The uptake of [8-14C]adenine and [2-14C]phenylalanine by rat-liver nuclei in vitro

Logan, R.; Ficq, Adrienne; Errera, Maurice Leo

doi:10.1016/0006-3002(59)90014-9

Cited by 45 publications

(7 citation statements)

References 11 publications

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“…Similar results were presented more recently for nuclei isolated from both animal (9,15,17,19,21,23) Isolation of Chromatin. Chromatin was isolated according to Huang and Bonner (I1) and purified by centrifugation at l00,000g for 2 h through a 1.5 M sucrose cushion.…”

Section: Introductionsupporting

confidence: 87%

Protein-Synthesizing Activity of Maize-Shoot Chromatin

Wielgat

Kleczkowski²

1986

Plant Physiol.

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The evidence presented in this paper suggests that purified plant chromatin, similar to mammalian (SR Umansky et al., Eur J Biochem 1980 105: 117-129), has the ability to incorporate amino acids into acid precipitable material. The polypeptide-synthesizing system of chromatin seems to differ substantially from the classical polyribosomal translation mechanism in cytoplasm. When chromatin purified from 5-day-old etiol-ated maize (Zea mays) shoots was incubated with 4(C-labeled amino acids, label was incorporated into the trichloroacetic acid precipitable product. Chloramphenicol, pactamycin, and actinomycin D inhibited the incorporation almost completely, whereas treatment with cycloheximide, puromycin, or aurintricarboxylic acid did not affect the labeling. Prein-cubation with pancreatic RNase was also without effect, but treatment of chromatin with DNase I caused about 25% depression of label incorporation. A wheat germ translation system or its single components have no effect on the chromatin polypeptide-synthesizing activity beyond that expected for a simple addition. The protein-synthesizing system is tightly bound to chromatin and could not be removed by dissociation in 1 molar NaCl. The mean molecular weight of the major protein fraction synthesized in the presence of chromatin was 21 to 24 kilodaltons. Thirty years ago Allfrey et al. (2) reported that isolated calf thymus nuclei were able to incorporate labeled amino acids into the TCA-precipitable material. Similar results were presented more recently for nuclei isolated from both animal (9, 15, 17, 19, 21, 23) and plant (3, 7) tissues. It has been postulated that nuclei are equipped with a protein-synthesizing system similar to the cytoplasmic one. This opinion was criticized on the ground that isolated nuclei are usually contaminated with cytoplasmic polyribosomes (18), particularly those associated with perinu-clear ER (20). It was suggested that polypeptides found in nuclei were synthesized in the cytoplasm and subsequently transported to the nucleus (1). However, evidence (17) has been presented that incorporation of amino acids into the isolated nuclei was insensitive to puromycin and cycloheximide (inhibitors of translation on polyribosomes) as well as ribonuclease, but slightly inhibited by deoxyribonuclease. It has also been shown (22) that both isolated rat liver nuclei as well as purified chromatin from the same source have an ability to incorporate amino acids into the TCA-precipitable product. In this paper we present data on the ability of isolated maize shoot chromatin to synthesize proteins. The evidence against participation of the polyribosomal translation system in this 'Supported by the Polish Academy of Sciences within the Project 09.7. process is provided. A preliminary communication concerning part of this work has been presented (12). This work is an extension of a similar paper with mammalian chromatin presented by Umansky et al. (22). MATERIALS AND METHODS Plant Material. Five-d-old etiolated maize (Zea mays, cv K-72) plants gr...

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Section: Introductionsupporting

confidence: 87%

Protein-Synthesizing Activity of Maize-Shoot Chromatin

Wielgat

Kleczkowski²

1986

Plant Physiol.

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“…It has been observed in nuclei from other cell types and from other species, e.g., mouse AKR lymphoma (1), rat liver (54), a rat hepatoma (55), pea seedlings (56), and insect salivary glands (57). Low magnification autoradiographs showing the nuclear localization of the isotope have been published (55,57,60,62) and they lend further support to the general conclusion that the presence of protein synthetic machinery within cell nuclei is a necessary and widespread phenomenon.…”

Section: Discussionmentioning

confidence: 63%

Methods for the Purification of Thymus Nuclei and Their Application to Studies of Nuclear Protein Synthesis

Allfrey

Littau

Mirsky

1964

The Journal of Cell Biology

172

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Procedures are described for the purification of calf thymus nuclei using mild hypotonit shock to break intact cells, and layering techniques to remove cytoplasmic debris. Ficolc (a high polymer of sucrose) was dissolved in isotonic sucrose to give dense solutions suitable for gradient centrifugation. The method yields nuclei which can incorporate amino acids in vitro. Thymus nuclei isolated under isotonic conditions were incubated with C14-amino acids and later purified by centrifugation through dense sucrose solutions. The distribution of radioactivity in different nuclear proteins was measured and it was found that isotopic amino acids are actively incorporated into characteristically chromosomal proteins, such as the arginine-rich and lysine-rich histones. Protein synthesis in the nucleus is markedly inhibited by puromycin and by agents, such as 2,4-dinitrophenol, which inhibit ATP synthesis. The synthesis of histones is also inhibited by puromycin, but the uptake of several amino acids into the lysine-rich histone fraction seems less sensitive to puromycin inhibition than is uptake into the arginine-rich histones or other proteins of the nucleus. High resolution autoradiography using tritiated leucine and observing grain distribution over thin sections of isolated nuclei and whole cells shows that amino acid incorporation occurs within the nucleus and is not due to cytoplasmic contamination.

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“…Confirmation of these results with preparations purified by centrifugation through sucrose or Ficoll layers (e.g., 8) gives additional weight to this argument. In addition, a number of studies with other preparations have shown similar results with other tissues where a cytoplasmic contamination should not be of major proportions (e.g., [17][18][19][20][21].…”

Section: Introductionmentioning

confidence: 63%

An Electron Microscope Study of Calf Thymus Nuclear Preparations Isolated in Sucrose Solutions

Kodama

Tedeschi

1963

The Journal of Cell Biology

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This work was carried out with the intent of developing a method capable of routinely evaluating calf thymus nuclear preparations with the electron microscope. Examination of small random samples, pre-embedded in agar after fixation with permanganate, were found to give results comparable to those obtained with much larger samples withdrawn randomly from pellets and embedded and sectioned conventionally. Results obtained by this pre-embedding technique with acrolein, osmium tetroxide, or permanganate fixations were equivalent. Calf thymus nuclear preparations isolated in sucrose by the method prevalently used (see 1) are contaminated only slightly with intact cells, to a degree which varies with each preparation. However, intact cells, damaged cells, or nuclei with some cytoplasm constitute together about 30 per cent of the preparation. Particles other than intact cells are not readily distinguishable from one another by light or phase microscope techniques. These preparations can be purified further by centrifuging through a dense sucrose layer. In our hands, however, contamination with some cytoplasm still remains in approximately 10 per cent of the particles. Incubation of the particles prepared without purification procedures, under conditions frequently used, results in the extensive breakdown of particles. Under at least one set of conditions, nuclei are selectively disrupted, leaving primarily damaged cells in the preparation.

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The uptake of [8-14C]adenine and [2-14C]phenylalanine by rat-liver nuclei in vitro

Cited by 45 publications

References 11 publications

Protein-Synthesizing Activity of Maize-Shoot Chromatin

Protein-Synthesizing Activity of Maize-Shoot Chromatin

Methods for the Purification of Thymus Nuclei and Their Application to Studies of Nuclear Protein Synthesis

An Electron Microscope Study of Calf Thymus Nuclear Preparations Isolated in Sucrose Solutions

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