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 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 Mg2+) 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 beta-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 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 transcription in isolated nuclei is better understood.
Strand
R
R
Department of Biochemistry and Molecular Biology, University of Bergen, Norway.
Bøe
R
R
Flatmark
T
T
D000818:Animals; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000187:drug effects / Q000201:enzymology / Q000235:genetics* / Q000648:ultrastructure*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D005260:Female; D005990:Glycerol / Q000494:pharmacology; D008099:Liver / Q000378:metabolism / Q000648:ultrastructure; D015636:Magnesium Chloride / Q000494:pharmacology; D008297:Male; D008830:Microbodies / Q000201:enzymology; D009994:Osmolar Concentration; D011189:Potassium Chloride / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D013395:Sucrose / Q000494:pharmacology; D014158:Transcription, Genetic
D003451:Cryoprotective Agents; D012333:RNA, Messenger; D015636:Magnesium Chloride; D013395:Sucrose; D011189:Potassium Chloride; D012321:DNA-Directed RNA Polymerases; D005990:Glycerol