Comparison and characterization of nuclear isolation procedures as applied to chick oviduct.
A number of methods for the preparation of chick oviduct nuclei have been compared. Nuclei have been isolated in hypertonic sucrose and citric acid and the product has been characterized with respect to cleanliness, ultrastructure, RNA polymerase activity, RNA integrity, and chromatin composition. The study demonstrates that the choice of oviduct nuclear isolation procedure will depend markedly on the purpose for which the nuclei are required. Thus, nuclei prepared entirely in high-molarity sucrose retain the highest levels of RNA polymerase. Those prepared rapidly in the presence of citric acid retain nuclear RNA in an essentially undegraded state. Finally, a bulk preparation is described which, because of its adaptability and high yield of morphologically intact nuclei using large amounts of tissue, is ideal for use in preparing chromatin. Conditions are described by which isolated nuclei can be stored for up to 6 months and retain their morphology, chemical characteristics, and RNA polymerase activity.
D000818:Animals; D002466:Cell Nucleolus / Q000032:analysis; D002467:Cell Nucleus / Q000032:analysis* / Q000166:cytology; D002645:Chickens / Q000033:anatomy & histology; D002712:Chlorides / Q000494:pharmacology; D002843:Chromatin / Q000032:analysis; D002951:Citrates / Q000494:pharmacology; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004054:Diethylstilbestrol / Q000008:administration & dosage; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006657:Histones / Q000032:analysis; D006982:Hypertonic Solutions; D008274:Magnesium; D008722:Methods; D008854:Microscopy, Electron; D010057:Oviducts / Q000166:cytology*; D011506:Proteins / Q000032:analysis; D012313:RNA / Q000032:analysis; D013395:Sucrose / Q000494:pharmacology; D013698:Templates, Genetic
D002712:Chlorides; D002843:Chromatin; D002951:Citrates; D006657:Histones; D006982:Hypertonic Solutions; D011506:Proteins; D013395:Sucrose; D012313:RNA; D004054:Diethylstilbestrol; D012321:DNA-Directed RNA Polymerases; D008274:Magnesium