Detection of small, single-copy genes on protein-G-banded chromosomes by electron microscopy

Fetni, Raouf; Lemieux, Nicole; Malfoy, Bernard; Dutrillaux, Bernard; Messier, Paul-Emil; Richer, Claude‐Lise

doi:10.1159/000133332

Cited by 9 publications

(9 citation statements)

References 5 publications

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“…T he slides were incubated w ith the secondary antibody diluted 1:10 in PBS-HSA-Tween 20 for 45 m in at 3 7°C and w ashed. T w o separate approaches were used to m ake the B rdU -substituted bands visible: (1) pro tein A (Pharm acia) diluted 1:10 in PBS-HSA-Tween 20 was added, and the specim ens were incubated for 30 m in at room tem perature (Fetni et a!., 1992). o r (2) protein A incubation was followed with incubation w ith an ti protein A antibody (Sigm a) diluted 1:100 in PBS-HSA-Tween 20 for 60 min at 37 °C .…”

Section: Immunohistocliemical Detection O F Brdumentioning

confidence: 99%

“…However, a sufficient accumu lation of antibodies and protein A can result in adequate con trast (Fetni et al" 1992). To demonstrate the presence of pro tein A on the positively stained bands and its absence on the negatively stained bands, anti-protein A antibodies were used, and the binding of these antibodies was revealed with a low concentration of protein A-gold complex.…”

Section: Accessibility To Anti-brdu Antibody: Comparative Analysis Ofmentioning

confidence: 99%

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Complementary replication R- and G-band patterns induced by cell blocking at the R-band/G-band transition, a possible regulatory checkpoint within the S phase of the cell cycle

Fetni

Drouin²,

Richer³

et al. 1996

Cytogenet Genome Res

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The characteristic band patterns of replication banding (dynamic banding) were analyzed. High-resolution (550–1,250 bands per haploid genome) G- and R-band patterns were obtained after 5-bromo-2’-deoxyuridine (BrdU) incorporation during early or late S phase. Thymidine-BrdU permutation culture methods, which arrest DNA synthesis at the R-band/G-band transition, allow complementary BrdU substitution. The RBI (R bands by BrdU using immunological staining) and GBI (G bands by BrdU using immunological staining) band patterns were complementary for all chromosomes. There was no overlapping, and every part of each chromosome was positively stained by one of the two banding procedures. Cornparative analysis of RBG (R bands by BrdU using Giemsa staining) and RBI band patterns, as well as GBG (G bands by BrdU using Giemsa staining) and GBI band patterns, showed good congruency, displaying a very good band-for-band match. The congruency and complementarity found for these band patterns show that high concentrations of both thymidine and BrdU blocked S-phase progression near the R-band to G-band replication transition within the S phase. They also prove that BrdU incorporation is complementary and, therefore, demonstrate the existence of the R/G transition; a possible regulatory checkpoint within the S phase of the cell cycle.

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Section: Immunohistocliemical Detection O F Brdumentioning

confidence: 99%

Section: Accessibility To Anti-brdu Antibody: Comparative Analysis Ofmentioning

confidence: 99%

Complementary replication R- and G-band patterns induced by cell blocking at the R-band/G-band transition, a possible regulatory checkpoint within the S phase of the cell cycle

Fetni

Drouin²,

Richer³

et al. 1996

Cytogenet Genome Res

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“…It was later shown that this gold-labeling method could be combined with in situ hybridization to produce immunochemical banding of chromosomes along with simultaneous visualization of hybridization signals by using different sizes of colloidal-gold particles (Fetni et al 1991). Further improvements in this technique have led to protein-G chromosome banding, which no longer requires gold labeling to disclose chromosome bands (Fetni et al 1992). The banding pattern observed allows accurate chromosome identification, good discrimination between bands, and produces an image more familiar to cytogeneticists.…”

Section: Introductionmentioning

confidence: 99%

“…In this paper, we have applied fluorescence ) and EMISH (Fetni et al 1992) methodologies to assess the level of resolution afforded by EM. This study of FISH and EMISH signals has been conducted with a variety of DNA sequences currently employed in both basic science and diagnostic clinical research.…”

Section: Introductionmentioning

confidence: 99%

Increased resolution of in situ hybridization signal by electron microscopy: A comparison with fluorescence microscopy

Fetni¹,

Scott²,

Tihy³

et al. 1999

Genome

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Cytogenetic studies by in situ hybridization (ISH) have proven to be valuable for gene mapping on banded chromosomes when combined with fluorescence microscopy (FISH). However, even under the best conditions, FISH technology has a resolving power inherent to light of just 0.2 µm. Its utilization is further limited by the diffusion of light coming from the fluorescent signal which covers an area considerably larger than the target DNA sequence. The development of new ISH protocols applied to electron microscopy (EMISH) should increase the resolution for cytogenetic mapping and fine chromosomal structure studies. Despite these advances, few attempts have been made which exploit this increased resolution. Here we present a detailed analysis of ISH signals obtained by fluorescence and electron microscopy methodologies to demonstrate and define the higher sensitivity obtainable by electron microscopy. This comparative study was conducted with probes of different origins: telomeric, classical satellite, alpha satellite, and single-copy DNA sequences, which provide a good reference point for later studies. We were also able to map a 200-bp cDNA probe by EMISH. This study assesses the nature of the resolution and the better definition of the EMISH signal, which confirms the greater resolution of electron microscopy as compared with that achieved with light microscopy. It also indicates that better delineation of two closely linked sequences is achieved at the electron microscopy level.Résumé : L'analyse cytogénétique par hybridation in situ (ISH) sur chromosomes marqués, combinée à la fluorescence (FISH), s'est avérée très utile pour la cartographie génique. Toutefois, il faut tenir compte du fait que la technologie du FISH possède un pouvoir de résolution de seulement 0.2 µm, inhérent à la lumière, et cela même dans les meilleures conditions. Son utilisation est aussi limitée par la diffusion de la lumière émanant du signal fluorescent qui couvre une surface beaucoup plus grande que la séquence d'ADN cible. L'utilisation de la microscopie électronique en conjonction avec l'ISH constitue une technologie (EMISH) mieux adaptée à la cartographie à haute résolution et à l'analyse de la structure fine des chromosomes. Malgré l'instauration de cette nouvelle technologie, peu de recherches l'ont utilisée pour tirer parti de son plus grand pouvoir de résolution. Afin de mettre en évidence cette résolution augmentée, nous présentons ici la comparaison de résultats obtenus tant en fluorescence qu'en microscopie électronique avec des sondes de différents types: télomériques, satellites classiques, alpha satellites ainsi que des séquences de ADN simple copie. Nous avons même pu localiser avec EMISH une séquence de ADNc mesurant seulement 200 paires de bases. Cette étude confirme le meilleur niveau de résolution obtenu avec EMISH. La localisation est plus précise et deux séquences rapprochées peuvent être mieux circonscrites et donc situées plus exactement. Nous espérons que ce travail puisse servir de référence pour des travaux ulté...

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“…Gold-labeled probes can be detected by electron microscopy with a fantastic lateral resolution of about 10 nm [7][8][9][10], and another candidate is atomic force microscopy (AFM): Putman et al [11] have used an AFM to demonstrate that morphological labels, such as precipitated diaminebenzidine (DAB) [12], can be visualized as pronounced bulges on top of the chromosome structure. Apart from difficulties with chromosome preparation, an important disadvantage of these techniques is the lack of multiplicity: one is not able to recognize different DNA probes on a single chromosome, in contrast to the detection of multiple colour FISH signals with fluorescence microscopy.…”

Section: Introductionmentioning

confidence: 99%

Detection of fluorescence in situ hybridization on human metaphase chromosomes by near-field scanning optical microscopy

et al. 1995

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Fluorescence in situ hybridization signals on human metaphase chromosomes are detected by a near-field scanning optical microscope. This makes it possible to localize and identify several fluorescently labeled genomic DNA fragments on a single chromosome with a resolution superior to traditional fluorescence microscopy. Several nucleic acid probes have been used. The hybridization signals are well resolved in the near-field fluorescence images, and the exact location of the probes can be correlated to the topography as it is afforded by the shear-force feedback.

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Detection of small, single-copy genes on protein-G-banded chromosomes by electron microscopy

Cited by 9 publications

References 5 publications

Complementary replication R- and G-band patterns induced by cell blocking at the R-band/G-band transition, a possible regulatory checkpoint within the S phase of the cell cycle

Complementary replication R- and G-band patterns induced by cell blocking at the R-band/G-band transition, a possible regulatory checkpoint within the S phase of the cell cycle

Increased resolution of in situ hybridization signal by electron microscopy: A comparison with fluorescence microscopy

Detection of fluorescence in situ hybridization on human metaphase chromosomes by near-field scanning optical microscopy

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