Differential synthesis of the genes for ribosomal RNA during amphibian oögenesis.

Abstract

Molecular hybridization experiments have shown that the genes for ribosomal RNA are located in or near the nucleolus organizer in Drosophila melanogasterl, 2 and in the toad Xenopus laevis.3' 4 Under normal circumstances the number of organizers per genome will be characteristic of the organism, and DNA from various tissues should contain the same proportion of ribosomal genes. This conclusion has been confirmed for several tissues of the chicken.2 However, cytological evidence has long suggested that the nucleolus organizer undergoes a differential replication in odcytes of certain animals. Data supporting this view will be summarized later in this article. If such a differential replication occurs, o6cyte DNA should be enriched with respect to the ribosomal genes. The cytological picture is particularly striking in ovaries of the toads Bufo and Xenopus, in which the differential synthesis occurs during pachytene. In recently metamorphosed toads the ovary contains a sufficiently high proportion of pachytene oocytes to permit detection of the differential synthesis by biochemical means.5 This communication describes the ovarian DNA of Xenopus and demonstrates that it contains an excess of sequences coding for rRNA. Recently Brown and co-workers6' I have studied the DNA from older o6cytes of Xenopus and have reached similar conclusions.Materials and Methods.-(1) Animals: Tadpoles of the African clawed toad Xenopus laevis were grown in the laboratory. During the second and through the fourth weeks after metamorphosis, a large number of obcytes are in the pachytene stage. At this time the testes and ovaries can be distinguished by gross inspection (cf. Figs. A and B in Blackler8). When the oocytes pass into diplotene and begin their growth phase, they have completed the differential DNA synthesis. If individual o6cytes are detectable with the dissecting microscope (10X-20X), the ovary will show little incorporation into the nucleolar DNA.(2) DNA extraction: Ovaries from 8-10 toads were dissected under sterile conditions and cultured 20-60 hr in a medium containing 9 Mc thymidine-C"4 (47 mc/mM) or 100 Mc

Nucleolar localization elements in U8 snoRNA differ from sequences required for rRNA processing

“…Using fluorescence microscopy, we have analyzed the sequences within U8 snoRNA necessary for its nucleolar localization+ In vitro transcripts of wild-type U8 snoRNA labeled with fluorescein-UTP were injected into Xenopus oocyte nuclei+ After 2 h, the nuclei were manually isolated, the nuclear envelope was removed, and the nuclear contents, including the multiple nucleoli, were centrifuged onto a microscope slide for direct analysis with a fluorescence microscope+ In Xenopus oocytes, there are multiple nucleoli due to rDNA amplification (Painter & Taylor, 1942;Brown & David, 1968;Gall, 1968;Perkowska et al+, 1968)+ Individual nucleoli can vary in size (Wu & Gall, 1997) and also can fuse into a multinucleolar cluster that sometimes has a vacuolated appearance (Shah et al+, 1996)+ As seen in Figure 1, nucleoli are brightly stained by fluoresceinlabeled U8 in foci that encompass the ribosomal DNA stained by DAPI+ This localization is specific, because nucleoli are unstained after injection of fluoresceinlabeled U2 snRNA (Fig+ 1), a small nuclear RNA that is part of the splicing machinery and is not present in nucleoli+ In addition, the lampbrush chromosome seen in Figure 2 does not stain with fluorescein, supporting the specificity of nucleolar localization of U8 snoRNA+…”

REPLICATION OF NUCLEOLUS-ASSOCIATED DNA DURING "G2 PHASE" IN PHYSARUM POLYCEPHALUM

“…polycephalum (6,24) . As nucleolus-associated DNA has a higher GC content than the bulk of the nuclear DNA (2,8,35), it is possible that the DNA which we find to be labeled in the region of the nucleolus at late interphase is identical with the DNA which was found to be predominantly transcribed during that time (6,24) . Hence, the possibility exists that the low rate of incorporation of thymidine-3H in the nucleolar region could be due to repair replication following transcription (29) rather than to net synthesis .…”

“…The few silver grains found over these nuclei and over most of the nuclei from here on throughout the remainder of the intermitotic period (Figs . [4][5][6][7][8] were concentrated over the nucleolar area .…”

“…Incorporation of thymidine-3H into nuclei as late as prophase has been found in grasshopper neuroblasts (9) and, during metamorphosis, in epidermal cells of the milkweed bug Oncopeltus fasciatus (21) . DNA synthesis also occurs during meiotic prophase in microsporocytes of Tradescantia (27), in male newts (36), in GC-rich regions of the chromosomes of lily anthers (18), and in oocytes of Xenopus (8,23) . In the latter, this DNA synthesis represents the production of extra copies of GC-rich DNA cistrons of the nucleolar region (5, 8) which code for ribosomal RNA.…”

Pachytene morphology of the chromosome complement in Cornus florida 'sweetwater'

“…Though it is better stained than the main nucleolus, the stainability suggests similarities to the latter one. The appearance of small nucleolus-like bodies during different meiotic stages has been reported by other investigators (Gall 1968, Jelenkovic and Harrington 1972, Jones 1957, Snoad 1956). However, such nucleoli vary greatly in size, number and position on the chromosomes.…”