Edmond Puvion

Nucleolar organization of HeLa cells as studied by in situ hybridization

The distribution of the ribosomal genes and their ribosomal RNA (rRNA) products in the different compartments of the nucleolus of HeLa cells was examined on thin sections of Lowicryl embedded material. The ribosomal nucleic acids were visualized after hybridization with a set of biotinylated double-stranded ribosomal DNA (rDNA) probes from different locations along the gene, followed by immunogold labelling of biotin. Ribosomal genes were detected over both the entire fibrillar centres (FCs) and some masses of intranucleolar condensed chromatin. As for the rRNA components, comparison of the signal levels obtained with the different probes provides some information about the compartmentalization of distinct stages of ribosome biogenesis. Thus a probe specific for the 5′ external transcribed spacer (5′ETS) portion of pre-rRNA labels almost exclusively the dense fibrillar component (DFC) and the border of the FCs, while the interior of the FCs appears devoid of any kind of rRNA species. By contrast, probes recognizing either 18S or 28S mature rRNA sequences label both the DFC and the granular component (GC). Moreover, mature 18S rRNA sequences are markedly under-respresented relative to mature 28S rRNA sequences in the GC, as compared with the other nucleolar compartments. Our observations are consistent with the view that DFCs contain elongating and 47S–45S precursor rRNA molecules whereas the subsequent various rRNA processing intermediates are mainly located within the GC. Since the border of FCs is the only site where both rDNA and newly synthesized pre-rRNA coexist, the transcription of ribosomal genes seems to take place at the periphery of the FCs, and not in the DFC, suggesting that elongating and newly completed transcripts are immediately transferred into the surrounding DFC where they transiently accumulate before undergoing processing reactions and transfer to the GC.

Early stages of pre-rRNA formation within the nucleolar ultrastructure of mouse cells studied by in situ hybridization with a 5'ETS leader probe.

The first cleavage in the processing of the rRNA primary transcript in mammals occurs within the 5′-terminal region of the 5′ external transcribed spacer (5′ETS), which makes the upstream portion of this spacer a selective marker of nascent transcripts. Moreover, short treatments with low doses of actinomycin D (AMD), which selectively suppress pre-rRNA synthesis and allow processing of preformed pre-rRNAs, result in the production of prematurely terminated transcripts essentially spanning the 5′ETS leader region. To gain further insight into the intranucleolar localization of early stages of preribosome formation we analyzed the distribution of this specific pre-rRNA segment by in situ hybridization at the ultrastructural level in AMD-treated or in control 3T3 mouse cells. In control cells, 5′ETS leader rRNA was detected at the border of the fibrillar centers and over the dense fibrillar component, in agreement with previous data suggesting that rRNA gene transcription takes place at the border of the fibrillar centers before a rapid transfer of the nascent transcript to the dense fibrillar component. Observation of cells subjected to a short treatment with low doses of AMD fully supports this conclusion, with the prematurely terminated 5′ETS leader-containing transcripts detected at the border of enlarged fibrillar centers. With prolonged periods of AMD treatment even the partial transcription of rRNA genes is blocked and fibrillar centers of typically segregated nucleoli show no positive signals with the 5′ETS leader probe. We also analyzed in parallel the intranucleolar distribution of U3 small nucleolar RNA, which is involved in 5′ETS processing, by hybridization with biotinylated antisense oligonucleotides. Distribution of U3 roughly paralleled that of 5′ETS leader rRNA in untreated cells. However, U3 RNA persisted in the dense fibrillar component of segregated nucleoli whatever the conditions of drug treatment, i.e., even after a thorough chase of the rRNA precursors from this nucleolar compartment.

Functional significance of perichromatin granule accumulation induced by cadmium chloride in isolated rat liver cells

Abstract The addition of cadmium chloride (CdCl 2 ) to the culture medium of isolated rat liver cells induced rapid nuclear lesions. As observed by biochemical analysis and high resolution autoradiography after labeling the cells with [5- 3 H]uridine, CdCl 2 induced a preferential inhibition of nucleolar RNA synthesis and a slowing down of the processing of both extranucleolar polyadenylated and nucleolar non-polyadenylated RNA molecules synthesized before the action of CdCl 2 . Inhibition of rRNA synthesis was accompanied by a condensation of nucleoli. Disturbance of hnRNA and rRNA processing induced accumulation of large numbers of perichromatin granules contiguous to and within extranucleolar and nucleolar condensed chromatin, respectively, with concomitant loss of interchromatin fibrils. Autoradiographs after 15 min labeling followed by 3 h chase in the presence of CdCl 2 revealed that all of the incorporated uridine remained over the perichromatin fibrils and granules at the border of the condensed chromatin. These observations support our hypothesis that perichromatin granules represent storage forms of recently synthesized RNA, and that there may be at least two types (hnRNA and rRNA) of perichromatin granules.

Intranuclear migration of newly synthesized extranucleolar ribonucleoproteins. A high resolution quantitative autoradiographical and cytochemical study.

Abstract Isolated rat liver cells were pulse-labeled with tritiated uridine after stimulation of transcription by hydrocortisone. The ribonucleoproteins (RNP) were revealed by ultrastructural cytochemistry and the radioactivity as visualized by high resolution autoradiography was measured by the hypothetical grain method. Immediately after labeling 70% of the nuclear radioactivity was concentrated in the dense chromatin border that also contained a large quantity of newly synthesized perichromatin fibrils. During chase, the perichromatin fibrils were shown to migrate towards the interchromatin space in parallel with the radioactivity, while labeling of the perichromatin border decreased to a very low level. The interpretation of these results is discussed in connection with our knowledge of the heterogeneous nuclear ribonucleoprotein (HnRNP) particles.

Analysis by in Situ hybridization and autoradiography of sites of replication and storage of single- and double-stranded adenovirus type 5 DNA in lytically infected HeLa cells

The distribution in the different compartments of infected nuclei of double-stranded (ds) and single-stranded (ss) adenovirus type 5 (Ad5) DNA and of the sites of viral DNA replication were examined on thin sections of Low-icryl-embedded material. The DNA is visualized with a biotinylated viral probe and immunogold labeling of biotin, and its replication is monitored by high-resolution autoradiography after short pulses with tritiated thymidine. The first detectable sites of viral DNA, named early replicative sites, contained all the ss and ds viral DNA and viral replicative activity. At a later stage of nuclear transformation, they gave rise to two new structures. The compact fibrillar ssDNA accumulation sites enlarged greatly and became transformed functionally to become a transient site of accumulation of large numbers of ss replicative intermediates. Double-stranded viral DNA and its replicative activity shifted primarily into immediately surrounding fibrillogranular peripheral replicative zones. Ad5 DNA replication continues in the ssDNA accumulation sites but it is intermittent, whereas in the peripheral replicative zones it is continuous. Still later in infection, a single, large, centrally located mass of dense fibrils, the viral genome storage site, developed in each nucleus which proved to be the main site of storage of nonreplicating, nonencapsidated, ds viral genomes. We discuss the possible distribution of the various viral DNA replicative intermediates among these virus-induced intranuclear structures.

Relationship between chromatin and perichromatin granules in cadmium-treated isolated hepatocytes

The relationship of perichromatin granules (PG) to transcribing DNA fibrils and nascent hnRNA perichromatin fibrils (PF) was studied in isolated rat liver cells treated with cadmium chloride (CdCl 2 ) in order to increase the number of PG. Demonstration of their in situ organization was made possible by submitting the CdCl 2 -treated cells to a procedure which induced a partial decondensation of nuclear components. These decondensed preparations were combined with cytochemical stains for DNA and RNP, enzymic digestions with DNase and RNase, and autoradiography after pulse labeling with [5- 3 H]uridine, and the results were compared with Miller spreads of completely decondensed chromatin. We found that many if not all PG were attached to DNA fibers by short RNP fibrils. On the basis of their labeling with 5-min pulses of [5- 3 H]uridine and the reported effect of CdCl 2 to block the processing but not the synthesis of hnRNA, we propose that the PG induced by CdCl 2 develop during the course of transcription by the coiling of nascent RNP fibrils.

Visualization of two different types of nuclear transcriptional complexes in rat liver cells

Transcriptional complexes were visualized by a modification of the method of Miller and Beatty in the chromatin of isolated and cultured rat liver cells. Under our experimental conditions, spread DNA molecules showing transcriptional activity were extremely rare. Nevertheless, two different patterns were observed in most preparations: (i) multiple “Christmas-tree”-like figures, comparable in length and symmetry to the now classical forms and probably related to ribosomal RNA transcription; (ii) twisted fibrils in single units along a DNA molecule, irregular both in the length of the transcriptional complex and particularly in the length of the side branches. These latter are thought to represent non-nucleolar transcription.

Sites of transcription of adenovirus type 5 genomes in relation to early viral DNA replication in infected HeLa cells. A high resolution in situ hybridization and autoradiographical study

Summary— The distribution of viral RNA molecules in HeLa cells infected with adenovirus type 5 (Ad5) was determined by in situ hybridization at the ultrastructural level at an intermediate stage of nuclear transformation, when viral DNA synthetic activities were maximal but progeny viruses were still sparse. Transcription sites of the viral DNA were localized by short pulse, high resolution autoradiography. Nascent viral RNA was found mainly within the nuclear compartment identified at the peripheral replicative zone, which is known to be the main replicative site of Ad5 viral genomes. Viral RNA molecules also were present, but to a markedly lesser extent, within the contiguous single‐stranded (ss) DNA accumulation site, another intranuclear virus‐induced structure in which some replication of viral genomes also occurs. Two other virus‐induced nuclear structures contained viral RNA, the occasional exceptionally enlarged clusters of interchromatin granules and the compact rings, both DNA‐free structures of unknown significance but which might play a role in the process of maturation of the Ad5 primary transcripts. Viral messenger RNA molecules were localized over the large areas of the cytoplasm which contain numerous ribosomes. Our analysis of the effects of various enzymatic pretreatments of the sections of infected cells on the revelation of nascent RNA by in situ hybridization is reviewed.

Human and mouse MOK2 proteins are associated with nuclear ribonucleoprotein components and bind specifically to RNA and DNA through their zinc finger domains.

The human and murine MOK2 ortholog genes that are preferentially expressed in brain and testis tissues encode two different Krüppel-like zinc finger proteins. In this paper, we show that the MOK2 proteins are mainly associated with nuclear ribonucleoprotein components, including the nucleoli and extranucleolar structures, and exhibit specific RNA homopolymer binding activities. Moreover, we have identified an identical 18-bp specific DNA binding sequence for both MOK2 proteins using a pool of random sequence oligonucleotides. The DNA binding domain is localized in the seven adjacent zinc finger motifs, which show 94% identity between human and murine proteins. Taken together, these results establish that the MOK2 proteins are able to recognize both DNA and RNA through their zinc fingers. This dual affinity and the subnuclear localization suggest that MOK2 may play roles in transcription, as well as in the posttranscriptional regulation processes of specific genes.

The nucleus of euglena. I. An Ultracytochemical study of the nucleic acids and nucleoproteins of synchronized Euglena gradlis Z.

The ultrastructure of the interphase nucleus of Euglena gracilis Z was studied with techniques of cryoultramicrotomy and specific or preferential staining of nucleoproteins. The usual nucleolar components including RNP granules and fibrils and DNA fibrils were found in the endosome, which therefore must be considered as a true nucleolus with a special structure. Three nucleoplasmic components were identified: a fibrillar RNP matrix, thicker U-shaped fibrils sensitive to double digestion by a protease, and RNase, and finally, dense granules. Neither perichromatin fibrils nor granules were seen. DNA links between the permanently condensed chromosomes were demonstrated. Functions and interrelations of the nuclear components are discussed.