Isolation and Properties of Nuclei from Control and Auxin-treated Soybean Hypocotyl

Abstract

A quick procedure for the isolation of nuclei with good yield from soybean hypocotyl (Glycine max var. Wayne) was developed. The isolated nuclei appeared to retain their structural integrity. They were typically ellipsoidal with minima and maxima diameter of about 6 and 8 to 10 micrometers. While the nuclei were similar in size, the nucleoli were significantly larger in nuclei from auxin-treated tissue. The DNA content per nucleus was 4 ± 1 picograms for both untreated and auxin-treated tissues. The DNA:RNA :protein ratio of isolated nuclei in untreated and auxin-treated tissues was 1: 3.1: 11 and 1: 5.4: 21.7, respectively. The purified nuclei were active in RNA synthesis; the level of RNA polymerase II activity expressed in the nuclei from untreated tissue was 50 to 60% higher than RNA polymerase I. The nuclei from auxin-treated tissues contained about 2.5 times as much RNA polymerase I activity as nuclei from untreated tissue. The purified nuclei from both untreated and auxin-treated tissues were also active in the incorporation of 3H-TTP into DNA.thesis. Isolated nuclei thus are essential to the study of in vitro RNA synthesis if conditions relating to the in vivo state are to be approached. Isolated nuclei from other systems have been used to great advantage in the study of several important problems: e.g. the study of the transcription of defined RNA species (15,23,25,30,34), the intranuclear metabolism and release of RNA (26), the post-transcriptional additions of poly(A) to HnRNA (16,32,33), the mechanisms involved in DNA replication (6,9,10,22), the modification of chromatin proteins and their role in gene expression (18,28,35,39), the preparation of high mol wt DNA (1), and enzyme localization (8,29). We have pursued the isolation of nuclei from soybean hypocotyl with the objective of isolating large quantities of "intact," biologically active nuclei to be used in studies on the auxin regulation of RNA synthesis. In this report, we describe a procedure for isolating nuclei from control and 2, 4-D-treated soybean hypocotyl. The attendant RNA and DNA synthetic activities, gross morphology, and macromolecular composition are described. MATERIALS AND METHODSStudies on the mechanism of plant hormone action have received much attention in recent years. Hormones are involved in the regulation of many different physiological processes; some lines of evidence indicate that in several cases this regulation may involve the control of the transcription or translation process (17). Because it is generally difficult to isolate plant nuclei in sufficient yield for extensive biochemical work, the isolation of plant chromatin has been used routinely in the study of the influence of auxin on the transcription of RNA (11,24). Recent studies with soybean chromatin isolated by conventional methodology (13)

Replication of DNA by Nuclei Isolated from Soybean Suspension Cultures

“…Incorporation of deoxytriphosphates into an acid-insoluble product has been reported previously (2). Here we describe a system for the in vitro synthesis of DNA by nuclei isolated from soybean cells growing in suspension culture.…”

Isolation of Nucleoli and Localization of Ribonucleic Acid Polymerase I from Soybean Hypocotyl

“…Thus we have utilized pH 6 buffer to develop methods for the isolation of highly purified nuclei and nucleoli from soybean. The isolation of nuclei has been described (2). By utilizing procedures which are less gentle (i.e., longer and more rigorous homogenization), nuclei rupture while the nucleoli remain intact.…”

“…RNA polymerases are compartmentalized within three organelles which include nuclei (2,18), chloroplast (1), and mitochondria (4). We have shown previously that soybean nuclei contain at least two forms of RNA polymerase (2,3).…”

“…RNA polymerases are compartmentalized within three organelles which include nuclei (2,18), chloroplast (1), and mitochondria (4). We have shown previously that soybean nuclei contain at least two forms of RNA polymerase (2,3). We have adopted the nomenclature introduced by Roeder and Rutter (14) and refer to these two forms as RNA polymerase I and RNA polymerase II, which relates to their order of elution from DEAE-cellulose.…”

Fractionation of Nucleoli from Auxin-Treated Soybean Hypocotyl into Nucleolar Chromatin and Preribosomal Particles

“…Auxin treatment of quiescent mature hypocotyl tissue of soybean leads to a dramatic increase in nucleolar volume in association with an 8-to 10-fold increase in the rate of rRNA synthesis (7,10,19). The very large increase in ribosome accumulation following auxin treatment makes the soybean system an attractive model for assessing the relationship between rRNA synthesis and the synthesis of ribosomal proteins (9,30) as well as for an analysis of the mechanism(s) operative in the modulation of RNA polymerase I activity and nucleolar function (20).…”