Protein incorporation by isolated amphibian oocytes III. Optimum incubation conditions

Wallace, Robin A.; Jared, Donald W.; Dumont, James N.; Sega, M.

doi:10.1002/jez.1401840305

Cited by 473 publications

(151 citation statements)

References 24 publications

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“…As a control, U2 snRNA alone was injected into oocytes. After overnight incubation in OR2 medium (Wallace et al, 1973) at room temperature, GVs were manually isolated in 5:1 KCl-NaCl isolation medium (Gall et al, 1991). RNA was extracted using the RNAqueous-micro kit (Ambion) and analyzed for 2Ј-O-methylation mapping.…”

Section: In Vitro Modification Assaysmentioning

confidence: 99%

Small Cajal Body–specific RNAs ofDrosophilaFunction in the Absence of Cajal Bodies

Deryusheva

Gall

2009

MBoC

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During their biogenesis small nuclear RNAs (snRNAs) undergo multiple covalent modifications that require guide RNAs to direct methylase and pseudouridylase enzymes to the appropriate nucleotides. Because of their localization in the nuclear Cajal body (CB), these guide RNAs are known as small CB-specific RNAs (scaRNAs). Using a fluorescent primer extension technique, we mapped the modified nucleotides in Drosophila U1, U2, U4, and U5 snRNAs. By fluorescent in situ hybridization (FISH) we showed that seven Drosophila scaRNAs are concentrated in easily detectable CBs. We used two assays based on Xenopus oocyte nuclei to demonstrate that three of these Drosophila scaRNAs do, in fact, function as guide RNAs. In flies null for the CB marker protein coilin, CBs are absent and there are no localized FISH signals for the scaRNAs. Nevertheless, biochemical experiments show that scaRNAs are present at normal levels and snRNAs are properly modified. Our experiments demonstrate that several scaRNAs are concentrated as expected in the CBs of wild-type Drosophila, but they function equally well in the nucleoplasm of mutant flies that lack CBs. We propose that the snRNA modification machinery is not limited to CBs, but is dispersed throughout the nucleoplasm of cells in general. INTRODUCTIONThe two major ribosomal RNAs (rRNAs) and the spliceosomal small nuclear RNAs (snRNAs) U1, U2, U4, U5, and U6 contain many posttranscriptionally modified nucleotides, which are crucial for RNA-RNA and RNA-protein interactions, as well as spliceosome function (Yu et al., 1998; Yu, 2004, 2007). The most abundant modifications in both rRNAs and snRNAs are 2Ј-O-methylation and pseudouridylation, which are directed by small box C/D and box H/ACA guide RNAs, respectively. Each guide RNA molecule is associated with a set of four core proteins: box C/D RNAs form RNP particles with fibrillarin (the methyltransferase), Nop56, Nop58, and a 15.5-kDa protein, whereas box H/ACA RNAs associate with NAP57/dyskerin (the pseudouridine synthase), GAR1, NHP2, and NOP10 proteins (reviewed in Yu et al., 2004;Matera et al., 2007). Most guide RNAs are concentrated in the nucleolus, where they are involved in posttranscriptional modifications of rRNA. Because of their localization, they are referred to as small nucleolar RNAs (snoRNAs). However, the guide RNAs that mediate modification of the snRNAs are preferentially found in another nuclear organelle, the Cajal body (CB). These guide RNAs are called small CB-specific RNAs (scaRNAs).The first scaRNA to be studied in detail was U85 scaRNA (Jády and Kiss, 2001;Darzacq et al., 2002). U85 is a remarkable double guide RNA that contains both box C/D and box H/ACA motifs. In a set of detailed experiments it was shown that human U85 scaRNA guides both 2Ј-O-methylation at C45 and pseudouridylation at U46 in U5 snRNA. Modification takes place in the nucleus after import of the U5 snRNA from the cytoplasm . CB localization of U85 scaRNA was originally demonstrated by fluorescent in situ hybridization (FISH) in mammalian and ...

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Section: In Vitro Modification Assaysmentioning

confidence: 99%

Small Cajal Body–specific RNAs ofDrosophilaFunction in the Absence of Cajal Bodies

Deryusheva

Gall

2009

MBoC

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“…Oocytes from adult Xenopus laevis were defolliculated for 2 h at room temperature in Ca 2ϩ -free OR2 (Wallace et al, 1973) containing 0.2% collagenase (type II, C6885; Sigma Chemical, St. Louis, MO). Stage IV-VI oocytes were collected and held in OR2 at 18°C.…”

Section: Oocyte Injection and Protein Extractsmentioning

confidence: 99%

Coilin Can Form a Complex with the U7 Small Nuclear Ribonucleoprotein

Bellini

Gall

1998

MBoC

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Coiled bodies (CBs) in the amphibian oocyte nucleus are spherical structures up to 10 m or more in diameter, much larger than their somatic counterparts, which rarely exceed 1 m. Oocyte CBs may have smaller granules attached to their surface or embedded within them, which are identical in structure and composition to the many hundreds of B-snurposomes found free in the nucleoplasm. The matrix of the CBs contains the diagnostic protein p80-coilin, which is colocalized with the U7 small nuclear ribonucleoprotein (snRNP), whereas the attached and embedded B-snurposomes contain splicing snRNPs. A few of the 50 -100 CBs in the oocyte nucleus are attached to lampbrush chromosomes at the histone gene loci. By coimmunoprecipitation we show that coilin and the U7 snRNP can form a weak but specific complex in the nucleoplasm, which is dependent on the special U7 Sm-binding site. Under the same conditions coilin does not associate with the U1 and U2 snRNPs. Coilin is a nucleic acid-binding protein, as shown by its interaction with single-stranded DNA and with poly r(U) and poly r(G). We suggest that an important function of coilin is to form a transient complex with the U7 snRNP and accompany it to the CBs. In the case of CBs attached to chromosomes at the histone gene loci, the U7 snRNP is thus brought close to the actual site of histone pre-mRNA transcription. INTRODUCTIONCoiled bodies (CBs) 1 are small nuclear organelles, generally 1 m or less in diameter, found in a variety of animal and plant cells. They are characterized by the presence of a unique protein, p80-coilin , along with components involved in three different RNA-processing pathways: splicing, prerRNA processing, and histone pre-mRNA processing (reviewed by Lamond and Carmo-Fonseca, 1993;Bohmann et al., 1995;Gall et al., 1995;Roth, 1995;Lamond and Earnshaw, 1998;Matera, 1998). Long before their homology to somatic CBs was recognized, CBs had been described from the oocyte nucleus or germinal vesicle (GV) of insects (Jö rgensen, 1913) and amphibians (Gall, 1954;Callan and Lloyd, 1960). In amphibians they have generally been known under the name spheres or sphere organelles (reviewed by Callan, 1986). GVs from mature or nearly mature Xenopus oocytes contain 50 -100 CBs, of which the majority are free in the nucleoplasm, with a few specifically attached to lampbrush chromosomes at the histone gene loci . CBs in the Xenopus GV consist of three parts, easily distinguished by phase contrast or differential interference microscopy (DIC): a nearly perfectly spherical body (hence the original name), one to many rounded structures on the surface of the sphere, and one to many inclusions ( Figure 1). The attached structures and inclusions are identical in morphology and composition to the thousands of socalled B-snurposomes found free in the nucleoplasm (Wu et al., 1991). The unusually large size of oocyte CBs, some of which have diameters greater than 10 m, permits more precise cytological localization of components than is possible with the much smaller somatic CBs....

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“…The females were anaesthesized in 1 % MS222 (3-aminobenzoic acid ethyl ester, Sigma) and sacrificed by decapitation. The ovary was cut into fine pieces, and oocytes were released from their follicle by digestion with 0.2 % collagenase in OR2 solution (82.5 ITIM NaCI, 2.5ITIM KCI, 1 ITIM Na 2 HPO 4 , 5mM Hepes, 5ITIM Polyvinylpyrrolidone; (Wallace et al, 1973) without calcium for 2-3 h at room temperature. Oocytes were centrifuged (400 g, 10 min, 19 °C) in plastic petri dishes to which a 0.8 mm-mesh plastic grid (Z rcher Beuteltuchfabrik, R schlikon, Switzerland) was glued to the bottom with a few drops of CHCI 3 ; this centrifugation brings the nuclei to the surface where their position is indicated by a white area in the pigmented animal hemisphere (Kressmann et al, 1978).…”

Section: Plasmid Constructionmentioning

confidence: 99%

Dramatic Changes in the Ratio of Homologous Recombination to Nonhomologous DNA-End Joining in Oocytes and Early Embryos ofXenopus laevis

Hagmann¹,

Adlkofer²,

Pfeiffer³

et al. 1996

Biological Chemistry Hoppe-Seyler

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We have developed a versatile plasmid vector (pReco80% of the homologously recombined product, whereas in eggs a highly efficient DMA-end joining activity predominates Both reactions, homologous recombination and DMA-end joining, are shown to occur quickly, with the majority of the respective products being formed within the first 20 minutes of incubation under optimal conditions. In fertilized eggs, up to 50% of all injected linear DMA molecules are recircularized by DMA-end joining. With high amounts of injected DMA per fertilized egg, DMA-end joining is reduced, presumably due to competition for essential factors, and homologous recombination becomes readily detectable.As there is a sequence of rapid cleavage divisions after fertilization of the egg, the fast and highly efficient DMA-end joining, even though it is error-prone at the junction site, seems to be best suited to cope 1 present address: Institut für Zellbiologie, Swiss Federal Institute of Technology, Zürich-Hönggerberg, CH-8093 Zürich, Switzerland with DMA double-strand breaks that might occur in the genome during early embryogenesis. On the other hand, the long-lived oocytes seem to repair DMA double-strand breaks via homologous recombination. This latter property may be exploited both in Xenopus and in other organisms to achieve homologous integration of exogenous DMA into germ cells for gene targeting.

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Protein incorporation by isolated amphibian oocytes III. Optimum incubation conditions

Cited by 473 publications

References 24 publications

Small Cajal Body–specific RNAs ofDrosophilaFunction in the Absence of Cajal Bodies

Small Cajal Body–specific RNAs ofDrosophilaFunction in the Absence of Cajal Bodies

Coilin Can Form a Complex with the U7 Small Nuclear Ribonucleoprotein

Dramatic Changes in the Ratio of Homologous Recombination to Nonhomologous DNA-End Joining in Oocytes and Early Embryos ofXenopus laevis

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