Association between the mammalian 110,000-dalton heat-shock protein and nucleoli.

Subjeck, John R.; Shyy, Thung-Tai; Jing, Shen; Johnson, Richard J.

doi:10.1083/jcb.97.5.1389

Cited by 112 publications

(49 citation statements)

References 27 publications

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“…It also seems to be different from the nucleolar protein of Mr 100000 of cultured Chinese hamster ovary cells which has been found in association with nascent pre-rRNA and preribosomes [20]. Whether the Mr 180000 protein of Xenopus is related to the much smaller mammalian heat shock protein of Mr 110000 which has been localized in the fibrillar component of segregated nucleoli of postconfluent Chinese hamster ovary cells [51] cannot be decided at the moment. The karyoskeletal nucleolar protein of Mr 145000 and an isoelectric pH of approx.…”

Section: Discussionmentioning

confidence: 90%

Identification and localization of a novel nucleolar protein of high molecular weight by a monoclonal antibody

Schmidt-Zachmann

Hügle

Scheer

et al. 1984

Experimental Cell Research

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A monoclonal murine antibody (No-I 14) is described which reacts specifically with a polypeptide of molecular weight (M,) 180000 present in low-speed nuclear pellets from oocytes and somatic cells of Xenopus laevis and X. borealis and in isolated amplified nucleoli. Two-dimensional gel electrophoresis has revealed the acidic nature of this polypeptide (isoelectric at pH of ca 4.2 in the presence of 9.5 M urea). A relatively large proportion of the protein is extracted at elevated ionic strength( i.e., at 0.4-0.5 M alkali salt) in a form sedimenting at approx. 7-8S , compatible with a monomeric state. It is also extracted by digestion with RNase but not with DNase. In immunofluorescence microscopy , antibody No-114 stains intensely nucleoli of oocytes and all somatic cells examined , including the residual nucleolar structure of Xenopus erythrocytes which are transcriptionally inactive. During mitosis the antigen does not remain associated with the nucleolar organizer regions (NOR) of chromosomes but is released and dispersed over the cytoplasm until telophase when it re-associates with the reforming interphase nucleoli. At higher resolution the immunofluorescent region is often resolved into a number of distinct subnucleolar components of varied size and shape. Immunoelectron microscopy using colloidal gold-coupled secondary antibodies reveals that the M, 180000 protein is confined to the dense fibrillar component of the nucleolus. This conclusion is also supported by its localization in the fibrillar part of segregated nucleoli of cells treated with actinomycin D. We conclude that nucleoli contain a prominent protein of M , 180000 which contributes to the general structure of the dense fibrillar component of the interphase nucleolus , independent of its specific transcriptional activity.In the past two decades considerable progress has been made in understanding the basic functional organization of the nucleolus. Combined morphological, biochemical, and auto radiographic data have established that the nucleolus is the site of the genes which code for pre-rRNAs, transcription of these genes, assembly of the pre-rRNA molecules with specific proteins into ribonucleoprotein complexes, and maturation of the preribosomal particles both at the RNA and protein level (for reviews see [1][2][3]). Based primarily on electron microscopic autoradiography of cells labeled with radioactive uridine, morphologically distinct components of nucleoli can be correlated with certain steps of the pathway of ribosome biogenesis [4][5][6][7][8][9]. Although nucleoli can display different shapes and arrangements of their compot)ept substructures, depending on the .specific cell type and metabolic condition of the cell, there is general agreement that primary transcripts containing pre-rRNA are located in the dense fibrillar part of the

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Section: Discussionmentioning

confidence: 90%

Identification and localization of a novel nucleolar protein of high molecular weight by a monoclonal antibody

Schmidt-Zachmann

Hügle

Scheer

et al. 1984

Experimental Cell Research

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“…Antibodies against the major ORPs are being prepared to localise these proteins. One possibility is localisation of some ORPs on the nucleolus to protect RNA production similar to heat shock proteins 70 and 110 (Welch & Feramisco, 1985;Subjeck et al, 1983).…”

Section: Resultsmentioning

confidence: 99%

Enhanced synthesis of stress proteins caused by hypoxia and relation to altered cell growth and metabolism

Heacock

Sutherland²

1990

Br J Cancer

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Summary Cultured cells maintained in very low oxygen levels alter their structure, metabolism and genetic expression. Culture conditions for cells were modified to minimise variation of nutrients and to allow normal survival levels after 24 h of hypoxic exposure. Under these hypoxic conditions, glucose consumption and lactate production rates were similar to aerobic rates until about 12 h after which the hypoxic rates increased. DNA and protein synthesis rates are continuously inhibited to about 48% or 55% of the respective aerobic rates. During this period of decreased protein synthesis, a set of proteins termed oxygen regulated proteins (ORPs), exhibits enhanced relative synthesis. The molecular weights of the five major ORPs are approximately 260, 150, 100, 80 and 33 kDa. While increased relative synthesis of oxygen regulated proteins is partly due to increased levels of mRNA which encode these proteins, the mechanism of enhanced synthesis of ORPs may be more complex.Hypoxia and related pathophysiologic environmental and cellular metabolic changes can be determining factors for the effectiveness of treatment of tumours using radiation therapy (Hall, 1978), hyperthermia (Gerweck et al., 1979) or chemotherapy (Teicher et al., 1981). Cells are resistant to irradiation virtually immediately upon introduction of severe hypoxia and are sensitised quickly after oxygen addition (Michael et al., 1973). Slower responses to hypoxia include increased hyperthermia and adriamycin resistance (Li & Shrieve, 1982;Smith et al., 1980).The most notable metabolic change during hypoxia is the increased rate of glucose consumption. Enhanced synthesis of selected proteins after hypoxia has been reported in plants (Guttman et al., 1980) and mammalian cell lines (Sciandra et al., 1984;Heacock & Sutherland, 1986). These stress proteins have been termed oxygen regulated proteins (ORPs) to denote proteins whose synthesis is enhanced during hypoxia and repressed after addition of oxygen. ORPs may be very similar to proteins synthesised after prolonged glucose deprivation (Sciandra et al., 1984). To understand the molecular basis of resistance to various treatments and rationally plan therapeutic treatments, the basic biochemical events occurring during hypoxia must be studied. (Chen, 1977). Additionally, rodent cell lines were free of human cell contamination by lactate dehydrogenase isozyme determination (Dietz & Lubrano, 1967).Experimental cultures were initiated 20-24 hours before each experiment, except where noted, such that approximately 106 cells existed per 100 mm diameter glass Petri dish. When dishes of differing size were used, the number of cells and volume of medium was changed proportionally to the measured surface area. Loosely attached and floating cells were removed by pipetting off the growth medium and BME rinse. Attached cells were removed by 5-O min incubation at 37'C with 5 ml of 0.1% trypsin (Worthington). Trypsin was inactivated by addition of 5 ml of growth medium before determination of cell number and cell vol...

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“…Previous studies in yeast cells (25) (44,48), which may play a role in recovery from heat shock (40). It has been hypothesized that HSPs function in the normal state to unfold proteins for transport through membranes (9,11) and function in the stressed state to unfold aggregated proteins to allow them to renature into their native states (41).…”

Section: Discussionmentioning

confidence: 99%

Analysis of pre-rRNAs in heat-shocked HeLa cells allows identification of the upstream termination site of human polymerase I transcription.

Parker¹,

Bond²

1989

Mol. Cell. Biol.

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Human rRNA precursors from normal or stressed HeLa cells were studied by Si nuclease mapping of unlabeled RNA and by antisense RNase mapping of RNA from cells that had been labeled in vivo with[32p]PO4. Heating cells to 43°C decreased the amount of newly synthesized rRNA to less than 5% of the control level and led to greater than 95% inhibition of transcription termination at a region 355 to 362 nucleotides downstream of the 3' end of 28S rRNA, with readthrough continuing into the next transcription unit. Heating of cells to 42°C led to 60% inhibition of termination at this site; 50% of transcripts that extended into the nontranscribed spacer ended in a region 200 to 210 nucleotides upstream of the polymerase I (Pol I) initiation site. This is presumed to be the human upstream transcription termination site because of the absence of RNAs with a 5' end corresponding to this region, the location relative to the Pol I initiation site (which is similar to the location of upstream terminators in other species), and the fact that it is 15 to 25 nucleotides upstream of the sequence GGGTTGACC, which has an 8-of-9 base identity with the sequence 3' of the downstream termination site. Surprisingly, treatment of cells with sodium arsenite, which also leads to the induction of a stress response, did not inhibit termination. Pol I initiation was decreased to the same extent as termination, which lends support to the hypothesis that termination and initiation are coupled. Although termination was almost completely inhibited at 43°C, the majority of the recently synthesized rRNAs were processed to have the correct 3' end of 28S. This finding suggests that 3'-end formation can involve an endonucleolytic cut and is not solely dependent on exonucleolytic trimming of correctly terminated rRNAs.

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Association between the mammalian 110,000-dalton heat-shock protein and nucleoli.

Cited by 112 publications

References 27 publications

Identification and localization of a novel nucleolar protein of high molecular weight by a monoclonal antibody

Identification and localization of a novel nucleolar protein of high molecular weight by a monoclonal antibody

Enhanced synthesis of stress proteins caused by hypoxia and relation to altered cell growth and metabolism

Analysis of pre-rRNAs in heat-shocked HeLa cells allows identification of the upstream termination site of human polymerase I transcription.

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