3'-end-dependent formation of U6 small nuclear ribonucleoprotein particles in Xenopus laevis oocyte nuclei.

We have identified and characterized a U6 small nuclear (sn) ribonucleoprotein particle (RNP) present in the nuclei of Xenopus laevis oocytes. The structure of this U6 snRNP was investigated by native gel shift analysis and a combination of RNA-protein UV cross-linking, RNase T1 fingerprinting, and immunoprecipitation assays. These analyses demonstrate that certain forms of U6 snRNA associate with the 50-kDa nuclear antigen La both in vivo and in vitro. The La protein binds the stretch of uridylates at the 3' hydroxyl end of newly synthesized U6 snRNA. La does not bind to mature U6 snRNAs that have 2',3'-cyclic phosphate (greater than p) groups at their 3' ends (E. Lund and J. E. Dahlberg, Science 255:327-330, 1992) or to U6 snRNAs in anti-Sm-precipitable U4/U6 snRNPs. We propose that 3'-end modification, including posttranscriptional UMP addition, modulates the binding of La protein to U6 snRNA which, in turn, may affect the function of this RNA.

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Nuclear transport of U1 snRNP in somatic cells: differences in signal requirement compared with Xenopus laevis oocytes.

The Journal of Cell Biology ◽

10.1083/jcb.125.5.971 ◽

1994 ◽

Vol 125 (5) ◽

pp. 971-980 ◽

Cited By ~ 27

Author(s):

U Fischer ◽

J Heinrich ◽

K van Zee ◽

E Fanning ◽

R Lührmann

Keyword(s):

Xenopus Laevis ◽

Nuclear Import ◽

Nuclear Transport ◽

Somatic Cells ◽

Xenopus Laevis Oocytes ◽

Xenopus Laevis Oocyte ◽

Nuclear Targeting ◽

Transport Pathway ◽

U1 Snrnp

The signal requirement for the nuclear import of U1 RNA in somatic cells from different species was investigated by microinjection of both digoxygenin-labeled wild type and mutant U1 RNA molecules and in vitro reconstituted U1 snRNPs. U1 RNA was shown to be targeted to the nucleus by a temperature-dependent process that requires the prior assembly of RNPs from the common proteins and the microinjected RNA. Competition in the cell between immunoaffinity-purified U1 snRNPs and digoxygenin-labeled U1 snRNPs reconstituted in vitro showed that the transport is saturable and should therefore be a mediated process. The transport of a karyophilic protein under the same conditions was not affected, indicating the existence of a U snRNP-specific transport pathway in somatic cells, as already seen in the Xenopus laevis oocyte system. Surprisingly, the signal requirement for nuclear transport of U1 snRNP was found to differ between oocytes and somatic cells from mouse, monkey and Xenopus, in that the m3GGpppG-cap is no longer an essential signaling component in somatic cells. However, as shown by investigation of the transport kinetics of m3GpppG- and ApppG-capped U1 snRNPs, the m3GpppG-cap accelerates the rate of U1 snRNP import significantly indicating that it has retained a signaling role for nuclear targeting of U1 snRNP in somatic cells. Moreover, our data strongly suggest that cell specific rather than species specific differences account for the differential m3G-cap requirement in nuclear import of U1 snRNPs.

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Purification of a p47 phosphoprotein from Xenopus laevis oocytes and identification as an in vivo and in vitro p34cdc2 substrate

FEBS Letters ◽

10.1016/0014-5793(89)81458-9 ◽

1989 ◽

Vol 251 (1-2) ◽

pp. 219-224 ◽

Cited By ~ 26

Author(s):

Odile Mulner-Lorillon ◽

Robert Poulhe ◽

Patrick Cormier ◽

Jean-Claude Labbe ◽

Marcel Doree ◽

...

Keyword(s):

Xenopus Laevis ◽

Xenopus Laevis Oocytes

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Characterization of the repressed 5S DNA minichromosomes assembled in vitro with a high-speed supernatant of Xenopus laevis oocytes

Molecular and Cellular Biology ◽

10.1128/mcb.8.10.4257-4269.1988 ◽

1988 ◽

Vol 8 (10) ◽

pp. 4257-4269

Author(s):

A Shimamura ◽

D Tremethick ◽

A Worcel

Keyword(s):

Xenopus Laevis ◽

Transcriptional Activation ◽

High Speed ◽

Histone H1 ◽

Xenopus Laevis Oocytes ◽

Xenopus Laevis Oocyte ◽

5S Rna ◽

Rna Transcription

We describe an in vitro system, based on the Xenopus laevis oocyte supernatant of Glikin et al. (G. Glikin, I. Ruberti, and A. Worcel, Cell 37:33-41, 1984), that packages DNA into minichromosomes with regularly spaced nucleosomes containing histones H3, H4, H2A, and H2B but no histone H1. The same supernatant also assembles the 5S RNA transcription complex; however, under the conditions that favor chromatin assembly, transcription is inhibited and a phased nucleosome forms over the 5S RNA gene. The minichromosomes that are fully loaded with nucleosomes remain refractory to transcriptional activation by 5S RNA transcription factors. Our data suggest that this repression is caused by a nucleosome covering the 5S RNA gene and that histone H1 is not required for regular nucleosome spacing or for gene repression in this system.

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Testosterone and Progesterone Rapidly Attenuate Plasma Membrane Gβγ-Mediated Signaling in Xenopus laevis Oocytes by Signaling through Classical Steroid Receptors

Molecular Endocrinology ◽

10.1210/me.2006-0301 ◽

2007 ◽

Vol 21 (1) ◽

pp. 186-196 ◽

Cited By ~ 26

Author(s):

Kristen Evaul ◽

Michelle Jamnongjit ◽

Bala Bhagavath ◽

Stephen R. Hammes

Keyword(s):

Xenopus Laevis ◽

G Protein ◽

G Proteins ◽

Oocyte Maturation ◽

Live Cells ◽

Inhibition Mechanism ◽

Xenopus Laevis Oocytes ◽

Xenopus Laevis Oocyte ◽

Potassium Influx

Abstract Many transcription-independent (nongenomic) steroid effects are regulated by G proteins. A well-established, biologically relevant example of steroid/G protein interplay is steroid-triggered oocyte maturation, or meiotic resumption, in Xenopus laevis. Oocyte maturation is proposed to occur through a release of inhibition mechanism whereby constitutive signaling by Gβγ and other G proteins maintains oocytes in meiotic arrest. Steroids (androgens in vivo, and androgens and progesterone in vitro) overcome this inhibition to promote meiotic resumption. To test this model, we used G protein-regulated inward rectifying potassium channels (GIRKs) as markers of Gβγ activity. Overexpression of GIRKs 1 and 2 in Xenopus oocytes resulted in constitutive potassium influx, corroborating the presence of basal Gβγ signaling in resting oocytes. Testosterone and progesterone rapidly reduced potassium influx, validating that steroids attenuate Gβγ activity. Interestingly, reduction of classical androgen receptor (AR) expression by RNA interference abrogated testosterone’s effects on GIRK activity at low, but not high, steroid concentrations. Accordingly, androgens bound to the Xenopus progesterone receptor (PR) at high concentrations, suggesting that, in addition to the AR, the PR might mediate G protein signaling when androgens levels are elevated. In contrast, progesterone bound with high affinity to both the Xenopus PR and AR, indicating that progesterone might signal and promote maturation through both receptors, regardless of its concentration. In sum, these studies introduce a novel method for detecting nongenomic steroid effects on G proteins in live cells in real time, and demonstrate that cross talk may occur between steroids and their receptors during Xenopus oocyte maturation.

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Xenopus laevis oocytes, eggs and tadpoles contain immunoactive insulin

Journal of Endocrinology ◽

10.1677/joe.0.1410123 ◽

1994 ◽

Vol 141 (1) ◽

pp. 123-129 ◽

Cited By ~ 6

Author(s):

F de Pablo ◽

R Dashner ◽

A R Shuldiner ◽

J Roth

Keyword(s):

Xenopus Laevis ◽

High Performance ◽

Immunoblot Analysis ◽

Reversed Phase ◽

Xenopus Laevis Oocytes ◽

Maternal Origin ◽

Insulin Mrna ◽

Low Levels

Abstract Insulin is a multifunctional polypeptide hormone that regulates metabolic processes and promotes mitogenesis and differentiation in vitro in the cells and tissues of several species. Its role in vivo during embryogenesis is still poorly understood. We have previously found insulin mRNA in mature Xenopus laevis oocytes and in embryos during neurulation (before organogenesis of the pancreas takes place). We have now measured insulin immunoactivity in mature oocytes, unfertilized eggs and day-2 tadpoles. Using reversed phase high performance liquid chromatography, we found low levels of insulin in extracts of oocytes (stage VI). Both Xenopus insulin I and II were detected in unfertilized eggs. The day-2 tadpoles (stages 31–33) also contained immunoactive insulin, and in swimming tadpoles (stage 46) a few clusters of cells containing insulin immunoactivity could be identified by indirect immunofluorescence. Immunoblot analysis was relatively insensitive, detecting insulin only in the adult Xenopus pancreas. In summary, insulin (from maternal origin and embryonic expression) appears to be present early enough in Xenopus laevis to influence developmental processes such as neurulation. Journal of Endocrinology (1994) 141, 123–129

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Characterization of recombination intermediates from DNA injected into Xenopus laevis oocytes: evidence for a nonconservative mechanism of homologous recombination

Molecular and Cellular Biology ◽

10.1128/mcb.11.6.3278-3287.1991 ◽

1991 ◽

Vol 11 (6) ◽

pp. 3278-3287 ◽

Cited By ~ 1

Author(s):

E Maryon ◽

D Carroll

Keyword(s):

Homologous Recombination ◽

Xenopus Laevis ◽

Kinetic Properties ◽

Xenopus Laevis Oocytes ◽

Xenopus Laevis Oocyte ◽

Two Dimensional Gel Electrophoresis ◽

Recombination Mechanism ◽

Single Strand Annealing ◽

Recombination Pathway

Homologous recombination between DNA molecules injected into Xenopus laevis oocyte nuclei is extremely efficient if injected molecules have overlapping homologous ends. Earlier work demonstrated that ends of linear molecules are degraded by a 5'----3' exonuclease activity, yielding 3' tails that participate in recombination. Here, we have characterized intermediates further advanced along the recombination pathway. The intermediates were identified by their unique electrophoretic and kinetic properties. Two-dimensional gel electrophoresis and hybridization with oligonucleotide probes showed that the intermediates had heteroduplex junctions within their homologous overlaps in which strands ending 3' were full length and those ending 5' were shortened. Additional characterization suggested that these intermediates had formed by the annealing of complementary 3' tails. Annealed junctions made in vitro were rapidly processed to products, indicating that they are on the normal recombination pathway. These results support a nonconservative, single-strand annealing mode of recombination. This recombination mechanism appears to be shared by many organisms, including bacteria, fungi, plants, and mammals.

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Accurate in vitro transcription of Xenopus laevis mitochondrial DNA from two bidirectional promoters

Molecular and Cellular Biology ◽

10.1128/mcb.6.7.2543-2550.1986 ◽

1986 ◽

Vol 6 (7) ◽

pp. 2543-2550

Author(s):

D F Bogenhagen ◽

B K Yoza

Keyword(s):

Mitochondrial Dna ◽

Xenopus Laevis ◽

Rna Polymerase ◽

Consensus Sequence ◽

In Vitro Transcription ◽

Xenopus Laevis Oocytes ◽

Mitochondrial Rna ◽

Bidirectional Promoters

The mitochondrial RNA polymerase from Xenopus laevis oocytes was partially purified by heparin-Sepharose chromatography and phosphocellulose chromatography. This RNA polymerase preparation specifically initiated the transcription of X. laevis mitochondrial DNA (mtDNA) from two bidirectional promoters contained within a 123-base-pair segment of the mtDNA between the heavy-strand replication origin and the rRNA cistrons. Transcription in vitro initiated from precisely the same start sites previously mapped as initiation sites for transcription in vivo. At each of the four sites, initiation occurred within a conserved nucleotide sequence, ACPuTTATA. This consensus sequence is not related to promoters for transcription of human mtDNA.

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Characterization of the repressed 5S DNA minichromosomes assembled in vitro with a high-speed supernatant of Xenopus laevis oocytes.

Molecular and Cellular Biology ◽

10.1128/mcb.8.10.4257 ◽

1988 ◽

Vol 8 (10) ◽

pp. 4257-4269 ◽

Cited By ~ 58

Author(s):

A Shimamura ◽

D Tremethick ◽

A Worcel

Keyword(s):

Xenopus Laevis ◽

Transcriptional Activation ◽

High Speed ◽

Histone H1 ◽

Xenopus Laevis Oocytes ◽

Xenopus Laevis Oocyte ◽

5S Rna ◽

Rna Transcription

We describe an in vitro system, based on the Xenopus laevis oocyte supernatant of Glikin et al. (G. Glikin, I. Ruberti, and A. Worcel, Cell 37:33-41, 1984), that packages DNA into minichromosomes with regularly spaced nucleosomes containing histones H3, H4, H2A, and H2B but no histone H1. The same supernatant also assembles the 5S RNA transcription complex; however, under the conditions that favor chromatin assembly, transcription is inhibited and a phased nucleosome forms over the 5S RNA gene. The minichromosomes that are fully loaded with nucleosomes remain refractory to transcriptional activation by 5S RNA transcription factors. Our data suggest that this repression is caused by a nucleosome covering the 5S RNA gene and that histone H1 is not required for regular nucleosome spacing or for gene repression in this system.

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Accurate in vitro transcription of Xenopus laevis mitochondrial DNA from two bidirectional promoters.

Molecular and Cellular Biology ◽

10.1128/mcb.6.7.2543 ◽

1986 ◽

Vol 6 (7) ◽

pp. 2543-2550 ◽

Cited By ~ 24

Author(s):

D F Bogenhagen ◽

B K Yoza

Keyword(s):

Mitochondrial Dna ◽

Xenopus Laevis ◽

Rna Polymerase ◽

Consensus Sequence ◽

In Vitro Transcription ◽

Xenopus Laevis Oocytes ◽

Mitochondrial Rna ◽

Bidirectional Promoters

The mitochondrial RNA polymerase from Xenopus laevis oocytes was partially purified by heparin-Sepharose chromatography and phosphocellulose chromatography. This RNA polymerase preparation specifically initiated the transcription of X. laevis mitochondrial DNA (mtDNA) from two bidirectional promoters contained within a 123-base-pair segment of the mtDNA between the heavy-strand replication origin and the rRNA cistrons. Transcription in vitro initiated from precisely the same start sites previously mapped as initiation sites for transcription in vivo. At each of the four sites, initiation occurred within a conserved nucleotide sequence, ACPuTTATA. This consensus sequence is not related to promoters for transcription of human mtDNA.

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