Mass Isolation of Pea Nuclei

Tautvydas, Kestutis J.

doi:10.1104/pp.47.4.499

Cited by 51 publications

(20 citation statements)

References 7 publications

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“…The relative DNA, RNA, and protein contents of purified nuclei isolated from untreated and auxin-treated tissue are given in Table I (38). Similar data to those for pea (38) The enlarged nucleoli might suggest that this large increase in RNA and protein reflects primarily an increase in ribonucleoprotein particles associated with rRNA synthesis and processing in the nucleoli.…”

Section: Methodssupporting

confidence: 61%

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

Chen¹,

Lin²,

Chang³

et al. 1975

Plant Physiol.

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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)

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Section: Methodssupporting

confidence: 61%

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

Chen¹,

Lin²,

Chang³

et al. 1975

Plant Physiol.

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“…Several methods have been described for the isolation of transcriptionally active plant nuclei (6,10,11,18,24,26,34,35,39). Although various criteria (e.g., microscopy, protein/RNA/DNA ratios, chromatin structure, specific activity and duration of RNA synthesis, and analysis of run-off transcription products) have been used to assess the purity, integrity, and transcriptional capacity of nuclei isolated from plants, none of the nuclear preparations has been shown to direct accurate initiation or termination of specific RNA transcripts.…”

Section: Introductionmentioning

confidence: 99%

Synthesis of 5S rRNA and putative precursor tRNAs in nuclei isolated from wheat embryos

1986

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Nuclei isolated from wheat embryos synthesize 4.5S precursor tRNAs and 5S rRNA in vitro. The precursor tRNAs can be processed to 4S tRNAs. Transcription of the tRNA and 5S rRNA genes is carried out by endogenous RNA polymerase III, and addition of exogenous wheat RNA polymerase III results in increased transcription on these genes. A number of experimental results suggest that proper initiation and accurate transcription of the tRNA and 5S rRNA genes occurs with isolated wheat nuclei.

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“…They were finally extracted by Laemmli's buffer and separated by SDS-PAGE without any further purification. The DNA and protein content of a plant nucleus is around 20% and 60% of the nucleus dry weight, respectively [Tautvydas, 1971]. Then, the total weight of 5 million G1 nuclei is estimated to 250 μg giving a total protein amount of 150 μg.…”

Section: Protein Extractionmentioning

confidence: 99%

Proteomic Analysis of Barley Cell Nuclei Purified by Flow Sorting

Petrovská

Jeřábková

Chamrád

et al. 2014

Cytogenet Genome Res

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Many proteins are present in the nucleus; some are involved with its structural and functional organization, some with gene expression, and some with cell division. The plant nuclear proteome has not been well explored. Its characterization requires extraction methods which minimize both the artifactual alteration of the proteins and the extent of contamination with non-nuclear proteins. The conventional multi-step fractionation procedure is both laborious and prone to contamination. Here, we describe a single-step method based on flow sorting. The method allows the separation of G1, S and G2 phase nuclei and minimizes the risk of contamination by non-nuclear proteins. Preliminary results obtained using G1 phase cell nuclei from barley root tips indicate that flow sorting coupled with a protein/peptide separation and mass spectrometry will permit a comprehensive characterization of the plant nuclear proteome.

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Mass Isolation of Pea Nuclei

Cited by 51 publications

References 7 publications

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

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

Synthesis of 5S rRNA and putative precursor tRNAs in nuclei isolated from wheat embryos

Proteomic Analysis of Barley Cell Nuclei Purified by Flow Sorting

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