New approaches to in situ detection of nucleic acids

Thiry, Marc

doi:10.1007/bf01451570

Cited by 12 publications

(10 citation statements)

References 70 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This co-localization, visualized directly from the isotope images, avoids the complications potentially introduced by immunochemical methods [33]. Localization of newly synthesized RNA requires distinguishing a local excess of 15 N over its natural occurrence.…”

Section: Resultsmentioning

confidence: 99%

High-resolution quantitative imaging of mammalian and bacterial cells using stable isotope mass spectrometry

Lechène

Hillion

McMahon

et al. 2006

J Biol

306

207

View full text Add to dashboard Cite

BackgroundSecondary-ion mass spectrometry (SIMS) is an important tool for investigating isotopic composition in the chemical and materials sciences, but its use in biology has been limited by technical considerations. Multi-isotope imaging mass spectrometry (MIMS), which combines a new generation of SIMS instrument with sophisticated ion optics, labeling with stable isotopes, and quantitative image-analysis software, was developed to study biological materials.ResultsThe new instrument allows the production of mass images of high lateral resolution (down to 33 nm), as well as the counting or imaging of several isotopes simultaneously. As MIMS can distinguish between ions of very similar mass, such as 12C15N- and 13C14N-, it enables the precise and reproducible measurement of isotope ratios, and thus of the levels of enrichment in specific isotopic labels, within volumes of less than a cubic micrometer. The sensitivity of MIMS is at least 1,000 times that of 14C autoradiography. The depth resolution can be smaller than 1 nm because only a few atomic layers are needed to create an atomic mass image. We illustrate the use of MIMS to image unlabeled mammalian cultured cells and tissue sections; to analyze fatty-acid transport in adipocyte lipid droplets using 13C-oleic acid; to examine nitrogen fixation in bacteria using 15N gaseous nitrogen; to measure levels of protein renewal in the cochlea and in post-ischemic kidney cells using 15N-leucine; to study DNA and RNA co-distribution and uridine incorporation in the nucleolus using 15N-uridine and 81Br of bromodeoxyuridine or 14C-thymidine; to reveal domains in cultured endothelial cells using the native isotopes 12C, 16O, 14N and 31P; and to track a few 15N-labeled donor spleen cells in the lymph nodes of the host mouse.ConclusionMIMS makes it possible for the first time to both image and quantify molecules labeled with stable or radioactive isotopes within subcellular compartments.

show abstract

Section: Resultsmentioning

confidence: 99%

High-resolution quantitative imaging of mammalian and bacterial cells using stable isotope mass spectrometry

Lechène

Hillion

McMahon

et al. 2006

J Biol

306

207

View full text Add to dashboard Cite

show abstract

“…The presence of DNA in the FCs was strongly suggested by data obtained by combining, at EM level, the silver staining procedure for the AgNOR proteins with the Feulgen-like osmium ammine staining specific for DNA (Derenzini et al 1981a;Hernandez-Verdun et al 1982): a constant overlapping of silver grains with DNA filaments was in fact observed in interphase cell nucleoli of human cell lines (Hernandez-Verdun et al 1984;Derenzini et al 1990). Subsequently, the presence of DNA within the FCs was unambiguously demonstrated in various cell types by different immunocytological approaches, in particular, using the terminal deoxynucleotidyl transferase-immunogold technique which allows the specific detection of DNA on ultrathin sections in very well-preserved cell structures, identified by routine uranium and lead staining (Thiry 1992a(Thiry ,b,1993(Thiry ,1995Thiry et al 1993). EM in situ hybridization studies ThiryBlaise 1989,1991;Puvion-Dutilleul et al 1991,1992 have further indicated that some rRNA genes are generally located in the intra-and perinucleolar chromatin but are also present in the FCs.…”

Section: Localization Of Ribosomal Genes Within the Nucleolar Componentsmentioning

confidence: 99%

Structural and Functional Organization of Ribosomal Genes within the Mammalian Cell Nucleolus

Derenzini

Pasquinelli

O’Donohue

et al. 2006

J Histochem Cytochem.

Self Cite

View full text Add to dashboard Cite

S U M M A R Y Data on the in situ structural-functional organization of ribosomal genes in the mammalian cell nucleolus are reviewed here. Major findings on chromatin structure in situ come from investigations carried out using the Feulgen-like osmium ammine reaction as a highly specific electron-opaque DNA tracer. Intranucleolar chromatin shows three different levels of organization: compact clumps, fibers ranging from 11 to 30 nm, and loose agglomerates of extended DNA filaments. Both clumps and fibers of chromatin exhibit a nucleosomal organization that is lacking in the loose agglomerates of extended DNA filaments. In fact, these filaments constantly show a thickness of 2-3 nm, the same as a DNA doublehelix molecule. The loose agglomerates of DNA filaments are located in the fibrillar centers, the interphase counterpart of metaphase NORs, therefore being constituted by ribosomal DNA. The extended, non-nucleosomal configuration of this rDNA has been shown to be independent of transcriptional activity and characterizes ribosome genes that are either transcribed or transcriptionally silent. Data reviewed are consistent with a model of control for ribosome gene activity that is not mediated by changes in chromatin structure. The presence of rDNA in mammalian cells always structurally ready for transcription might facilitate a more rapid adjustment of the ribosome production in response to the metabolic needs of the cell. (J Histochem Cytochem 54:131-145, 2006)

show abstract

“…Later, around anaphase, many of these components form small condensed cytoplasmic structures, which increase in size and number as the cells pass from anaphase to telophase (44 -49). These structures, which re-enter the nuclei around telophase, have been called MIGs, as ultrastructural studies have shown that they are equivalent to interphasic IGCs (48,50,51). During mitosis, the recombinant SRcyp, overproduced in human U2OS cells, behaved like factors involved in the regulation of transcription and splicing of pre-mRNA.…”

Section: Fig 3 the Cdc2-cyclin B Complex Phosphorylates The Cad Framentioning

confidence: 99%

The Human Nuclear SRcyp Is a Cell Cycle-regulated Cyclophilin

Dubourg

Kamphausen

Weiwad

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

) are found only in organisms of the animal kingdom and share several structural and enzymatic features. The presence of serine/arginine (S/R) dipeptide repeats in their C-terminal tail suggests that these enzymes belong to the SR protein family involved in the regulation of gene expression. The function of this group of cyclophilins is currently unknown. However, their C-terminal tails contain a highly conserved polypeptide signature segment (the moca domain), which may well be involved in the functional regulation of these proteins. We report here the identification of five Cdc2-type phosphorylation sites gathered in and around the moca domain of SRcyp, a human cyclophilin belonging to the Moca family. The segment of SRcyp containing the identified sites is specifically phosphorylated in mitotic cells. This mitosis-specific phosphorylation was inhibited by treatment of the cells with roscovitine, a specific inhibitor of cyclin-dependent kinases, suggesting that the unknown activity of the moca domain of SRcyp requires mitotic regulation by the Cdc2-cyclin B kinase complex. The Cdc2-cyclin B complex was found to phosphorylate four of the five identified phosphorylation sites in vitro, providing further support for this possibility. Like many factors stored in nuclear speckles and involved in the regulation of gene expression, this nuclear cyclophilin displays a predominantly diffuse cytoplasmic distribution at the onset of mitosis. Only in late telophase is SRcyp recruited to the newly formed nuclei. The transit of SRcyp through mitotic interchromatin granule clusters, before re-entering the nucleus, suggests that the timing of the appearance of this cyclophilin in the telophasic nuclei is tightly coordinated with post-mitotic events. Human SRcyp is the first cell cycle-regulated cyclophilin to be described.

show abstract

New approaches to in situ detection of nucleic acids

Cited by 12 publications

References 70 publications

High-resolution quantitative imaging of mammalian and bacterial cells using stable isotope mass spectrometry

High-resolution quantitative imaging of mammalian and bacterial cells using stable isotope mass spectrometry

Structural and Functional Organization of Ribosomal Genes within the Mammalian Cell Nucleolus

The Human Nuclear SRcyp Is a Cell Cycle-regulated Cyclophilin

Contact Info

Product

Resources

About