Isolation and Properties of Liver Cell Nucleoli

Monty, Kenneth J.; Litt, M.; Kay, Ernest; Dounce, Alexander L.

doi:10.1083/jcb.2.2.127

Cited by 58 publications

(7 citation statements)

References 28 publications

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“…To test the extent to which FANCI is localized to the NO, we turned to the well-established technique (47,60,63) of subcellular fractionation to isolate nucleoplasm (NP) and NO from HeLa cells. Equal masses of total protein from WCE, NP, and NO were separated by SDS/PAGE and analyzed by Western blotting.…”

Section: Resultsmentioning

confidence: 99%

Fanconi anemia protein FANCI functions in ribosome biogenesis

Sondalle

Longerich

Ogawa

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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Fanconi anemia (FA) is a disease of DNA repair characterized by bone marrow failure and a reduced ability to remove DNA interstrand cross-links. Here, we provide evidence that the FA protein FANCI also functions in ribosome biogenesis, the process of making ribosomes that initiates in the nucleolus. We show that FANCI localizes to the nucleolus and is functionally and physically tied to the transcription of pre-ribosomal RNA (pre-rRNA) and to large ribosomal subunit (LSU) pre-rRNA processing independent of FANCD2. While FANCI is known to be monoubiquitinated when activated for DNA repair, we find that it is predominantly in the deubiquitinated state in the nucleolus, requiring the nucleoplasmic deubiquitinase (DUB) USP1 and the nucleolar DUB USP36. Our model suggests a possible dual pathophysiology for FA that includes defects in DNA repair and in ribosome biogenesis.

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Section: Resultsmentioning

confidence: 99%

Fanconi anemia protein FANCI functions in ribosome biogenesis

Sondalle

Longerich

Ogawa

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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“…The only structure visible in the resting nucleus is the nucleolus, and so the method for fractionation was begun with the isolation of this subnuclear component from disrupted nuclei. The isolation of nucleoli from disrupted rat-liver nuclei has been described by Monty, Litt, Kay & Dounce (1956). The procedure, however, involved a sedimentation step of over 12 hr.…”

Section: Discussionmentioning

confidence: 99%

The metabolism of isolated rat-liver nucleoli and other subnuclear fractions. The active site of amino acid incorporation in the nucleus

Rees¹,

Rowland²,

Varcoe³

1963

Biochemical Journal

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It was shown by Rees & Rowland (1961) that rat-liver nuclei isolated in 025 M-sucrose incorporated amino acids into protein, and nucleotides into RNA. Although the incorporation was inhibited to varying degrees by anoxia and by several inhibitors of oxidative phosphorylation, it was not affected by detergents, by freezing and thawing the nuclei, or by disruption of the nuclei by ultrasonic vibration (Rees, Ross & Rowland, 1961). We have now studied isolated nucleoli and other subnuclear fractions in order to investigate the active site of synthesis of proteins and nucleic acids within the nucleus. In addition the enzymic and chemical composition of the various fractions has been examined. Part of this work has been published in a preliminary form (Rowland, Rees & Varcoe, 1962). MATERIALS AND METHODS Animals. Male albinorats,weighing200-250g., were used. Reagent8. These were as described by Rees & Rowland (1961). In addition [1-34C]valine and [1-14C]leucine were obtained from The Radiochemical Centre, Amersham, Bucks. Nuclear and8ubnuclearpreparation&. Nuclei were isolated from rat liver in 0*25M-sucrose as described by Rees & Rowland (1961). Scheme 1 shows the method used for isolating nucleoli and other subnuclear fractions. Batches of isolated nuclei, suspended in 15-20 ml. of 0 25m-sucrose, were subjected to ultrasonic vibration (20 keyc./sec.) at 20 in a MSE ultrasonic disintegrator (60w) with a titanium probe of 1 cm. diam. until all nuclei were disrupted (usually 7 min.). The resulting suspension was centrifuged for 30 sec. at 2500g to remove probe debris and coagulated protein. The nucleoli I Sediment Recentrifuged twice in water (see text) Nucleoli (fraction A) Disrupted nuclei Centrifuged at 412 x 104g,^min.

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“…Chemical studies have not been able to shed any more light on this problem. In essence, radioisotope studies on isolated and fractionated nuclei {Lilt et al [1952], Monty et al [1956], Vincent [1952) have also dem onstrated early labelled RNA in the. nucleolus.…”

Section: Thymidinementioning

confidence: 99%