TAFII70 protein in Cajal bodies of the amphibian germinal vesicle

Genome ◽  
2001 ◽  
Vol 44 (6) ◽  
pp. 1100-1103 ◽  
Author(s):  
Stefania Bucci ◽  
Letizia Giani ◽  
Giorgio Mancino ◽  
Mario Pellegrino ◽  
Matilde Ragghianti
Keyword(s):  
Monoclonal Antibody ◽  
Germinal Vesicle ◽  
Tata Binding Protein ◽  
Cajal Body ◽  
Cajal Bodies ◽  
Oocyte Nucleus ◽  
Lampbrush Chromosomes ◽  
Rna Transcription ◽  
Associated Factor ◽  
Oocyte Cytoplasm

The localization of the TATA-binding protein (TBP) associated factor II70 (TAFII70) in the germinal vesicle (GV) of newt oocytes was investigated. In spreads of GV content, anti-hTAFII70 monoclonal antibody (mAb) stained Cajal bodies (CBs) that were either attached to specific sites on the lampbrush chromosomes or free in the nucleoplasm. To confirm this localization the PwTAFII70 cDNA was cloned and myc-tagged transcripts injected into the oocyte cytoplasm. Newly translated PwTAFII70 protein was detected a few hours later in the Cajal bodies. These data support the hypothesis that Cajal bodies are the assembly sites of the transcription machinery of the oocyte nucleus. TAFII70 protein can play a role in lampbrush transcription; alternatively TAFII70 can be considered a component in the subset of TFIID complexes that do not function during oogenesis, but are accumulated in the oocyte for later use during early development.Key words: TAFII70, Cajal body, lampbrush chromosomes, RNA transcription and processing, newts, Pleurodeles.

Heat shock induces mini-Cajal bodies in the Xenopus germinal vesicle

2002 ◽  
Vol 115 (10) ◽  
pp. 2011-2020 ◽  
Author(s):  
Korie E. Handwerger ◽  
Zheng'an Wu ◽  
Christine Murphy ◽  
Joseph G. Gall
Keyword(s):  
Heat Shock ◽  
Germinal Vesicle ◽  
Model System ◽  
Cajal Body ◽  
Self Organization ◽  
Cajal Bodies ◽  
Oocyte Nucleus ◽  
Evolutionarily Conserved ◽  
And Behavior ◽  
Heat Shock Induction

Cajal bodies are evolutionarily conserved nuclear organelles that are believed to play a central role in assembly of RNA transcription and processing complexes. Although knowledge of Cajal body composition and behavior has greatly expanded in recent years, little is known about the molecules and mechanisms that lead to the formation of these organelles in the nucleus. The Xenopus oocyte nucleus or germinal vesicle is an excellent model system for the study of Cajal bodies, because it is easy to manipulate and it contains 50-100 Cajal bodies with diameters up to 10 μm. In this study we show that numerous mini-Cajal bodies (less than 2 μm in diameter) form in the germinal vesicle after oocytes recover from heat shock. The mechanism for heat shock induction of mini-Cajal bodies is independent of U7 snRNA and does not require transcription or import of newly translated proteins from the cytoplasm. We suggest that Cajal bodies originate by self-organization of preformed components, preferentially on the surface of B-snurposomes.

scholarly journals RNA Polymerase III in Cajal Bodies and Lampbrush Chromosomes of the Xenopus Oocyte Nucleus

2002 ◽  
Vol 13 (10) ◽  
pp. 3466-3476 ◽  
Author(s):  
Christine Murphy ◽  
Zhengxin Wang ◽  
Robert G. Roeder ◽  
Joseph G. Gall
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Cajal Bodies ◽  
Oocyte Nucleus ◽  
Xenopus Laevis Oocyte ◽  
Lampbrush Chromosomes ◽  
Oocyte Cytoplasm ◽  
Pol Iii

We used immunofluorescence to study the distribution and targeting of RNA polymerase (pol) III subunits and pol III transcription factors in the Xenopus laevis oocyte nucleus. Antibodies against several of these proteins stained Cajal bodies and ∼90 specific sites on the lampbrush chromosomes. Some of the chromosomal sites had been identified previously by in situ hybridization as the genes for 5S rRNA. The remaining sites presumably encode tRNAs and other pol III transcripts. Pol III sites were often resolvable as loops similar to the much more abundant pol II loops, but without a matrix detectable by phase contrast or differential interference contrast. This morphology is consistent with the transcription of short repeated sequences. Hemagglutinin-tagged transcripts encoding core subunits and transcription factors were injected into the oocyte cytoplasm, and the distribution of newly translated proteins inside the nucleus was monitored by immunostaining. Cajal bodies were preferentially targeted by these proteins, and in some cases the chromosomal sites were also weakly stained. The existence of pol III subunits and pol III transcription factors in Cajal bodies and their targeting to these organelles are consistent with a model of Cajal bodies as sites for preassembly of the nuclear transcription machinery.

scholarly journals Cajal Bodies, Nucleoli, and Speckles in the Xenopus Oocyte Nucleus Have a Low-Density, Sponge-like Structure

2005 ◽  
Vol 16 (1) ◽  
pp. 202-211 ◽  
Author(s):  
Korie E. Handwerger ◽  
Jason A. Cordero ◽  
Joseph G. Gall
Keyword(s):  
Xenopus Oocyte ◽  
Aqueous Media ◽  
Germinal Vesicle ◽  
Physical Structure ◽  
Cajal Bodies ◽  
Oocyte Nucleus ◽  
Refractive Indices ◽  
Low Density ◽  
Penetration Data ◽  
Fibrillar Region

Nuclear organelles, unlike many cytoplasmic organelles, lack investing membranes and are thus in direct contact with the surrounding nucleoplasm. Because the properties of the nucleoplasm and nuclear organelles influence the exchange of molecules from one compartment to another, it is important to understand their physical structure. We studied the density of the nucleoplasm and the density and permeability of nucleoli, Cajal bodies (CBs), and speckles in the Xenopus oocyte nucleus or germinal vesicle (GV). Refractive indices were measured by interferometry within intact GVs isolated in oil. The refractive indices were used to estimate protein concentrations for nucleoplasm (0.106 g/cm3), CBs (0.136 g/cm3), speckles (0.162 g/cm3), and the dense fibrillar region of nucleoli (0.215 g/cm3). We determined similar protein concentrations for nuclear organelles isolated in aqueous media, where they are no longer surrounded by nucleoplasm. To examine the permeability of nuclear organelles, we injected fluorescent dextrans of various molecular masses (3–2000 kDa) into the cytoplasm or directly into the GV and measured the extent to which they penetrated the organelles. Together, the interferometry and dextran penetration data show that organelles in the Xenopus GV have a low-density, sponge-like structure that provides access to macromolecules from the nucleoplasm.

Nonhistone Proteins of the Oocyte Nucleus of the Newt

1974 ◽  
Vol 15 (1) ◽  
pp. 145-161
Author(s):  
R. J. HILL ◽  
K. MAUNDRELL ◽  
H. G. CALLAN
Keyword(s):  
Nuclear Membrane ◽  
Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Germinal Vesicle ◽  
Oocyte Nucleus ◽  
Lampbrush Chromosomes ◽  
Nonhistone Proteins ◽  
Aerial Oxidation ◽  
A Minor

Evidence has been obtained which indicates that disulphide bond crosslinks contribute to the morphological integrity of isolated lampbrush chromosomes (both chromomeres and lateral loops) and nucleoli. It is suggested that the progressive formation of these bonds in vitro by aerial oxidation may provide the basis for the previously recognized time-dependent hardening or ‘denaturing’ of these structures. Manually isolated germinal vesicle nuclei have been massed and fractionated by low-speed centrifugation into nucleoplasm and chromatin. Phase-contrast microscopy demonstrates the chromatin to consist of nucleoli, lampbrush chromosomes and nuclear membranes. Urea gel electrophoresis has been employed to resolve the reduced and S-carboxymethylated proteins of whole nuclei into some 12 components, negatively charged at pH 8. The nucleoplasm alone gives an essentially similar pattern, but with the distinct depletion of one component and slight depletion of another. Both of these components are much enriched in the chromatin pellet where they predominate over all other proteins. The total chromatin has been subfractionated by microdissection, taking advantage of the differential attachment of nucleoli to the nuclear membrane at different stages of oogenesis. It is concluded that the nuclear membrane per se does not contribute to the major chromatin proteins. The two major polypeptides are components of the nucleoli. Preparations of isolated lampbrush chromosomes have not, to date, provided sufficient material to give a distinctive electropherogram; only one faint band, a major component of whole nuclei, was apparent. Sodium dodecyl sulphate gel electrophoresis has resolved some 25 components in whole nuclei, and again demonstrates the enrichment of the two major species in the total chromatin fraction. The apparent molecular weights of these two species are 43 kilodaltons and 110 kilodaltons. Approximately 20 minor species are also present in the chromatin and are obviously good candidates as components of the nucleolar and chromosomal structures. Histones, at most, make only a minor contribution to the overall chromatin protein population.

scholarly journals Assembly of the Nuclear Transcription and Processing Machinery: Cajal Bodies (Coiled Bodies) and Transcriptosomes

1999 ◽  
Vol 10 (12) ◽  
pp. 4385-4402 ◽  
Author(s):  
Joseph G. Gall ◽  
Michel Bellini ◽  
Zheng’an Wu ◽  
Christine Murphy
Keyword(s):  
Germinal Vesicle ◽  
Cajal Bodies ◽  
Oocyte Nucleus ◽  
Electron Microscopic ◽  
Pol Ii ◽  
Processing Machinery ◽  
Small Nuclear Ribonucleoprotein ◽  
Coiled Bodies ◽  
Pol I ◽  
Pol Iii

We have examined the distribution of RNA transcription and processing factors in the amphibian oocyte nucleus or germinal vesicle. RNA polymerase I (pol I), pol II, and pol III occur in the Cajal bodies (coiled bodies) along with various components required for transcription and processing of the three classes of nuclear transcripts: mRNA, rRNA, and pol III transcripts. Among these components are transcription factor IIF (TFIIF), TFIIS, splicing factors, the U7 small nuclear ribonucleoprotein particle, the stem–loop binding protein, SR proteins, cleavage and polyadenylation factors, small nucleolar RNAs, nucleolar proteins that are probably involved in pre-rRNA processing, and TFIIIA. Earlier studies and data presented here show that several of these components are first targeted to Cajal bodies when injected into the oocyte and only subsequently appear in the chromosomes or nucleoli, where transcription itself occurs. We suggest that pol I, pol II, and pol III transcription and processing components are preassembled in Cajal bodies before transport to the chromosomes and nucleoli. Most components of the pol II transcription and processing pathway that occur in Cajal bodies are also found in the many hundreds of B-snurposomes in the germinal vesicle. Electron microscopic images show that B-snurposomes consist primarily, if not exclusively, of 20- to 30-nm particles, which closely resemble the interchromatin granules described from sections of somatic nuclei. We suggest the name pol II transcriptosome for these particles to emphasize their content of factors involved in synthesis and processing of mRNA transcripts. We present a model in which pol I, pol II, and pol III transcriptosomes are assembled in the Cajal bodies before export to the nucleolus (pol I), to the B-snurposomes and eventually to the chromosomes (pol II), and directly to the chromosomes (pol III). The key feature of this model is the preassembly of the transcription and processing machinery into unitary particles. An analogy can be made between ribosomes and transcriptosomes, ribosomes being unitary particles involved in translation and transcriptosomes being unitary particles for transcription and processing of RNA.

scholarly journals Dynamic Nature of Cleavage Bodies and Their Spatial Relationship to DDX1 Bodies, Cajal Bodies, and Gems

2006 ◽  
Vol 17 (3) ◽  
pp. 1126-1140 ◽  
Author(s):  
Lei Li ◽  
Ken Roy ◽  
Sachin Katyal ◽  
Xuejun Sun ◽  
Stacey Bléoo ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Actin Polymerization ◽  
Spatial Relationship ◽  
S Phase ◽  
Nuclear Bodies ◽  
Cajal Bodies ◽  
Rna Transcription ◽  
Nuclear Structures ◽  
The Relationship ◽  
Striking Effect

DDX1 bodies, cleavage bodies, Cajal bodies (CBs), and gems are nuclear suborganelles that contain factors involved in RNA transcription and/or processing. Although all four nuclear bodies can exist as distinct entities, they often colocalize or overlap with each other. To better understand the relationship between these four nuclear bodies, we examined their spatial distribution as a function of the cell cycle. Here, we report that whereas DDX1 bodies, CBs and gems are present throughout interphase, CPSF-100-containing cleavage bodies are predominantly found during S and G2 phases, whereas CstF-64-containing cleavage bodies are primarily observed during S phase. All four nuclear bodies associate with each other during S phase, with cleavage bodies colocalizing with DDX1 bodies, and cleavage bodies/DDX1 bodies residing adjacent to gems and CBs. Although inhibitors of RNA transcription had no effect on DDX1 bodies or cleavage bodies, inhibitors of DNA replication resulted in loss of CstF-64-containing cleavage bodies. A striking effect on nuclear structures was observed with latrunculin B, an inhibitor of actin polymerization, resulting in the formation of needlelike nuclear spicules made up of CstF-64, CPSF-100, RNA, and RNA polymerase II. Our results suggest that cleavage body components are highly dynamic in nature.

Differential cytolocalization of prosomes in axolotl during oogenesis and meiotic maturation

1988 ◽  
Vol 90 (4) ◽  
pp. 543-553 ◽  
Author(s):  
J. Gautier ◽  
J.K. Pal ◽  
M.F. Grossi de Sa ◽  
J.C. Beetschen ◽  
K. Scherrer
Keyword(s):  
Mammalian Cells ◽  
De Novo ◽  
Germinal Vesicle ◽  
Immunoblot Analysis ◽  
Ambystoma Mexicanum ◽  
Meiotic Maturation ◽  
Crescent Formation ◽  
Oocyte Growth ◽  
Oocyte Cytoplasm

The prosomes, a novel type of small RNA-protein complex previously characterized in avian and mammalian cells, were isolated from axolotl (Ambystoma mexicanum) oocytes and identified by sedimentation analysis and protein composition. The prosomal nature of these particles was further ascertained by immunoblot analysis with anti-duck prosome monoclonal antibodies. By in vitro [35S]methionine labelling, de novo synthesis of prosomal proteins could be detected neither during oogenesis nor meiotic maturation. The results obtained by both indirect immunofluorescence and immunoblot analyses demonstrated a dramatic change in the localization of prosomal antigens during oocyte development. They were initially detected in the oocyte cytoplasm, during oocyte growth. At the end of vitellogenesis (stages V-VI), they entered the nucleus (germinal vesicle) and were accumulated there to the highest concentration. During oocyte maturation, after nuclear envelope breakdown, prosomal antigens were found to be localized again in the cytoplasm, until fertilization. No specific localization of prosomal antigens in mature oocytes, unfertilized and fertilized eggs was observed within the oocyte cytoplasm in relation to the cytoplasmic rearrangements leading to grey crescent formation.

Injected nuclei in frog oocytes: fate, enlargement, and chromatin dispersal

Development ◽  
1976 ◽  
Vol 36 (3) ◽  
pp. 523-540
Author(s):  
J. B. Gurdon
Keyword(s):  
Bovine Serum Albumin ◽  
Xenopus Laevis ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Oocyte Nucleus ◽  
Oocyte Cytoplasm ◽  
Nuclear Injection

A method is described by which nuclei associated with some cytoplasm can be rapidly prepared from a suspension of cells. The method involves the use of lysolecithin and bovine serum albumin. Oocytes of Xenopus laevis were injected with about 200 nuclei prepared from human He La cells by this method. Nuclei were deposited in oocyte cytoplasm, in the oocyte nucleus, or in the dispersed contents of a ruptured oocyte nucleus. Injected He La nuclei enlarge up to several hundred times in volume in the course of a few days. Their enlargement is associated with chromatin dispersion, increased binding of an acidic dye, and with the reduction in size, and eventual disappearance, of nucleoli. The amount of He La nucleus enlargement is much greater when the oocyte nucleus is ruptured. The fate of injected nuclei was followed by the use of HeLa nuclei whose DNA had been previously la belled with [3H] thymidine. La belled DNA does not pass from injected He La nuclei into the oocyte nucleus. Injected nuclei appear not to fuse with each other or with the oocyte nucleus. Nuclei prepared by the above method look morphologically healthy in oocytes cultured in vitro for up to one month after nuclear injection. Nuclei prepared by other methods, such as those involving the use of detergents, undergo deterioration within a few days after injection into oocytes.

Origin and spatial distribution of maternal messenger RNA during oogenesis of an insect, Oncopeltus fasciatus

1979 ◽  
Vol 39 (1) ◽  
pp. 63-76
Author(s):  
D.G. Capco ◽  
W.R. Jeffery
Keyword(s):  
Spatial Distribution ◽  
Messenger Rna ◽  
Germinal Vesicle ◽  
Oncopeltus Fasciatus ◽  
Follicle Cells ◽  
Rna Molecules ◽  
Maternal Mrna ◽  
Chorion Formation ◽  
Oocyte Cytoplasm ◽  
Rna Accumulation

In order to investigate the origin and spatial distribution of maternal mRNA during oogenesis, in situ hybridization with [3H]-poly(U) was utilized for the detection of poly(A)-containing RNA [poly(A)+RNA] in histological sections of Oncopeltus fasciatus ovaries. In the germarium poly(A)+RNA was found to accumulate in the trophocyte cytoplasm concomitant with the maturation of these cells. Poly(A)+RNA was also detected in the trophic cores and nutritive tubes suggesting that these channels participate in the transport of trophocyte-derived mRNA to the oocytes. Although large amounts of poly(A)+RNA were also detected in the cytoplasm of the follicle cells, particularly during late vitellogenesis when pseudopod-like processes projected into the ooplasm, no evidence was obtained for the transport of poly(A)+RNA from these processes to the oocytes. The content of poly(A)+RNA in the oocyte cytoplasm continually increased during oogenesis. In stage 2–4 oocytes poly(A)+RNA accumulation occurred in the apparent absence of transcriptional activity in the germinal vesicle nuclei suggesting that most maternal mRNA molecules synthesized during early oogenesis are of trophocyte origin. Poly(A)+RNA also continued to accumulate after chorion formation, when the nutritive tubes are longer active in RNA transport. This implies that other sources of maternal mRNA may exist during late oogenesis. The distribution of poly(A)+RNA molecules in the oocyte cytoplasm appeared to be uniform throughout oogenesis with one exception. During late vitellogenesis poly(A)+RNA activity was significantly enhanced in the anterior and posterior periplasmic cytoplasms relative to the lateral periplasm and the endoplasm. After chorion formation these variations disappeared. The results suggest that maternal mRNA molecules arise from at least 2 sources during oogenesis. During late vitellogenesis these molecules appear to be subject to differential localization in the polar perimeters of the oocyte cytoplasm.

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