Characterization of protein synthesis initiation factor 2 from Xenopus laevis oocytes

The protein synthesis initiation factor 2 (eIF2) from Xenopus laevis oocytes has been extensively purified and characterized. Depending upon the purification scheme, eIF2 containing three subunits (alpha, beta and gamma) with Mr of 160,000, or two subunits (alpha and gamma) with Mr 90,000 can be obtained. The key step for obtaining the three subunit factor is the addition of 30 mM benzamidine to the initial homogenization, since this compound protects the highly sensitive beta subunit from proteolytic degradation. Subunit alpha of the oocyte eIF2 can be phosphorylated by the specific kinase from rabbit reticulocytes, whereas subunit beta is phosphorylated by oocyte casein kinase II. The oocyte eIF2 has a KD of 7.2 X 10(-8) M for GDP and 3.8 X 10(-6) M for GTP. The purified three subunit eIF2 has 0.4 mol of GDP bound/mol of factor. The crude preparations of eIF2 are not affected by Mg2+ in their exchange of guanine nucleotides or in the formation of ternary complexes with GTP and methionyl-tRNA, but these reactions are strongly inhibited by Mg2+ when the highly purified preparations are used.     

Mitochondria DNA synthesis in oocytes of Xenopus laevis

The process of attachment was studied in primary mouse kidney epithelial cell cultures by means of reflexion contrast microscopy, a method developed for studying the cell membrane-substrate relationship. The first in a series of events is simple adherence to the substrate, called close contact. This phenomenon is associated with the greatest extension of lamellar cytoplasm and the fewest number of cell nuclei/unit area. The nuclei of such cells are in close contact with the bottom portion of the cell membrane. Approx. 24 h after planting, as the cultures become more crowded, cells develop a different kind of attachment to the substrate—focal contacts—that are correlated with a decrease in lamellar cytoplasm. Cells detached from the substrate after close contact formation readily reattach, while cells detached after formation of focal contacts do not reattach. After incubation for periods greater than 5 days, the dense cultures degenerate and cells lose their attachment to the glass surface.     

The effect of cytochalasin D on protein synthesis in Xenopus laevis oocytes

Defolliculated fully grown oocytes of Xenopus laevis were treated with cytochalasin D (10 micrograms/ml) and their protein synthesis was studied by labelling with S-35 methionine. This treatment brought about an alteration in pigment pattern as well as a reduction in amino acid uptake by the oocytes. However, the radioactive amino acid taken by cytochalasin-treated oocytes was incorporated into protein in the same proportion as in untreated oocytes. These results suggested that subcortical pigment distribution and amino acid uptake in fully grown oocytes were microfilament-dependent processes, whereas protein synthesis in the oocyte was not.     

Effect of diadenosine tetraphosphate microinjection on heat shock protein synthesis in Xenopus laevis oocytes.

Bisnucleosides polyphosphates are thought to be chemical messengers signalling to the cell the onset of various stresses. Diadenosine tri- and tetraphosphates (respectively, Ap3A and Ap4A) accumulate in prokaryotic and eukaryotic cells under heat shock conditions, suggesting they could trigger the synthesis of heat shock proteins (hsps). In this study, Ap4A, Ap3A and, as a control, Ap4 (adenosine tetraphosphate) were injected into Xenopus oocytes. Whereas none of these compounds is able to trigger the synthesis of hsps in the absence of hyperthermic treatment, nuclear microinjection of Ap4A after a mild heat shock specifically enhances the synthesis of the 70-kd hsp, which is involved in the regulation and possibly the termination of the heat shock response. The microinjection of Ap4A prior to the hyperthermic treatment results in a strong inhibition of hsps synthesis (with the exception of the 70-kd hsp) suggesting that Ap4A is involved in the regulation and/or termination of the heat shock response. Ap3A and Ap4 do not induce any detectable modification of hsps expression.     

Antibodies to phosphotyrosine injected in Xenopus laevis oocytes modulate maturation induced by insulin/IGF-I.

Xenopus oocytes carry IGF-I receptors, and undergo meiotic maturation in response to binding of IGF-I or insulin to the IGF-I receptor. Maturation is initiated upon activation of the IGF-I receptor tyrosine kinase and requires tyrosine dephosphorylation of p34cdc2, the kinase component of maturation promoting factor (MPF). To further evaluate the role of tyrosine phosphorylation in the signalling pathway triggered by insulin/IGF-I, we have injected antibodies to phosphotyrosine into oocytes and examined their effects on oocyte maturation. Antibodies at a low concentration (40 ng/oocyte, corresponding to a concentration of 40 micrograms/ml), enhanced specifically insulin-, but not progesterone-induced maturation. In contrast, at 150 ng/oocyte, the same antibodies decreased maturation induced by insulin, progesterone, or microinjected MPF. In cell-free systems, antibodies to phosphotyrosine recognized the oocyte IGF-I receptor and modulated its ligand-induced tyrosine kinase activity in a biphasic manner, with a stimulation at 40 micrograms/ml and an inhibition at higher concentrations. Moreover, antibodies at 150 ng/oocyte neutralized the kinase activity of a crude MPF extract. This neutralization was not accompanied by a rephosphorylation of p34cdc2, but by a decrease in tyrosine phosphorylation of a 60-kDa protein, which was present in M phase extracts and undetectable in G2-arrested oocytes. Taken together, these results point to at least two levels of anti-phosphotyrosine antibody action: (i) the IGF-I receptor signalling system, and (ii) a regulatory step of MPF activation, which might be distinct of the well-documented inactivating phosphorylation of p34cdc2.     

DNA synthesis in ocytes and eggs of Xenopus laevis injected with DNA.

Single-stranded calf thymus DNA injected into preovulation oocytes, postovulation oocytes or eggs of Xenopus laevis induces synthesis of double-stranded DNA of similar base composition. In contrast, native (double-stranded) calf thymus DNA injected into oocytes does not stimulate DNA synthesis, though it does do so in eggs. The buoyant density of normal or IUdR-substituted newly-synthesized DNA on neutral or alkaline CsCl gradients suggests that the injected DNA is replicated.The amount of synthesis induced by injecting single-stranded DNA is five times greater in eggs than in oocytes. The maximum synthesis observed in eggs injected with native DNA is 50 pg/hr; this is sufficient for nuclear DNA replication in uninjected fertilised eggs, but not in midcleavage. However in vitro studies (reported elsewhere) indicate the presence of a large store of DNA polymerase activity in eggs. We conclude that only a small proportion of the total DNA polymerase activity in an egg is available for DNA synthesis during the first 2 hr of development.     

Cyclic AMP synthesis in Xenopus laevis oocytes: inhibition by progesterone.

[alpha-32P]ATP was microinjected into Xenopus oocyte and neosynthesized cyclic AMP was isolated. Cholera toxin inhibited progesterone-induced maturation and stimulated after 3 h of preincubation the amount of neosynthesized cyclic AMP. Progesterone decreased the neosynthesis of cyclic AMP during the first hour following addition of the hormone.     

Changes in protein phosphorylation accompanying maturation of Xenopus laevis oocytes

Protein phosphorylation has been measured after injection of [³²P]phosphate into oocytes of Xenopus laevis undergoing progesterone-induced meiotic maturation. As oocytes mature, there is a burst of nonyolk protein phosphorylation several hours after progesterone exposure and shortly before germinal vesicle breakdown (GVBD). This burst is not due to changes in the specific activity of the phosphate or ATP pool. Enucleated oocytes exposed to progesterone also experience the burst, indicating the cytoplasmic location of phosphoprotein formation. When an oocyte receives an injection of cytoplasm containing the maturation-promoting factor (MPF), a burst of protein phosphorylation occurs immediately, and GVBD occurs shortly thereafter, even in the presence of cycloheximide. Under a variety of conditions promoting or blocking maturation, oocytes which undergo GVBD are the only ones to have experienced the phosphorylation burst. The results suggest that the protein phosphorylation burst is a necessary step in the mechanism by which MPF promotes GVBD.     

Mitochondrial DNA synthesis in large and mature oocytes of Xenopus laevis

Deoxynucleosides are incorporated into mitochondrial DNA (mtDNA) of large oocytes; the rate of incorporation is about 2% of the mtDNA amount per 24 hr. When oocytes have been induced to mature in vitro with human chorionic gonadotropin (HCG), uptake and actual incorporation of thymidine decrease, although phosphorylation is enhanced. An examination of mtDNA replication shows that HCG treatment induces an increase in the relative synthesis of E-strands and an accumulation of D-loops. A similar effect is obtained by ethidium bromide treatment. Thus, gonadotropin appears to delay E-strand elongation and to synchronize mtDNA molecules at the begining of their replication cycle.