Localization of nucleolar organizer region proteins and ribonucleoproteins

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7"1(;- May 1985

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PrentkJ, P. and Krisch, H. M. (1984) Gene29, 303 313

Localization of nucleolar organizer region proteins and ribonucleoproleins

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Specific one-step Ag-NOR (Nucleolar Organizer Region) silver staining followed by acetylation can be used for the precise localization of the AgNOR proteins during all phases of mitosis. When sections are contrasted with uranyl acetate and lead citrate, the contrast of the chromatin is high whereas that of RNP structures (nucleolus, peri- and inter-chromatin fibrils and granules) is low. The regular silver dots ( 5 0 - 1 0 0 ) t in diameter) are specifically localized within fibdllar centres and part of the dense fihrillar component. The granular component, which is easily localized, is not stained.

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Fig. 1. General strategy/or in vitro mutagenesis of circular DNA using the Omega t ~ fragment The plasmid fi)r mutagenesis is linearized and its ends made blunL The 2 kb Sma/Omega fragment i.~th~a added and blunt end liga. tion is poforrned Every Sm'/Spc" transformant carries an in.~,rtion of ft. and is a mutant whose phenotype can be analysed in vivo or in vitro. When necessary, the antibiotic-resistance gene (thick line) as well as the tran.wnption terminators (arrows) can be removed by Hind///digestion and religation. The mutant plasmid thus obtained diffi'rs from the original one by a 52 bp palindromic insertion containing TGA translation termination codons on both DNA strands in all three phases, and a H indlll and two BamHl sites. The translation terminators can in turn be remot~d by BamHl digestion and religation, leaving behind a 10 bp insertion (containing a Barn///site) at the position at which the lineagtzatien occurred. The BamH/recircularization can also be performed on the original fl mutant

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Fig. 2. L~calization of the Ag-NOR imdeins during prophase m hunmn breast cancerous tissues (M. The bleached arndenslng chromosomes (c) are preferenflail)' localized along with the nuclear enoek,pe. Nucleoli trd linked set~,ul c h ~ . The Ag-NOR proteins are seen within the nucleolu.~(arrowhead) or in contact with the c h ~ (arrows). EDTA regressive staining, x 16000. (B and 0. 7"u~ serial but ru)t consez'utivesections of contrast between a nucleolus and c h ~ . (8 The Ag-NOR proteins are krcalized within the R N P fibrillar component of the nucleolus (an'owheadL or exlmded from the nuc.leolus and integrated within the c h ~ s (arrow,). EDTA regrez,sive staining, x 46000. (OOnthistangentialsectionthe Ag-NORproteinsareseen within contorled zones that extend on or into the chrmnosome. ED TA regressive staining, x 46000. (D) The Ag-NOR proteins are localized within irregular ~trusions of (hefibn'llar comporu'nt in contact with the chromosomes (arrou'.s) The rest of the nucleolus was not silver stained and shows micro-segregation and disorganization (appearance of a central vacuole) (v). Umnyl and lead staining. x 46000 (Reproduced by permi.~qionfrom Ploton et al. }

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When sections are preferentially contrasted for RNP by EDTA regressive staining, the chromatin clumps are well bleached whereas RNP components show a high contrast. Silver dots are not displaced by the EDTA treatment. The bleaching of the chromosomes greatly enhances the precision of the localization of the AgNOR proteins and allows them

to be studied, particularly during nucleolar disintegration and reformation (see Fig. 2). Ploton, D., Menager, M aml Adnet, ]. J. Simultaneous ultrastructural localization of Ag-NOR (Nucleolar Organizer Region) proteins and ribenucleoproteins during mitoses, in human breast cancerous ti~ues. J. Cell Sci 74, April, (in press).