Nonspecific effects of oligodeoxynucleotide injection in Xenopus oocytes: a reevaluation of previous D7 mRNA ablation experiments

Development ◽  
1990 ◽  
Vol 110 (3) ◽  
pp. 769-779
Author(s):  
R.C. Smith ◽  
W.M. Bement ◽  
M.A. Dersch ◽  
E. Dworkin-Rastl ◽  
M.B. Dworkin ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Xenopus Oocytes ◽  
Cell Metabolism ◽  
Germinal Vesicle ◽  
Annulate Lamellae ◽  
Maternal Mrna ◽  
Nonspecific Effects ◽  
Abnormal Accumulation ◽  
Cellular Mrnas

Microinjection of oligodeoxynucleotides (ODNs) complementary to cellular mRNAs has been advanced as an experimental approach to degrade target mRNAs in vivo and thereby obtain information as to the function of their cognate proteins. It is shown here that ODNs can induce a variety of aberrations in cell metabolism and structure when injected into Xenopus oocytes. Examination of histological sections of ODN-injected oocytes revealed the frequent abnormal accumulation of heavily staining basophilic material in the area of the germinal vesicle (gv). Ultrastructural analysis detected further abnormalities including blebbing of the plasma membrane, anomalous cytoskeletal structures, hyperorganised annulate lamellae, hyperinvagination of the gv, and formation of irregular nucleoli within the gv. Analysis of newly synthesised proteins by [35S]methionine radiolabelling of oocytes demonstrated that ODN injection can trigger a general decrease in both label uptake and protein synthesis. Qualitative effects on protein synthesis could also be observed, particularly a decrease in synthesis of high molecular weight proteins. The severity of ODN-induced effects is dose-dependent and highly variable from ODN to ODN. The previously reported delay in progesterone-induced maturation observed in oocytes depleted of the maternal mRNA D7 by ODN-directed degradation (Smith R. C., Dworkin M. B. and Dworkin-Rastl E. (1988) Genes and Devpt. 2, 1296–1306) is most likely a result of nonspecific ODN effects in the oocyte. Oocytes injected with effective antisense D7 ODNs that do not display detectable side effects matured with normal kinetics.

scholarly journals Studies on the mechanism for entry of vesicular stomatitis virus glycoprotein g mRNA into membrane-bound polyribosome complexes

1977 ◽  
Vol 74 (1) ◽  
pp. 43-57 ◽  
Author(s):  
MJ Grubman ◽  
JA Weinstein ◽  
DA Shafritz
Keyword(s):  
Protein Synthesis ◽  
Vesicular Stomatitis Virus ◽  
Cytosol Fraction ◽  
Initial Event ◽  
Glycoprotein G ◽  
Nonspecific Effects ◽  
Membrane Bound ◽  
Vesicular Stomatitis

Glycoprotein mRNA (G mRNA) of vesicular stomatitis virus is synthesized in the cytosol fraction of infected HeLa cells. Shortly after synthesis, this mRNA associates with 40S ribosomal subunits and subsequently forms 80S monosomes in the cytosol fraction. The bulk of labeled G mRNA is then found in polysomes associated with the membrane, without first appearing in the subunit or monomer pool of the membrane-bound fraction. Inhibition of the initiation of protein synthesis by pactamycin or muconomycin A blocks entry of newly synthesized G m RNA into membrane-bound polysomes. Under these circumstances, labeled G mRNA accumulates into the cytosol. Inhibition of the elongation of protein synthesis by cucloheximide, however, allows entry of 60 percent of newly synthesized G mRNA into membrane-bound polysomes. Furthermore, prelabeled G mRNA associated with membrane-bound polysomes is released from the membrane fraction in vivo by pactamycin or mucomycon A and in vitro by 1mM puromycin - 0.5 M KCI. This release is not due to nonspecific effects of the drugs. These results demonstrate that association of G mRNA with membrane-bound polysomes is dependent upon polysome formation and initiation of protein synthesis. Therefore, direct association of the 3' end of G mRNA with the membrane does not appear to be the initial event in the formation of membrane-bound polysomes.

A new role for Mos in Xenopus oocyte maturation: targeting Myt1 independently of MAPK

Development ◽  
2002 ◽  
Vol 129 (9) ◽  
pp. 2129-2139 ◽  
Author(s):  
Marion Peter ◽  
Jean-Claude Labbé ◽  
Marcel Dorée ◽  
Elisabeth Mandart
Keyword(s):  
Protein Kinase ◽  
Oocyte Maturation ◽  
Xenopus Oocyte ◽  
Xenopus Oocytes ◽  
Mapk Pathway ◽  
Germinal Vesicle ◽  
Mapk Cascade ◽  
Germinal Vesicle Breakdown ◽  
Mapk Activation

The resumption of meiosis in Xenopus arrested oocytes is triggered by progesterone, which leads to polyadenylation and translation of Mos mRNA, then activation of MAPK pathway. While Mos protein kinase has been reported to be essential for re-entry into meiosis in Xenopus, arrested oocytes can undergo germinal vesicle breakdown (GVBD) independently of MAPK activation, leading us to question what the Mos target might be if Mos is still required. We now demonstrate that Mos is indeed necessary, although is independent of the MAPK cascade, for conversion of inactive pre-MPF into active MPF. We have found that Myt1 is likely to be the Mos target in this process, as Mos interacts with Myt1 in oocyte extracts and Mos triggers Myt1 phosphorylation on some sites in vivo, even in the absence of MAPK activation. We propose that Mos is involved, not only in the MAPK cascade pathway, but also in a mechanism that directly activates MPF in Xenopus oocytes.

scholarly journals Molecular cloning of an amphibian insulin receptor substrate 1-like cDNA and involvement of phosphatidylinositol 3-kinase in insulin-induced Xenopus oocyte maturation.

1995 ◽  
Vol 15 (7) ◽  
pp. 3563-3570 ◽  
Author(s):  
X J Liu ◽  
A Sorisky ◽  
L Zhu ◽  
T Pawson
Keyword(s):  
Insulin Receptor ◽  
Oocyte Maturation ◽  
Xenopus Oocytes ◽  
Germinal Vesicle ◽  
Insulin Receptor Substrate ◽  
Phosphorylation Sites ◽  
Glutathione S Transferase ◽  
Germinal Vesicle Breakdown

An insulin receptor substrate 1 (IRS-1)-like cDNA was isolated from a Xenopus ovary cDNA library by low-stringency hybridization using rat IRS-1 cDNA as a probe. The deduced amino acid sequence encoded by this cDNA (termed XIRS-L) is 67% identical (77% similar) to that of rat IRS-1. Significantly, all the insulin-induced tyrosine phosphorylation sites identified in rat IRS-1, including those responsible for binding to the Src homology domains of phosphatidylinositol (PI) 3-kinase, Syp and Grb2, are conserved in XIRS-L. Both mRNA and protein corresponding to the cloned XIRS-L can be detected in immature Xenopus oocytes. Recombinant XIRS-L protein produced in insect cells or a bacterial glutathione S-transferase fusion protein containing the putative PI 3-kinase binding site can be phosphorylated in vitro by purified insulin receptor kinase (IRK) domain, and the IRK-catalyzed phosphorylation renders both proteins capable of binding PI 3-kinase in Xenopus oocyte lysates. Another glutathione S-transferase fusion protein containing the C terminus of XIRS-L and including several putative tyrosine phosphorylation sites is also phosphorylated by IRK in vitro, but it failed to bind PI 3-kinase. Insulin stimulation of immature Xenopus oocytes activates PI 3-kinase in vivo [as indicated by an elevation of PI(3,4)P2 and PI(3,4,5)P3] as well as oocyte maturation (as indicated by germinal vesicle breakdown). Pretreatment of these oocytes with wortmannin inhibited insulin-induced activation of PI 3-kinase in vivo. The same treatment also abolished insulin-induced, but not progesterone-induced, germinal vesicle breakdown. These results (i) identify an IRS-1-like molecule in immature Xenopus oocytes, suggesting that the use of IRS-1-like Scr homology 2 domain-docking proteins in signal transduction is conserved in vertebrates, and (ii) strongly implicate PI 3-kinase as an essential effector of insulin-induced oocyte maturation.

Differential effects of 6-DMAP, olomoucine and roscovitine on Xenopus oocytes and eggs

Zygote ◽  
2000 ◽  
Vol 8 (1) ◽  
pp. 3-14 ◽  
Author(s):  
Stéphane Flament ◽  
Jean-François Bodart ◽  
Marc Bertout ◽  
Edith Browaeys ◽  
Arlette Rousseau ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Xenopus Oocytes ◽  
Kinase Inhibitors ◽  
Germinal Vesicle ◽  
Cyclin B ◽  
Cyclin Dependent Kinase ◽  
Germinal Vesicle Breakdown ◽  
Lower Permeability ◽  
New Compounds

The effects of the new cyclin-dependent kinase inhibitors, roscovitine and olomoucine, on oocytes and eggs of Xenopus laevis were investigated and compared with those of 6-dimethylamino purine (6-DMAP). The inhibitory properties of 6-DMAP, olomoucine and roscovitine towards p34cdc2-cyclin B isolated from Xenopus eggs revealed K-IC50 values of 300, 40 and 10 μM respectively. The three compounds inhibited progesterone-induced maturation with M-IC50 values of 200, 100 and 20 μM. These values were consistent with the K-IC50 values but the ratio M-IC50/K-IC50 was higher for roscovitine and olomoucine than for 6-DMAP. The disappearance of spindle and condensed chromosomes without pronucleus formation was observed when 1 mM 6-DMAP was applied for 4 h at germinal vesicle breakdown or at metaphase II, whereas no effect was observed using 1 mM olomoucine or 50 μM roscovitine. Changes in the electrophoretic mobility of p34cdc2 and erk2 were observed only in homogenates of matured oocytes or eggs exposed for 4 h to 1 mM 6-DMAP. When the drugs were microinjected into matured oocytes, olomoucine (100 μM) and roscovitine (50 μM) induced pronucleus formation more efficiently than did 6-DMAP (100 μM). Taken together, these results demonstrate that Xenopus oocytes possess a lower permeability to olomoucine and roscovitine and that these new compounds are suitable for in vivo studies after germinal vesicle breakdown provided they are microinjected.

scholarly journals SIP/SHIP inhibits Xenopus oocyte maturation induced by insulin and phosphatidylinositol 3-kinase.

1997 ◽  
Vol 17 (5) ◽  
pp. 2559-2565 ◽  
Author(s):  
M Deuter-Reinhard ◽  
G Apell ◽  
D Pot ◽  
A Klippel ◽  
L T Williams ◽  
...  
Keyword(s):  
Xenopus Oocytes ◽  
Germinal Vesicle ◽  
Sh2 Domain ◽  
Mitogen Activated Protein Kinase ◽  
Phosphatidylinositol 3 Kinase ◽  
Germinal Vesicle Breakdown ◽  
Inositol Phosphatase ◽  
Phosphatidylinositol 3

SIP (signaling inositol phosphatase) or SHIP (SH2-containing inositol phosphatase) is a recently identified SH2 domain-containing protein which has been implicated as an important signaling molecule. SIP/SHIP becomes tyrosine phosphorylated and binds the phosphotyrosine-binding domain of SHC in response to activation of hematopoietic cells. The signaling pathways and biological responses that may be regulated by SIP have not been demonstrated. SIP is a phosphatidylinositol- and inositol-polyphosphate 5-phosphatase with specificity in vitro for substrates phosphorylated at the 3' position. Phosphatidylinositol 3'-kinase (PI 3-kinase) is an enzyme which is involved in mitogenic signaling and whose phosphorylated lipid products are predicted to be substrates for SIP. We tested the hypothesis that SIP can modulate signaling by PI 3-kinase in vivo by injecting SIP cRNAs into Xenopus oocytes. SIP inhibited germinal vesicle breakdown (GVBD) induced by expression of a constitutively activated form of PI 3-kinase (p110*) and blocked GVBD induced by insulin. SIP had no effect on progesterone-induced GVBD. Catalytically inactive SIP had little effect on insulin- or PI 3-kinase-induced GVBD. Expression of SIP, but not catalytically inactive SIP, also blocked insulin-induced mitogen-activated protein kinase phosphorylation in oocytes. SIP specifically and markedly reduced the level of phosphatidylinositol (3,4,5) triphosphate [PtdIns(3,4,5)P3] generated in oocytes in response to insulin. These results demonstrate that a member of the phosphatidylinositol polyphosphate 5-phosphatase family can inhibit signaling in vivo. Further, our data suggest that the generation of PtdIns(3,4,5)P3 by PI 3-kinase is necessary for insulin-induced GVBD in Xenopus oocytes.

Respiratory energy requirements and rate of protein turnover in vivo determined by the use of an inhibitor of protein synthesis and a probe to assess its effect

1994 ◽  
Vol 92 (4) ◽  
pp. 585-594 ◽  
Author(s):  
T. J. Bouma ◽  
R. De Visser ◽  
J. H. J. A. Janssen ◽  
M. J. De Kock ◽  
P H. Van Leeuwen ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Turnover ◽  
Energy Requirements ◽  
Inhibitor Of Protein Synthesis

Rapid in vivo induction of leukocyte tissue factor mRNA and protein synthesis following low dose endotoxin administration to rabbits

2001 ◽  
Vol 2 (3) ◽  
pp. 188-195 ◽  
Author(s):  
Tara C Brutzki ◽  
Myron J Kulczycky ◽  
Leslie Bardossy ◽  
Bryan J Clarke ◽  
Morris A Blajchman
Keyword(s):  
Protein Synthesis ◽  
Tissue Factor ◽  
Low Dose ◽  
Endotoxin Administration ◽  
In Vivo Induction
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