEAR MORPHOLOGY AND IN-VITRO TRANSCRIPTION

Standard protocols for in vitro transcription assay (nuclear run-off) include 10-40% (v/v) glycerol (of various ionic strength) in the mediu m used for resuspension/storage of the isolated nuclei. In the present work the morphological and functional properties of nuclei isolated f rom rat liver have been studied as a function of the content of glycer ol, sucrose and inorganic ions (K+ and Mg2+) in the resuspension mediu m. In contrast to earlier reports, glycerol was found not to be essent ial to maintain morphological integrity and RNA polymerase activity in frozen/stored nuclei. Nuclear pellets, resuspended and stored in isoo smotic sucrose media, were found to give morphologically intact and tr anscriptionally active nuclei. Furthermore, these nuclei displayed a h igher specific hybridization signal for the differentially expressed g enes encoding peroxisomal beta-oxidation enzymes, relative to the tota l 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 Mg2+, 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 t ranscription in isolated nuclei is better understood.