RNA-DNA Hybrids at the Cytological Level

John, Huw A.; Birnstiel, Max L.; Jones, K. W.

doi:10.1038/223582a0

Cited by 539 publications

(150 citation statements)

References 15 publications

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“…A powerful tool for localizing specific DNA sequences within the cell is the in situ hybridization technique performed on cytological preparations (John et al, 1969;Gall and Pardue, 1969). The application of in situ hybridization at the light microscopic level has revealed the presence of rDNA in the nucleoli of numerous cell types (for review see Hennig, 1973).…”

Section: Rdna Localization By In Situ Hybridizationmentioning

confidence: 99%

Localization of nucleolar chromatin by immunocytochemistry and in situ hybridization at the electron microscopic level

Thiry

Scheer

Goessens

1991

Electron Microscopy Reviews

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Abstract-Nucleoli are the morphological expression of the activity of a defined set of chromosomal segments bearing rRNA genes. The topological distribution and composition of the intranucleolar chromatin as well as the definition of nucleolar structures in which enzymes of the rDNA transcription machinery reside have been investigated in mammalian cells by various immunogold labelling approaches at the ultrastructural level. The precise intranucleolar location of rRNA genes has been further specified by electron microscopic in situ hybridization with a non-autoradiographic procedure.Our results indicate that the fibrillar centers are the sole nucleolar structures where rDNA, core histones, RNA polymerase I and DNA to po isomerase I are located together.Taking into account the potential value and limitations of immunoelectron microscopic techniques, we propose that transcription of the rRNA genes takes place within the confines of the fibrillar centers, probably close to the boundary regions to the surrounding dense fibrillar component.

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Section: Rdna Localization By In Situ Hybridizationmentioning

confidence: 99%

Localization of nucleolar chromatin by immunocytochemistry and in situ hybridization at the electron microscopic level

Thiry

Scheer

Goessens

1991

Electron Microscopy Reviews

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“…4D nucleome | telomeres | pericentromeric DNA | chromosomes A major advance in molecular cell biology occurred with the introduction of in situ nucleic acid hybridization in cytological preparations (1)(2)(3). The importance of this method, which has evolved into ever-more sensitive versions over the years, cannot be overstated.…”

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confidence: 99%

Multicolor CRISPR labeling of chromosomal loci in human cells

Naseri

Reyes-Gutierrez

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

378

350

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The intranuclear location of genomic loci and the dynamics of these loci are important parameters for understanding the spatial and temporal regulation of gene expression. Recently it has proven possible to visualize endogenous genomic loci in live cells by the use of transcription activator-like effectors (TALEs), as well as modified versions of the bacterial immunity clustered regularly interspersed short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system. Here we report the design of multicolor versions of CRISPR using catalytically inactive Cas9 endonuclease (dCas9) from three bacterial orthologs. Each pair of dCas9-fluorescent proteins and cognate single-guide RNAs (sgRNAs) efficiently labeled several target loci in live human cells. Using pairs of differently colored dCas9-sgRNAs, it was possible to determine the intranuclear distance between loci on different chromosomes. In addition, the fluorescence spatial resolution between two loci on the same chromosome could be determined and related to the linear distance between them on the chromosome's physical map, thereby permitting assessment of the DNA compaction of such regions in a live cell.4D nucleome | telomeres | pericentromeric DNA | chromosomes

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“…Initial reports of in situ hybridization achieved denaturation either with high temperatures or chemically, with NaOH or salts (John et al, 1969;Buongiorno-Nardelli and Amaldi, 1970;Barsacchi and Gall, 1972;Gall, 2016). Subsequent research favored the use of formamide at a concentration of 50-70 % in the hybridization buffer, in order to lower hybridization temperature and efficiently denature DNA/RNA (see for example (Barbera et al, 1979;Bauman et al, 1980;Gerhard et al, 1981;Hafen et al, 1983;Levine et al, 1983;Braissant and Wahli, 1998;Brown, 1998).…”

Section: Introductionmentioning

confidence: 99%

A safer, urea-based in situ hybridization method improves detection of gene expression in diverse animal species

Sinigaglia

Thiel

Hejnol

et al. 2018

Developmental Biology

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In situ hybridization is a widely employed technique allowing spatial visualization of gene expression in fixed specimens. It has proven to be essential to our understanding of biological processes, including developmental regulation. In situ protocols are today routine in numerous laboratories, and although details might change, they all include a hybridization step, where specific antisense RNA or DNA probes anneal to the target nucleic acids strand. This step, in general, is carried out at high temperatures and in a denaturing solution, the hybridization buffer, commonly containing 50% (v/v) formamide. An important drawback is that hot formamide poses a significant health risk and so must be handled with great care.We were prompted to test alternative hybridization solutions for in situ detection of gene expression in the medusa of the hydrozoan Clytia hemisphaerica, where traditional protocols caused extensive deterioration of the morphology and texture during hybridization, hindering observation and interpretation of results. Inspired by optimized protocols for Northern and Southern blot analysis, we substituted the 50% formamide with an equal volume of 8 M urea solution in the hybridization buffer. The new protocol yielded better morphologies and consistency of tissues, and also notably improved the resolution of the signal, allowing more precise localization of gene expression, as well as reduced staining at non-specific sites. Given the improved results using a less toxic hybridization solution, we tested the urea protocol on a number of other metazoans: two brachiopod species (Novocrania anomala and Terebratalia transversa) and the worm Priapulus caudatus, obtaining a similar reduction of aspecific probe binding. Overall, substitution of formamide by urea in in situ hybridization offers safer alternative protocols, potentially useful in research, medical and teaching contexts. We encourage other workers to test this approach on their study organisms, and hope that they will also obtain better sample preservation, more precise expression patterns and fewer problems due to aspecific staining, as we report here for Clytia medusae and Novocrania and Terebratalia developing larvae.

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RNA-DNA Hybrids at the Cytological Level

Cited by 539 publications

References 15 publications

Localization of nucleolar chromatin by immunocytochemistry and in situ hybridization at the electron microscopic level

Localization of nucleolar chromatin by immunocytochemistry and in situ hybridization at the electron microscopic level

Multicolor CRISPR labeling of chromosomal loci in human cells

A safer, urea-based in situ hybridization method improves detection of gene expression in diverse animal species

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