Isolation and Characterization of Nuclei from Rice Embryos.

Abstract

ABSTRACT. A method has been developed to isolate pure preparations of nuclei in high yield from commercially available viable rice embryos (germ), employing extraction with buffer solution containing glycerol (without detergent) and polyamine, followed by centrifugation on a 30% Percoll cushion. The intactness of the isolated nuclei was confirmed by light microscopy as well as electron microscopy. The protein profiles of both whole nuclei and nuclear extracts obtained by SDS-PAGE, organellar marker enzymeactivities, DNAand RNA analyses, and in vitro RNAsynthesis, all indicate that the highly purified nuclei are isolated from rice embryos.The biosynthesis of proteins encoded by the nuclear genomeconsists of sequentially oriented networks compartmentalized in the cell; DNAtranscription and RNA processing occur in the nuclei and subsequent RNA transport and translation of mRNA in the cytoplasm. Although the detailed mechanisms of each of these steps has been the subject of intensive cellular and molecular biological studies, RNAsynthesis in the nuclei is of particular interest and importance because of the role of proteins specifically involved in the transcriptional regulation. In recent years muchresearch interest has been focused on elucidating the mechanismsof transcriptional regulation using isolated nuclei of plant origin (2, 8, 17). One can thus readily recognize that isolation of intact nuclei is of prime importance to study the import mechanismacross the nuclear envelopes of various regulatory proteins engaged in the transcriptional activities of the nuclear genome. Since signal sequence(s) of amino acids relating to the import of nuclear proteins have been characterized in the mammalian system (4, 18), it is desirable to construct a feasible experimental system using plant materials to advance our knowledge concerning the mechanism(s) operating * To whomcorrespondence should be sent.

Periodic DNA Methylation in Maize Nucleosomes and Demethylation by Environmental Stress

“…Mononucleosomal DNA Assay-Roots from 13-day-old seedlings were harvested, and a pure preparation of nuclei was obtained by centrifugation on a 30% Percoll cushion (Amersham Biosciences) (27). A 60-g of nuclei suspension was digested with 4 units of micrococcal nuclease (Takara) at 37°C for 4 min and fractionated on a 2.5% NuSieve 3:1 agarose gel (BioWhittaker Molecular Applications, Rockland, ME).…”

Proteomic Analysis of Barley Cell Nuclei Purified by Flow Sorting

“…However, continuing technical improvements have largely overcome these difficulties in various explants, including the embryo [Masuda et al, 1991;Yamaguchi et al, 1992;Busk and Pages, 1997], endosperm [Ferreira et al, 2006;Li et al, 2008], seed [Riggs et al, 1989], seed coat [Renouard et al, 2012], leaf [e.g. Cushman, 1995;Zhang et al, 1995;Abdalla et al, 2009Abdalla et al, , 2010Sikorskaite et al, 2013], and root meristem [Silva et al, 2010], and in in vitro cultured cells [Willmitzer and Wagner, 1981].…”

Isolation of nuclear proteins from flax (Linum usitatissimum L.) seed coats for gene expression regulation studies

“…The CelLytic PN extraction kit from Sigma, a commercial adaptation of a protocol described to isolate nuclear proteins from A. thaliana leaves [12], yielded up to 168.5 μg proteins per gram fresh weight (Table 1). The presence of nuclear proteins in this fraction was confirmed by protein blot using an anti-histone H1 antibody (Figure 2), suggesting that the preparation is enriched in nuclear proteins.…”

“…The presence of contaminating proteins was estimated using alcohol dehydrogenase (ADH) activity assay [12] as an indication of cytosolic protein presence.…”

“…NADH production from NAD was measured by increase in absorbance at 340 nm at 25°C [12]. The reaction mixture contained 0.1 M Tris-HCl (pH 9.0), 2 mmol NAD and 1% (v/v) ethanol in a final volume of 5 ml.…”