Rabies mRNA translation in Xenopus laevis oocytes.

Two rabies virus-specific mRNA species were identified by analysis of their encoded proteins after translation of the partially purified species in Xenopus laevis oocytes. One of these coded for the virion surface glycoprotein (G protein), and the other coded for the major structural protein of the virion nucleocapsid (N protein). The G-mRNA sedimented in a sucrose density gradient at about 18S, and the N-mRNA had a sedimentation coefficient of approximately 16S. Their respective translation products were identified in a radioimmunoassay with specific monoclonal antibody probes that recognized only G or N proteins. Immunoprecipitates formed between the radiolabeled viral antigens synthesized in programmed oocytes and their respective monoclonal antibodies were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The glycoprotein antigen translated from G-mRNA in oocytes migrated in the gel ahead of the virion G protein with a migration rate that was similar to that of nonglycosylated intracellular glycoproteins from virus-infected cells. The results suggested that the branched-chain carbohydrate of G protein was not required for recognition by the particular monoclonal antibody used. The nucleocapsid antigen translated from N-mRNA in oocytes migrated to the same position in the gel as marker virion N protein. Both the electrophoretic mobility of virus-specific antigens in sodium dodecyl sulfate-polyacrylamide gel and the antibody concentration dependence for immunoprecipitations were criteria for identifying the individual viral proteins encoded by the two rabies mRNA's.     

Identification of a murine cysteinyl leukotriene receptor by expression in Xenopus laevis oocytes

We report the identification of an EST encoding a murine cysteinyl leukotriene (mCysLT) receptor. LTD4, LTC4 and LTE4 but not LTB4 or various nucleotides activated Ca2+-evoked Cl- currents in mCysLT1 expressing Xenopus laevis oocytes. The response to LTD4 was blocked by MK-571, reduced by pretreatment with pertussis toxin (PTX), and was partly dependent on extracellular Ca2+. The identified murine CysLT1 receptor differs from the hCysLT1 receptor with regard to PTX sensitivity, receptor-mediated Ca2+ influx, and antagonist sensitivity.     

Amino acid uptake in Xenopus laevis oocytes.

The isolated oocytes from Xenopus laevis are able to take up radioactive amino acids from the exogenous medium. Most amino acids tested are taken up to reach concentrations higher than the extracellular medium. The initial uptake velocities vary with the external amino acid concentration in a Michaelis-Menten fashion. Aspartic acid requires concentrations an order of magnitude higher than the five other amino acids tested to reach half the maximal uptake velocity. The uptake mechanism seems to be specific for groups of analogous amino acids, as can be determined by competition studies. The amino acid groups for which there is some evidence of uptake specificity would be aromatic, aliphatic, acidic and basic. Amino acid pools of oocytes show that these cells can concentrate amino acids from Xenopus blood, as well as from artificial media.     

Repair of UV-induced lesions in Xenopus laevis oocytes.

We characterized a DNA repair system in frog oocytes by comicroinjection of UV-irradiated pBR322 DNA and radiolabeled nucleotides. Repair synthesis was monitored by incorporation of label into recovered pBR322 DNA and by a novel method in which the removal of UV photoproducts was determined from the shift of DNA topoisomers that occurs during gel electrophoresis upon repair of these lesions. We investigated the effects of several drugs in the oocyte system and found that although novobiocin, an inhibitor of topoisomerase II, was an effective inhibitor of repair, VM-26, another inhibitor of topoisomerase II, was not. In addition, the topoisomerase I inhibitor camptothecin had no effect on repair in this system. Finally, circular DNA (either supercoiled or nicked circular) was repaired at least 50 times more rapidly than linear DNA.     

Endogenous L-glutamate transport in oocytes of Xenopus laevis.

The existence of an endogenous Na(+)-glutamate cotransporter in the oocytes of Xenopus laevis is demonstrated. The transporter does not accept D-glutamate as substrate. The dependence on substrate displays two saturating components with low (K1/2 = 9 mM) and high (K1/2 = 0.35 microM) affinities for L-glutamate. The dependence on external Na+ exhibits a saturating component with a K1/2 value of about 5 mM and a component that has not saturated up to 110 mM Na+. In voltage-clamped oocytes, it is possible to demonstrate that Na(+)-dependent L-glutamate transport is directly coupled to countertransport of Rb+. The analysis of the voltage dependence of the Na+,K(+)-dependent L-glutamate uptake suggests that positive charges are moved inwardly during the transport cycle.     

Evidence for a mitochondrial chromosome in Xenopus laevis oocytes

When Xenopus laevis mitochondria are gently lysed at physiologic ionic strength, mitochondrial DNA (mitDNA) is extracted associated with proteins. Sedimentation and buoyant density studies indicate that proteins are bound to mitDNA at a ratio of about 1/1. This DNA-protein complex visualized by electron microscopy after fixation with glutaraldehyde appears as a relaxed circular molecule consisting of an average of 48 globular particles interconnected by a thin filament.     

Identification of multiple RNases in Xenopus laevis oocytes and their possible role in tRNA processing.

A survey of RNases in Xenopus laevis oocytes has been carried out to identify potential tRNA-processing enzymes in this system. Using a variety of specific and nonspecific substrates, we have shown that oocytes contain multiple RNases with various specificities. Three activities that could cleave the extraneous residues from the artificial tRNA precursor, tRNA-C-[14C]U-C, to generate a substrate for -C-C-A addition by tRNA nucleotidyltransferase were identified. One of these was a cytoplasmic exonuclease which generated predominantly tRNA-C, whereas the other two were nuclear endonucleases which cleaved the precursor to generate tRNA-N. The possible involvement of these activities in 3' tRNA processing in oocytes is discussed.     

Export of proteins from oocytes of Xenopus laevis.

When human lymphoblastoid mRNA was microinjected into X. laevis oocytes, titers of interferon rapidly reached a maximum inside the oocyte while accumulation of interferon continued in the incubation medium for at least 45 hr. If interferon protein was injected into oocytes it was rapidly inactivated. Significantly, newly synthesized interferon but not injected interferon was found to be membrane-associated. Further experiments involving the co-injection of mRNAs coding for secretory proteins (guinea pig milk proteins and human interferon) and nonsecretory proteins (rabbit globin) revealed that only the secretory proteins were exported from the oocyte. Moreover, different proteins were exported at different rates. A distinct subclass of newly synthesized oocyte proteins of unknown function also accumulated in the incubation medium. Since the information encoded in the messenger RNAs of secretory proteins is sufficient to specify synthesis, compartmentation and secretion of these proteins, the oocyte may provide a complete system for the analysis of the secretory process.     

Polyadenylic acid-containing RNA in Xenopus laevis oocytes

The quantity of poly(A)-containing RNA is measured in Xenopus laevis oocytes as a function of developmental stage. The amount of poly(A)-containing RNA per oocyte, 0.7 to 1.0% of the total RNA, remains relatively constant from early vitellogenesis until ovulation. It is largely present in the cytoplasm of the oocyte in the form of a ribonucleoprotein complex. The poly(A) sequence is approximately 100 bases in length and is attached to molecules of heterogeneous sedimentation coefficients.     

Activin incorporation into vitellogenic oocytes of Xenopus laevis.

Activin uptake into Xenopus oocytes was studied by several complementary methods. Immunocytochemistry of adult ovary localized activin and follistatin in the cytoplasm of vitellogenic oocytes and surrounding follicle cells. Surface plasmon resonance analysis of protein interaction kinetics indicated that while follistatin or a complex of activin-follistatin bound to yolk vitellogenin, activin alone did not. Radioactive tracer analysis measured specific incorporation of activin by viable oocytes in vitro. Together, the results suggest that vitellogenic oocytes can import activins from follicle cells and that follistatin may act as a chaperone for binding activin to vitellogenin in yolk platelets.     

Soluble cytokeratins in Xenopus laevis oocytes and eggs

Xenopus oocytes contain a radial network of cytokeratins which seems to fragment during meiosis reinitiation (maturation). The mature egg contains only a cortical network of cytokeratins. We have looked for the presence of soluble cytokeratins in oocytes and unfertilized eggs and have found them in both cases. However, the proportion of soluble to insoluble cytokeratins is slightly higher in the egg than in the oocyte. Soluble cytokeratins incorporate 35S-methionine at a high rate in the oocyte but to a lesser extent in the egg. This suggests that they are biosynthetic intermediates in the oocyte. In the egg, at least a fraction of the soluble cytokeratins may arise from the fragmentation of the polymer which seems to occur during the maturation process. Insoluble cytokeratins are strongly labeled with 32P both in oocytes and eggs. On the other hand only the soluble keratins of the egg incorporate 32P. Since the isoelectric point of soluble and insoluble cytokeratins is the same in oocytes and eggs, their absolute level of phosphorylation probably remains relatively constant. This suggests that: i) phosphate turnover is very slow in oocyte soluble cytokeratins, ii) phosphorylation is not a major way of changing the structural state of cytokeratins in amphibian oocytes and eggs.     

Repair and recombination of X-irradiated plasmids in Xenopus laevis oocytes.

Plasmid DNA substrates were X-irradiated and injected into the nuclei of Xenopus laevis oocytes. After incubation for 20 h, DNA was recovered from the oocytes and analyzed simultaneously for repair and for intermolecular homologous recombination by electrophoresis and bacterial transformation. Oocyte-mediated repair of DNA strand breaks was observed with both methods. Using a repair-deficient mutant Escherichia coli strain and its repair-proficient parent as hosts for the transformation assay, we also demonstrated that oocytes repaired oxidative-type DNA base damage induced by X-rays. X-irradiation of a circular DNA stimulated its potential to recombine with a homologous linear partner. Recombination products were detected directly by Southern blot hybridization and as bacterial transformant clones expressing two antibiotic resistance markers originally carried separately on the two substrates. The increase in recombination was dependent on X-ray dose. There is some suggestion that lesions other than double-strand breaks contribute to the stimulation of oocyte-mediated homologous recombination. In summary, oocytes have considerable capacity to repair X-ray-induced damage, and some X-ray lesions stimulate homologous recombination in these cells.     

Modulation of the substrate specificity of the polycation-stimulated protein phosphatase from Xenopus laevis oocytes

A polycation-stimulated (PCS) protein phosphatase was isolated in high yield (280 micrograms/100 g ovaries) from Xenopus laevis oocytes through a procedure involving a tyrosine-agarose hydrophobic chromatography. The 220-kDa enzyme contains a 35-kDa and a 62-kDa subunit. It was identified as the low-Mr polycation-stimulated (PCSL) protein phosphatase. The labile p-nitrophenyl phosphatase activity, copurifying with the phosphorylase phosphatase activity, can be increased severalfold by preincubating the purified enzyme with ATP, its analogues or PPi. This activation is time-dependent and accompanied by a parallel decrease of the phosphorylase phosphatase activity. Although the stimulation was antagonized by metal ions during the preincubation, the basal and ATP-stimulated p-nitrophenyl phosphatase requires Mg2+ or Mn2+ in the assay, with pH optima of 8.5-9 and 7.5 respectively.     

Induction of maturation in small Xenopus laevis oocytes

The competence of Xenopus laevis oocytes in various stages of growth to respond to progesterone treatment was investigated. Full-grown (stage 6) oocytes undergo nuclear membrane dissolution and resume meiosis in response to progesterone exposure, while smaller oocytes (stages 3-5; less than 1100 micron in diameter) do not. The defect which prevents 750- to 1050-micron oocytes from responding to progesterone can be overcome by microinjecting cytoplasm withdrawn from a stage 6 oocyte. Germinal vesicle breakdown in these small oocytes occurs on a timetable similar to that of stage 6 oocytes exposed to progesterone and is accompanied by a twofold increase in protein synthesis as well as the activation of MPF. The results argue that a cytoplasmic factor(s) which probably first appears at late stage 5 is required for progesterone responsiveness. The identity and role of the factor(s) in the development of maturation competence and the regulation of maternal mRNA translation are discussed.