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

FEBS Letters ◽  
1989 ◽  
Vol 251 (1-2) ◽  
pp. 219-224 ◽  
Author(s):  
Odile Mulner-Lorillon ◽  
Robert Poulhe ◽  
Patrick Cormier ◽  
Jean-Claude Labbe ◽  
Marcel Doree ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Xenopus Laevis Oocytes

Xenopus laevis oocytes, eggs and tadpoles contain immunoactive insulin

1994 ◽  
Vol 141 (1) ◽  
pp. 123-129 ◽  
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

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

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.

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

1992 ◽  
Vol 12 (7) ◽  
pp. 3032-3040 ◽  
Author(s):  
M P Terns ◽  
E Lund ◽  
J E Dahlberg
Keyword(s):  
Xenopus Laevis ◽  
Nuclear Antigen ◽  
Xenopus Laevis Oocytes ◽  
Xenopus Laevis Oocyte ◽  
U6 Snrna ◽  
Small Nuclear Ribonucleoprotein ◽  
La Protein ◽  
Shift Analysis

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.

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

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.

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

1992 ◽  
Vol 12 (7) ◽  
pp. 3032-3040
Author(s):  
M P Terns ◽  
E Lund ◽  
J E Dahlberg
Keyword(s):  
Xenopus Laevis ◽  
Nuclear Antigen ◽  
Xenopus Laevis Oocytes ◽  
Xenopus Laevis Oocyte ◽  
U6 Snrna ◽  
Small Nuclear Ribonucleoprotein ◽  
La Protein ◽  
Shift Analysis

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.

A cDNA clone for a polyadenylated RNA-binding protein of Xenopus laevis oocytes hybridizes to four developmentally regulated mRNAs

1985 ◽  
Vol 5 (10) ◽  
pp. 2697-2704
Author(s):  
L J Lorenz ◽  
J D Richter
Keyword(s):  
Xenopus Laevis ◽  
Cdna Clone ◽  
Rna Binding ◽  
Binding Protein ◽  
Single Gene ◽  
In Vitro Translation ◽  
Xenopus Laevis Oocytes ◽  
Cdna Insert ◽  
Alternative Processing

Xenopus laevis oocytes contain a unique group of proteins which decrease during oogenesis, bind poly(A) RNA, and possibly play a role in the regulation of translation. A monoclonal antibody generated against one of these proteins was used to screen an expression vector cDNA library. A cDNA clone was isolated and confirmed to code for the binding protein by in vitro translation of hybrid-selected RNA followed by immunoprecipitation. This cDNA, when used in RNA gel blots, hybridized to four transcripts of 2.0, 1.7 (two transcripts of similar size), and 1.2 kilobases. All of the transcripts decreased in amount during oogenesis and were not evident in somatic cells. In addition, the fraction of the transcripts associated with polysomes decreased during oogenesis. Digestion of the cDNA insert with PstI generated two fragments of 220 and 480 base pairs which, when used as probes in an RNA gel blot, hybridized to unique as well as common transcripts. Genomic Southern blots suggested the presence of a single gene, indicating that these transcripts arose by alternative processing.

scholarly journals Actions of Agonists, Fipronil and Ivermectin on the Predominant In Vivo Splice and Edit Variant (RDLbd, I/V) of the Drosophila GABA Receptor Expressed in Xenopus laevis Oocytes

PLoS ONE ◽  
2014 ◽  
Vol 9 (5) ◽  
pp. e97468 ◽  
Author(s):  
Kristin Lees ◽  
Maria Musgaard ◽  
Siros Suwanmanee ◽  
Steven David Buckingham ◽  
Philip Biggin ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Gaba Receptor ◽  
Xenopus Laevis Oocytes

Further studies on the melanophores of periodic albino mutant of Xenopus laevis

Development ◽  
1986 ◽  
Vol 91 (1) ◽  
pp. 65-78
Author(s):  
T. Fukuzawa ◽  
H. Ide
Keyword(s):  
Xenopus Laevis ◽  
Microscopic Level ◽  
Wild Type ◽  
Light Microscopic Level ◽  
In Vitro Proliferation ◽  
Albino Mutant ◽  
Site Of Action ◽  
Periodic Albino

It is still unknown why dermal melanophores disappear during larval development, and why no or very few epidermal melanophores appear during and after metamorphosis, in Xenopus laevis showing periodic albinism (ap). To elucidate these points, we investigated (1) the occurrence of depigmentation in mutant (ap/ap) melanophores during in vitro proliferation and (2) the incidence of melanophore differentiation from mutant melanoblasts in the skin in vitro. During in vitro proliferation of mutant melanophores, ap-type melanosomes decreased in number gradually and instead the number of premelanosomes increased in the cells, which caused depigmentation at the light microscopic level in the culture. Depigmentation was observed only in mutant melanophores, and not in wild-type (+/+) melanophores. These results suggest that autonomous depigmentation of mutant dermal melanophores is the cause of the disappearance of these cells in vivo. Dopa-positive melanoblasts were demonstrated in both wild-type and mutant skins. However, the melanoblasts of metamorphosed mutant froglets did not differentiate in vitro, while those of wild-type froglets did. These results suggest that mutant melanoblasts in the skin of froglets lose the potency to differentiate into melanophores, and that this causes the lack of mutant melanophores in the froglets. The site of action of the ap gene is also discussed.

Electron microscopic studies on the structure of motile primordial germ cells of Xenopus laevis in vitro

Development ◽  
1978 ◽  
Vol 46 (1) ◽  
pp. 119-133
Author(s):  
Janet Heasman ◽  
C. C. Wylie
Keyword(s):  
Xenopus Laevis ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Structural Features ◽  
Culture Conditions ◽  
Structural Basis ◽  
Electron Microscopic ◽  
Microscopic Studies

Primordial germ cells (PGCs) of Xenopus laevis have been isolated from early embryos and kept alive in vitro, in order to study the structural basis of their motility, using the transmission and scanning electron microscope. The culture conditions used mimicked as closely as possible the in vivo environment of migrating PGCs, in that isolated PGCs were seeded onto monolayers of amphibian mesentery cells. In these conditions we have demonstrated that: (a) No significant differences were found between the morphology of PGCs in vitro and in vivo. (b) Structural features involved in PGC movement in vitro include (i) the presence of a filamentous substructure, (ii) filopodial and blunt cell processes, (iii) cell surface specializations. These features are also characteristic of migratory PGCs studied in vivo. (c) PGCs in vitro have powers of invasion similar to those of migrating PGCs in vivo. They occasionally become completely surrounded by cells of the monolayer and, in this situation, bear striking resemblance to PGCs moving between mesentery cells to the site of the developing gonad in stage-44 tadpoles. We conclude that as far as it is possible to assess, the behaviour of isolated PGCs in these in vitro conditions mimics their activities in vivo. This allows us to study the ultrastructural basis of their migration.

Uptake of biotin by native Xenopus laevis oocytes

1990 ◽  
Vol 259 (3) ◽  
pp. C397-C401 ◽  
Author(s):  
H. M. Said ◽  
L. Polenzani ◽  
S. Khorchid ◽  
D. Hollander ◽  
R. Miledi
Keyword(s):  
Xenopus Laevis ◽  
Mammalian Cells ◽  
Xenopus Oocytes ◽  
Sulfhydryl Group ◽  
Maximum Velocity ◽  
Significant Inhibition ◽  
Metabolic Inhibitors ◽  
Xenopus Laevis Oocytes ◽  
Uptake System

The present study examined biotin uptake by Xenopus laevis oocytes in vitro. Uptake of low (0.03 microM) and high (10 microM) concentrations of biotin was linear with time for up to 4 h of incubation and occurred with little initial binding to oocytes. Uptake of biotin was dependent on extracellular Na+ concentration [Na+]o and was severely inhibited when Na+ was replaced by other monovalent cations [choline, tetraethylammonia, Li+, and tris(hydroxymethyl)aminomethane]. The initial rate of biotin uptake was saturable as a function of concentration with an apparent Michaelis constant of 3.9 +/- 0.5 microM and maximum velocity of 1,559 +/- 70 fmol.oocyte-1.h-1. Addition to the incubation medium of biotin structural analogues desthiobiotin and thioctic acid caused significant and concentration-dependent inhibition in the uptake of [3H]biotin. This inhibition was found to be competitive in nature with inhibition constant values of 9 and 17.5 microM. In contrast, neither the structural analogue biocytin nor biotin methyl ester (compounds in which the carboxyl group of the valeric acid moiety is blocked) showed any effect on the uptake of [3H]biotin. Biotin uptake was significantly blocked by the metabolic inhibitors dinitrophenol, cyanide, and azide and by incubation at 4 degrees C. Also, the sulfhydryl group blocker p-(chloromercuri)phenylsulfonate caused significant inhibition in biotin uptake. These results demonstrate that Xenopus oocytes possess an uptake system for biotin in its cell membrane that is Na+, energy, and temperature dependent. These characteristics of biotin uptake are similar to those reported in mammalian cells. It is suggested that Xenopus oocytes might be a useful in vitro model system to study the details of the mechanisms and regulation of biotin movement across biological membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

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