Ultrastructural Methods for Nucleic Acid Detection by Immunocytology

Thiry, Marc

doi:10.1016/s0079-6336(99)80008-4

Cited by 13 publications

(9 citation statements)

References 217 publications

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“…For immunogold labeling, strains were fixed for 16 h at 4°C in 4% formaldehyde in 0.1 M phosphate buffer (0.1 M KH 2 PO 4 , 0.1 M K 2 HPO 4 [pH 6.5]) containing 10% sucrose, dehydrated through graded acetone solutions, and embedded in Lowicryl K4 M. Immunogold detection was performed according to classical procedures (28). Nop1p was detected with monoclonal antibody mA66 (a kind gift of J. Aris, University of Florida) diluted 1:20.…”

Section: Methodsmentioning

confidence: 99%

The Small Nucle(ol)ar RNA Cap Trimethyltransferase Is Required for Ribosome Synthesis and Intact Nucleolar Morphology

Colau

Thiry

Leduc

et al. 2004

Molecular and Cellular Biology

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Nucleolar morphogenesis is a poorly defined process. Here we report that the Saccharomyces cerevisiae nucleolar trimethyl guanosine synthase I (Tgs1p), which specifically selects the m 7 G cap structure of snRNAs and snoRNAs for m 2,2,7 G conversion, is required not only for efficient pre-mRNA splicing but also for pre-rRNA processing and small ribosomal subunit synthesis. Mutational analysis indicates that the requirement for Tgs1p in pre-mRNA splicing, but not its involvement in ribosome synthesis, is dependent upon its function in cap trimethylation. In addition, we report that cells lacking Tgs1p showed a striking and unexpected loss of nucleolar structural organization. Tgs1p is not a core component of the snoRNP proteins; however, in vitro, the protein interacts with the KKD/E domain present at the carboxyl-terminal ends of several snoRNP proteins. Strains expressing versions of the snoRNPs lacking the KKD/E domain were also defective for nucleolar morphology and showed a loss of nucleolar compaction. We propose that the transient and functional interactions of Tgs1p with the abundant snoRNPs, through presumed interactions with the KKD/E domain of the snoRNP proteins, contribute substantially to the coalescence of nucleolar components. This conclusion is compatible with a model of self-organization for nucleolar assembly.The driving forces that govern the dynamic behavior of cellular organelles (i.e., their shape and size and their emergence at specific cellular foci) are unknown. The concept of self-organization that describes the ability of a macromolecular complex, or organelle, to determine its own structure on the basis of the functional and transient interactions of its constituents is particularly attractive (reviewed in reference 17). In a self-organized system, a steady-state structure is generated from highly dynamic components, i.e., "ever-changing partners," without the need for a rigid architectural framework. Classically, selforganization is opposed to self-assembly, where the physical associations of molecules generate a stable, static structure that reaches a "true equilibrium" (e.g., viruses and phages).Ribosome synthesis is a dynamic, multistep process that takes place largely in the nucleolus, a specialized region of the nucleus that is highly enriched in RNA-processing factors (RRPs) and further compartmentalized in individual domains, which accounts for the vectorial formation of the ribosomes (reviewed in references 21 and 26). The nucleolus appears as a steady-state compartment rather than a static structure. Indeed, during pre-ribosome assembly, pre-rRNAs, ribosomal proteins (RPs), and several hundred trans-acting factors, including the small nucleolar RNAs (snoRNAs), interact transiently (reviewed in references 2 and 10). Furthermore, major nucleolar antigens, such as fibrillarin and nucleolin are continuously exchanged with the surrounding nucleoplasm (1, 22; reviewed in reference 18).The spliceosomal RNAs U1, U2, U4, and U5, as well as several snoRNAs, are individually transcribe...

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Section: Methodsmentioning

confidence: 99%

The Small Nucle(ol)ar RNA Cap Trimethyltransferase Is Required for Ribosome Synthesis and Intact Nucleolar Morphology

Colau

Thiry

Leduc

et al. 2004

Molecular and Cellular Biology

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“…However, to facilitate the identification of completely or partially labelled cells in mixtures, our in vivo incorporation of BrdU into bacterial DNA was combined with the in situ terminal deoxynucleotidyl transferase (TdT)-immunogold labelling procedure, a method allowing study of the general distribution of DNA in biological material with high specificity and high sensitivity (Thiry, 1999). In the TdT technique, TdT in the presence of modified nucleotides elongates DNA ends generated by sectioning.…”

Section: Microscopical Visualization Of Cell Fusionmentioning

confidence: 99%

“…A large variety of immunocytological methods are now available for studying DNA in situ in biological material (Thiry, 1999). These methods, combined or not with molecular biotechnology, are powerful tools for studying the structure and function of DNA.…”

Section: Cell Fusion: a Likely Mechanismmentioning

confidence: 99%

Spontaneous zygogenesis in Escherichia coli, a form of true sexuality in prokaryotes

Gratia¹,

Thiry

2003

Microbiology

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A new type of mating, differing from classic conjugation and previously observed in a certain strain of Escherichia coli K-12, has also been found in strains derived from ordinary F " cells of E. coli K-12 exposed to an exogenous factor originating in an E. coli clinical isolate.Immunofluorescence and electron microscopy after single and double labelling of DNA were used to produce evidence in favour of a novel mating mechanism by cell contact at the poles of the bacterial rod. These findings are supported by genetic analyses indicating complete genetic mixing. Unstable complementing diploids were formed, which throw off phenotypically haploid cells, of which some showed a parental phenotype and some were true genetic recombinants. Recombination was observed even when one parent was a UV-inactivated F " RecA " strain. The name 'spontaneous zygogenesis' (Z-mating, for short) is proposed for this kind of mating.

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“…Bromodeoxyuridine, a nonradioactive thymidine analogue, is incorporated into nuclear DNA during its replication and can subsequently be detected by immunocytochemistry (Thiry, 1995). Bromodeoxyuridine, a nonradioactive thymidine analogue, is incorporated into nuclear DNA during its replication and can subsequently be detected by immunocytochemistry (Thiry, 1995).…”

Section: Chromosome Segregationmentioning

confidence: 99%

Nucleus behavior during the closed mitosis of Tritrichomonas foetus

Ribeiro

Mariante

Coutinho

et al. 2002

Biology of the Cell

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We present observations on the fine structure and the division process of the nucleus in the protist Tritrichomonas foetus, parasite of the urogenital tract of cattle. The nucleus was followed by immunofluorescence and electron microscopy during interphase and mitosis. Conventional karyotyping coupled to image processing and bright field Panotic staining were used to follow nucleus modifications, chromosome number and condensation pattern along the whole cell cycle. Confocal laser scanning microscopy (CLSM) using DNA fluorescent probes, followed by image processing in the SURF-Driver program, produced three-dimensional reconstruction data of the mitotic nucleus under each phase of the division process. Immunocytochemistry in thin-sections revealed the chromosome spatial arrangement after bromodeoxyuridine incorporation and immunogold labeling using anti-DNA monoclonal antibodies. Our results indicate that: (1) the nucleus assumes different size and shapes along mitosis: it appears oval in interphase, becoming lobed or concave in prophase, then undergoing torsion and constriction, displaying an 'S' shape (metaphase). Next, it becomes elongated and it is finally separated in two nuclei at the transition of anaphase to telophase; (2) T. foetus nucleus harbors five chromosomes; (3) chromosomes become condensed in a pre-mitotic phase; (4) the nucleolus persists during the mitosis.

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Ultrastructural Methods for Nucleic Acid Detection by Immunocytology

Cited by 13 publications

References 217 publications

The Small Nucle(ol)ar RNA Cap Trimethyltransferase Is Required for Ribosome Synthesis and Intact Nucleolar Morphology

The Small Nucle(ol)ar RNA Cap Trimethyltransferase Is Required for Ribosome Synthesis and Intact Nucleolar Morphology

Spontaneous zygogenesis in Escherichia coli, a form of true sexuality in prokaryotes

Nucleus behavior during the closed mitosis of Tritrichomonas foetus

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