Monoclonal antibodies against nuclear matrix detect nuclear antigens in mammalian, insect and plant cells: An immunofluorescence study

Chaly, Nathalie; Sabour, M.P.; Silver, Julie C.; Aitchison, William A.; Little, Judy E.; Brown, David L.

doi:10.1016/0309-1651(86)90037-8

Cited by 22 publications

(22 citation statements)

References 12 publications

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“…A number of studies have shown that such an immunofluorescence labeling pattern is produced by pore complex-specific antibodies (Davis and Blobe11986, 1987;Hanover et al 1987;Park et al 1987;Snow et al 1987). The nucleoplasmic labeled patches detected in previous work using PI1 hybridoma culture supernatant, or unpurified PI1 ascites fluid (Chaly et al 1984(Chaly et al , 1986 were not observed in this study. The loss of internal nuclear labeling in PI1 antibody fractionated on an hydroxylapatite column is being investigated.…”

Section: Characterization and Localization Of The Antigen Recognized contrasting

confidence: 94%

“…3 a). A similar pattern of fluorescence at the nuclear periphery had been noted when other mammalian cells and insect cells were analyzed with the same antibody (Chaly et al 1984(Chaly et al , 1986. A number of studies have shown that such an immunofluorescence labeling pattern is produced by pore complex-specific antibodies (Davis and Blobe11986, 1987;Hanover et al 1987;Park et al 1987;Snow et al 1987).…”

Section: Characterization and Localization Of The Antigen Recognized supporting

confidence: 59%

“…Murine monoclonal antibody PIl (IgM) was raised against a mouse Iymphocyte nuclear matrix fraction and has been characterized in detail (Chaly et al 1984(Chaly et al , 1986. Experiments were carried out using PIl ascites fluid purified by hydroxylapatite chromatography, concentrated and lyophilized as previously described (Stanker et al 1985).…”

Section: Methodsmentioning

confidence: 99%

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Functional role of newly formed pore complexes in postmitotic nuclear reorganization

et al. 1989

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Abstract. Many nuclear proteins are released into the cytoplasm at prometaphase and are transported back into the daughter nuclei at the end of mitosis. To determine the role of this reentry in nuclear remodelling during early interphase, we experimentally manipulated nuclear protein uptake in dividing cells. Recently we and others have shown that signal-dependent, pore complex-mediated uptake of nuclear protein is blocked in living cells on microinjection of the lectin wheat germ agglutinin (WGA), or of antibodies such as PI1 that are directed against WGA-binding pore complex glycoproteins. In the present study, we microinjected mitotic PtK z cells with WGA or antibody PIt and followed nuclear reorganization of the daughter cells by immunofluorescence and electron microscopy. The inhibitory effect on nuclear protein uptake was monitored by co-injection of the karyophilic protein nucleoplasmin. When injected by itself early in mitosis, nucleoplasmin became sequestered into the daughter nuclei as they entered telophase. In contrast, nucleoplasmin was excluded from the daughter nuclei in the presence of WGA or antibody PI1 . Although PtK z cells with blocked nuclear protein uptake completed cytokinesis, their nuclei showed a telophaselike organization characterized by highly condensed chromatin surrounded by a nuclear envelope containing a few pore complexes. These findings suggest that pore complexes become functional as early as telophase, in close coincidence with nuclear envelope reformation. They further indicate that the extensive structural rearrangement of the nucleus during the telophase-G1 transition is dependent on the influx of karyophilic proteins from the cytoplasm through the pore complexes, and is not due solely to chromosome-associated components.

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Section: Characterization and Localization Of The Antigen Recognized contrasting

confidence: 94%

Section: Characterization and Localization Of The Antigen Recognized supporting

confidence: 59%

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Functional role of newly formed pore complexes in postmitotic nuclear reorganization

et al. 1989

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“…6B) and FS-4 (Fig. 6E) cells and is typical of nuclear matrix-associated and hnRNP antigens (16,36). Little, if any, hnRNP C staining was observed in the cytoplasm of either cell type.…”

Section: Resultsmentioning

confidence: 92%

Purification, characterization, and cDNA cloning of an AU-rich element RNA-binding protein, AUF1.

Zhang¹,

Wagner²,

Ehrenman³

et al. 1993

Mol. Cell. Biol.

506

483

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The degradation of some proto-oncogene and lymphokine mRNAs is controlled in part by an AU-rich element (ARE) in the 3' untranslated region. It was shown previously (G. Brewer, Mol. Cell. Biol. 11:2460-2466) that two polypeptides (37 and 40 kDa) copurified with fractions of a 130,000 x g postribosomal supernatant (S130) from K562 cells that selectively accelerated degradation of c-myc mRNA in a cell-free decay system. These polypeptides bound specifically to the c-myc and granulocyte-macrophage colony-stimulating factor 3' UTRs, suggesting they are in part responsible for selective mRNA degradation. In the present work, we have purified the RNA-binding component of this mRNA degradation activity, which we refer to as AUFl. Using antisera specific for these polypeptides, we demonstrate that the 37-and 40-kDa polypeptides are immunologically cross-reactive and that both polypeptides are phosphorylated and can be found in a complex(s) with other polypeptides. Immunologically related polypeptides are found in both the nucleus and the cytoplasm. The antibodies were also used to clone a cDNA for the 37-kDa polypeptide. This cDNA contains an open reading frame predicted to produce a protein with several features, including two RNA recognition motifs and domains that potentially mediate protein-protein interactions. These results provide further support for a role of this protein in mediating ARE-directed mRNA degradation.The c-myc gene is important for the control of cellular growth, differentiation, and transformation (reviewed in references 17 and 41). It belongs to the class of immediateearly genes whose expression is required to drive cells from Go to G1 following stimulation of quiescent cells by growth factors. However, the c-myc gene is not unique in terms of having an essential role in cellular growth processes. It has been known for decades that specific and timely changes in the expression of multiple genes are required for proper embryonic development and cell maturation (20). Expression of genes such as c-myc seems to be regulated not only at the levels of transcription, attenuation, nuclear processing, and translation but also at the level of mRNA turnover (reviewed in reference 41). Indeed, direct half-life measurements indicated that c-myc mRNA has a half-life of 15 to 40 min (19). These and other studies (41) demonstrated that the control of c-myc mRNA turnover might be an important means of regulating both the level and the timing of c-myc expression.Many proto-oncogene mRNAs are very unstable. The rapid turnover of c-myc mRNA is controlled by sequences in the 3' untranslated region (3'UTR) or by coding region sequences (reviewed in references 33 and 60). A common feature of many labile mRNAs, such as those for c-myc, c-fos, and granulocyte-macrophage colony-stimulating factor (GM-CSF), is the presence of an AU-rich element (ARE) in the 3'UTR which is one cis-acting element responsible for their rapid degradation (reviewed in reference 3, 48, and 60). It AUUUA (70). This might be important because...

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“…Among the WGA-binding pore complex glycoproteins of Xenopus oocytes we have recently identified a major component of Mr 68,000 (p68) which is confined to the pore channel and crucially involved in nuclear protein transport processes (Dabauvalle et al 1988 b; see also Featherstone et al 1988). This pore complex glycoprotein is evolutionarily highly conserved and has been detected in a variety of species ranging from insects to mammals by means ofimmunofluorescence and immunoblotting studies employing the monoclonal antibody Pl1 (Chaly et al 1984(Chaly et al , 1986Benavente et al 1989 a, b;Dabauvalle et al 1988 b). Furthermore, microinjection of antibody PI1 inhibited nuclear protein uptake not only in Xenopus oocytes (Dabauvalle et al 1988 b) but also in mammalian cells (Benavente et al 1989 a, b).…”

Section: Introductionmentioning

confidence: 99%

Identification of a soluble precursor complex essential for nuclear pore assembly in vitro

1990

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Abstract. We analysed the soluble form in which the nudear pore complex protein p68 is stored in Xenopus /acpis eggs and its involvement in pore complex assembly processes, We have shown previously that p68, which is the major wheat germ agglutinin (WGA)-binding glycoprotein 01' nudear pore complexes from Xcnopus 00-cytes, is located in the pore channel and partieipates in mediated transport 01' karyophilic proteins. Using a monodonal antibody direeted against p68 (PlI) we re11l0ved this protein from XCIlOPUS egg extract by immunoadsorption, On addition 01' lambda DNA the immunodepleted extraet supported reconstitution ofnudei which were surrounded by a continuous double-membrane envdope but lacked pore complexes and were unable to import karyophilic pro teins such as nudeoplasmin or lamin L m , Essentially identical results were obtained with extract depleted 01' WGA-binding proteins. Our finding that both the anti-p68 antibody and WGA efficiently removed components from the extraet neeessary lor pore complex assembly but did not interfere with nudear membrane formation demonstrates that these processes are independent 01' each other. Analysis 01' the immunoprecipitate on silver-stained SDS-polyaerylamide gels indicated that the antibody adsorbed other proteins besides p68, notably two high molecular weight components. By sucrose gradient centrifugation and gel Illtration we showed that p68 together with associated protein(s) forms a stable, approximately globular eomplex with an M, 01' 254,000, a Stokes radius 01' 5.2 nm and a sedimentation coelTicient 01' 11.3 S. Our tinding that p68 occurs in the form 01' larger maeromolecular assemblies offers an explanation for the distinetly punctale immunofluorescence pattern observed in the eytoplasm 01' mitotic cells after staining with antibodies to p68.

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Monoclonal antibodies against nuclear matrix detect nuclear antigens in mammalian, insect and plant cells: An immunofluorescence study

Cited by 22 publications

References 12 publications

Functional role of newly formed pore complexes in postmitotic nuclear reorganization

Functional role of newly formed pore complexes in postmitotic nuclear reorganization

Purification, characterization, and cDNA cloning of an AU-rich element RNA-binding protein, AUF1.

Identification of a soluble precursor complex essential for nuclear pore assembly in vitro

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