Mass Isolation of Pea Nuclei

Abstract

Improvements in conventional filtration and centrifugation procedures made it possible to increase the yield of intact cytoplasm-free pea (Pisum sativum) nuclei from the usual 3 to 10% to 32% (6 x 10(8) nuclei per 7 grams fresh weight of pea apices) and to complete the isolation in 80 to 90 minutes. The isolated nuclei appeared to retain their structural integrity as revealed in electron photomicrographs, and remained intact for at least 5 hours at 20 Celsius.The DNA:RNA: protein ratio of isolated pea nuclei was found to be 3.1:1:9.9. Their RNA polymerase activity, monitored by incorporation of (14)C into RNA from (14)C-UTP, was linear for about 10 minutes, and then gradually declined over the next 15 to 20 minutes.