Localization by situ hybridization of amplified ribosomal DNA during Xenopus laevis oocyte maturation (a light and electron microscopy study).

Abstract

The presence of ribosomal DNA has been demonstrated, by light and electron microscopy study of in situ hybridization with 125I4labeled ribosomal RNA, in the Feulgen-positive bodies which appear during maturation in the cytoplasm of Xenopus laevis oocytes. The ultrastructure of these bodies is described. Many years ago (1-3), one of us described the appearance of Feulgen-positive granules in the cytoplasm of amphibian oocytes undergoing in vivo maturation. More recently, a cytochemical and ultrastructural study of in vitro maturation led to the conclusion that these granules probably originate from the nucleolar organizers (4, 5).The Feulgen-positive granules become detectable in the nucleoli when they begin to desintegrate; they then fuse together and migrate towards the animal pole, where they can be detected by the Feulgen reaction until the formation of the first meiotic spindle. For electron microscopy, the oocytes were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) at room temperature for 30 min. The vegetal hemispheres of the oocytes were then cut with a razor blade and discarded. Fixation was continued for another 30 min. Some oocytes of each batch were then postfixed for 1 hr with 1% OS04 in 0.1 M cacodylate buffer (pH 7.2) at room temperature and embedded in epoxy resin according to Spurr (7). The remaining oocytes were embedded in glycolmethacrylate following Leduc and Bernhard (8). Ultrathin sections of about 1000 A were cut with a Reichert OMU3 ultramicrotome and collected on Formvar-carbon-coated gold grids. The grids were processed for in situ hybridization according to Jacob et al. (9), with small modifications: two different temperatures of hybridization have been used, namely 700 or 800; SSC (standard saline-citrate) solutions were replaced by SET solu'ons (0.15 M NaCl, 50 mM Tris-HCl, 5 mM EDTA, pH 8 X). Ilford L4 emulsion was applied on the grids by the loop method (10). Autoradiograms were developed with Microdol X diluted 1:1 with water, after 6 weeks' to 5 months' exposure. The grids were stained with uranyl acetate in -Michaelis buffer and lead citrate, and were then observed with an AEI electron microscope EM6B.In Situ Hybridization for Light Microscopy. The sections were treated with ribonuclease (RNase) for 1 hr at 370[100,Mg/ml of RNase A in 2 X SET (pH 8)]. Denaturation was performed in 95% formamide in 0.1 X SET (pH 7) for 2.5 hr at 650 (11). The slides were dipped in cold 70% ethanol, passed through 95% ethanol, and air dried.

Ultrastructural localization of defined sequences of viral RNA and DNA by in situ hybridization of biotinylated DNA probes on sections of herpes simplex virus type 1 infected cells

“…Until recently, hybrid detection at the ultrastructural level was accomplished by autoradiography following hybridization of radioactive nucleic acid probes either in pre-embedding techniques (Croissant et al, 1972;Geuskens and May, 1974) or post-embedding techniques (Jacob et al, 1971;Steinert et al, 1976). It is now markedly improved by labeling of the probes with a biotinylated nucleotide and its subsequent detection with cytochemical techniques (Binder et al, 1986;Escaig-Haye et al, 1989;Hutchison et al, 1982;Manuelidis et al, 1982;McFadden, 1989;Puvion-Dutilleul and Puvion, 1989;Thiry and Thiry-Blaise, 1989;Wolber et al, 1988Wolber et al, , 1989.…”

Non-isotopic electron microscope in situ hybridization for studying the functional sub-compartmentalization of the cell nucleus

“…1988Trembleau etal. , 1990Wolber et al 1988), and on ultrathin sections of hydrosoluble resin-embedded material (Binder et al 1986;Jacob et al 1971;Le Guellec et al 1990;Olmedilla et al 1993;Steinert et al 1976;Testillano et al 1995;Yi et al 1995). This list of the contributions of in situ hybridization studies to the topological features of nucleic acids is not exhaustive.…”

Localization of adenovirus DNA by in situ hybridization electron microscopy

“…In particular, in the field of developmental biology this technique has shown to be the most direct way of answering questions of gene expression and its modulation at the individual cell level during developmental transitions or gene expression in somatic cells (Akam and Carlson, 1985;Cox et al, 1984;Hafen et al, 1983;Lawrence and Singer, 1986;Venezky et al, 1981). Attempts have been undertaken to extend this technique to the electron microscope (EM) level (Binder et al, 1986;Erickson et al, 1990;Geuskens and May, 1974;Hutchison et al, 1982;Jacob et al, 1971;Manning et al, 1975;Singer et al, 1989;Steinert et al, 1976Steinert et al, , 1984Thiry and Thiry-Blaise, 1989;Wachtler et al, 1990;Webster et al, 1987).…”