Nonaqueous isolation of nuclei from lily pollen and an examination of their histones

Abstract

Summary Pollen of Lilium longiflorum was suspended in cold carbon tetrachloride:cyclohexane (CTC) mixtures and released from a French pressure cell. Nuclei were isolated by adjusting the suspension to a specific gravity of 1.390 at 0°C. The pollen nuclei were separated into several fractions of increasing density by a step-wise increase in the CTC density followed by centrifugation. The lighter nuclear fraction was enriched for vegetative nuclei and the heavier fractions were enriched for generative nuclei. Histones were extracted from deoxyribonucleoprotein (DNP) fibers which were prepared from each of the nuclear fractions. The DNP was isolated by the use of 2 M NaCl extraction of the nuclei. The histones were examined by acrylamide gel and amino acid analysis. Histones from lighter nuclei contained only the arginine-rich histones while the heavier nuclei contained these histones and also the more lysine-rich histones and the meiotic histone ( Sheridan and Stern , 1967) known to be present in the pollen grain. These observations suggest that the generative nucleus may contain lysine-rich and arginine-rich histones while the vegetative nucleus may contain only the more arginine-rich histones. Such a difference in histone distribution is in agreement with the observations of Sauter , 1969 in his cytochemical studies on nuclear differentiation in pollen grains of Paoenia .