High‐resolution autoradiography as a tool for the localization of nucleic acid synthesis and distribution in the mammalian cell nucleus

Abstract

Several examples of the application of high resolution autoradiography to the study of nucleic acid distribution in ultrathin sections of fixed and plastic embedded or frozen material are presented. Newly-synthesized DNA, labelled by very short pulses of 3H-thymidine is found to be localized throughout the nucleus. In the blastomeres of early mouse embryos developing after fertilization by 3H-thymidine-labelled spermatozoa, the labelled paternal DNA is distributed non-homogeneously in the nucleus. Finally some results obtained by using a cytochemical enzymatic technique for visualization of DNA directly in ultrathin sections of fixed and embedded cells are demonstrated. Concerning the distribution of RNA, perichromatin localization of rapidly transcribed RNA is described in different cell systems. This label can be attributed to perichromatin fibrils. In cells labelled for longer periods of time, sometimes followed by prolonged postincubations, a residual label is always found over the nucleus. Clusters of interchromatin granules are usually only weakly labelled or unlabelled. These structures probably contain a limited amount of rather slowly labelled RNA. The present results are discussed in the context of some biochemical evidence and of the data described by other investigators.

Dynamics and three-dimensional localization of ribosomal RNA within the nucleolus

“…The approach that we have employed uses natural cellular mechanisms to introduce nucleotide analogs into intact cells+ We show that FuGene-6, in addition to functioning as a transfectant for plasmids, efficiently and rapidly mediates the uptake of the halogenated base analog BrUTP into cultured cells+ Although other lipid transfection reagents have been successfully used as vectors for the uptake and incorporation of BrUTP into growing cells (Haukenes et al+, 1997;Kanestrom et al+, 1998), we demonstrate that lipofection with BrUTP can be a useful tool for studying transport of newly labeled rRNAs in intact cells either within the nucleus or from the nucleus to the cytoplasm+ In summary, this nondestructive method presents several decisive advantages which permits (1) the complementary ultrastructural and optical investigations of the sites in which rRNA elongation and processing take place by using the same labeling conditions; (2) a more resolved ultrastructural identification of the nucleolar components containing the BrUTP-labeled RNAs, compared to previous autoradiographic studies (Fakan, 1978; (3) the detailed analysis of the volumic organization of the nucleolar components containing the BrUTP-labeled RNAs and of their modification along time by using confocal microscopy, three-dimensional reconstruction, and visualiza- Our ultrastructural data demonstrate that ribosome biogenesis starts in the FC and in the inner part of the DFC, then migrates rapidly in the outer part of the DFC and in the GC+ The three-dimensional reconstruction and visualization, by giving a global view, clearly indicate that the transport of rRNAs within the nucleolus occurs neither randomly nor along tracks as for some mRNA (Lawrence et al+, 1989;Smith et al+, 1999)+ On the contrary, we demonstrate that transport of rRNA follows a volumic, well-defined pathway from the inner part of the nucleolus toward its periphery+ This migration can be summarized by four typical labeling patterns, which could correspond to four main functional domains+…”

“…Since it became evident that cell nuclei are compartmentalized organelles (Spector, 1993;Strouboulis & Wolffe, 1996;Singer & Green, 1997;Lamond & Earnshaw, 1998), one of the major goals in cell biology has been to understand their functional organization+ In particular, the nucleolus provides the fascinating possibility of linking morphologically distinct structures such as those seen in the electron microscope with biochemical features of the synthesis of rRNAs and stepwise maturation of ribosomes+ In eukaryotic cells, the synthesis and maturation of cytoplasmic rRNAs mainly take place in the nucleolus (Hadjiolov, 1985;Sollner-Webb et al+, 1996)+ The 18S, 5+8S, and 25/28S rRNAs are synthesized as simple precursor RNAs (pre-rRNAs), which contain long external and internal transcribed spacers+ The primary transcripts are processed through a complex pathway of endonucleolytic cleavages, exonucleolytic digestions, and covalent modifications+ These pre-rRNAs are assembled with ribosomal and nonribosomal proteins in addition to the separately transcribed 5S rRNAs+ In this way, the large and small preribosomal particles are formed and subsequently released from the nucleolus into the cytoplasm, where they assembled into fully functional ribosomes+ Using cytological methods, considerable efforts have been devoted to the identification of the nucleolar sites at which the various biochemical events in rRNA synthesis take place+ In the mammalian cell nucleolus, three major substructures have been visualized by electron microscopy: the fibrillar center (FC), the dense fibrillar component (DFC), and the granular component (GC) + Most studies dealing with the nucleolus have been focused on the identification of rDNA transcription sites (Shaw & Jordan, 1995;Scheer & Hock, 1999)+ There is a general consensus that transcription occurs in the fibrillar regions of the nucleolus, but whether it is within the DFC, or at the border of the DFC and the FC, or even within the FC, remains a matter of much debate+ Kinetic information concerning the synthesis and maturation of pre-rRNA molecules within the nucleolus of intact, active cells is equally sparse+ Autoradiographic studies (Fakan, 1978) have consistently shown that when rRNAs are labeled with tritiated uridine for short periods of time, the signal is uniquely localized on the fibrillar part of the nucleolus+ The GC only becomes labeled after longer periods of radioactive incubation and/or after a cold chase+ In complementary biochemical studies (Daskal et al+, 1974;Royal & Simard, 1975), 45S pre-rRNAs appear restricted to the fibrillar component while 32S (i+e+, pre-28S) rRNAs are also located in the GC+ Together, these data suggest a migration of pre-rRNA molecules from the fibrillar component into the GC+ However, autoradiography displays a projection onto a plane of the three-dimensional data, thus discarding the possibility of dealing with the question of the spatial organization of the labeling+…”

New approaches to in situ detection of nucleic acids

“…Furthermore, the 5' end of single-stranded DNA present at the surface of ultrathin sections can, in principle, be labeled. Unlike autoradiography after uptake of labeled ribonucleotides (Bouteille et al 1975;Fakan 1976Fakan , 1978, the in situ-in vitro transcription technique avoids the danger of displacement of newly synthetized molecules from the site of synthesis to the site of accumulation. With the latter approach, however, as with the in situ nick translation-immunogold technique (Thiry 1991a, b), it is not possible to distinguish transcriptionally active from potentially active regions.…”

“…Compared to all the other techniques used so far to detect DNA, the in situ TdT method also shows a more general distribution of nucleic acids in biological materials. The autoradiographic techniques allow detection of only a fraction of the DNA, that which was freshly synthetized by replication during a certain period of time (Bouteille et al 1975;Fakan 1976Fakan , 1978. Likewise, the DNA configuration affects the labeling pattern obtained with DNase I-gold complexes (Bendayan 1981a(Bendayan , b, 1984.…”

Ultrastructural Autoradiographic Analysis of RNA in Isolated Human Lung Mast Cells during Secretion and Recovery from Secretion

“…Extensive literature exists on the nature of rapidly labeled and slowly labeled RNA-rich cellular organelles and domains using kinetic studies of the incorporation of radiolabeled uridine [5, 9, 10, 11, 12, 17, 54, 55, 56]. Generally, the most rapidly labeled RNA is heterogeneous nuclear RNA, some of which, after splicing and polyadenylation to form mRNA, is exported to the cytoplasm.…”