Kinesin II mediates Vg1 mRNA transport in Xenopus oocytes

The subcellular localization of specific mRNAs is a widespread mechanism for regulating gene expression. In Xenopus oocytes microtubules are required for localization of Vg1 mRNA to the vegetal cortex during the late RNA localization pathway. The factors that mediate microtubule-based RNA transport during the late pathway have been elusive. Here we show that heterotrimeric kinesin II becomes enriched at the vegetal cortex of stage III/IV Xenopus oocytes concomitant with the localization of endogenous Vg1 mRNA. In addition, expression of a dominant negative mutant peptide fragment or injection of a function-blocking antibody, both of which impair the function of heterotrimeric kinesin II, block localization of Vg1 mRNA. We also show that exogenous Vg1 RNA or Xcat-2, another RNA that can use the late pathway, recruits endogenous kinesin II to the vegetal pole and colocalizes with it at the cortex. These data support a model in which kinesin II mediates the transport of specific RNA complexes destined for the vegetal cortex.     

Phospholemman expression induces a hyperpolarization-activated chloride current in Xenopus oocytes.

A new type of chloride channel has been identified by functional expression of phospholemman, a 72-amino acid cardiac sarcolemmal protein with a single transmembrane domain. Xenopus oocytes injected with phospholemman RNA developed a chloride-selective current, which was activated by hyperpolarizing pulses. The current activated very slowly with a pronounced sigmoidal delay, did not inactivate, and increased in amplitude with trains of pulses, depolarized holding potentials, and low extracellular pH. Point mutations within the single transmembrane region abolished the sigmoidal delay of expressed currents. Phospholemman appears to be the smallest plasma membrane channel protein yet known. The structure is dissimilar to any chloride channel described thus far.     

Functional expression of renal organic anion transport in Xenopus laevis oocytes

Secretion of organic anions by the kidney plays a critical role in the elimination of toxic agents from the body. Recent findings in isolated membranes and intact tissue have demonstrated the participation of multiple transport proteins in this process. As a first step toward molecular characterization of these proteins through expression cloning, the studies reported below demonstrate functional expression of both fumarate- and lithium-sensitive glutarate and probenecid-sensitive p-aminohippurate transport in Xenopus oocytes injected with rat kidney poly(A)⁺RNA. Maximal increase in substrate uptake over buffer-injected controls was reached by 5 days after mRNA injection. Expression of size-fractionated mRNA indicated that the active species with respect to both transport activities were in the range of 1.8 to 3.5 kb.     

Gene expression in Xenopus oocytes

1. Gene expression in Xenopus oocytes is now an integral part of many molecular cloning strategies. 2. For some genes, such as those encoding the ion channels, this system has emerged as the only available means to authenticate and examine the biological activities of the cloned DNA. 3. This review discusses some of the current applications of Xenopus oocytes in modern molecular biology.     

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.     

An endogenous monocarboxylate transport in Xenopus laevis oocytes

We investigated the existence of an endogenous system for lactate transport in Xenopus laevis oocytes. (36)Cl-uptake studies excluded the involvement of a DIDS-sensitive anion antiporter as a possible pathway for lactate movement. L-[(14)C]lactate uptake was unaffected by superimposed pH gradients, stimulated by the presence of Na(+) in the incubating solution, and severely reduced by the monocarboxylate transporter inhibitor p-chloromercuribenzenesulphonate (pCMBS). Transport exhibited a broad cation specificity and was cis inhibited by other monocarboxylates, mostly by pyruvate. These results suggest that lactate uptake is mediated mainly by a transporter and that the preferred anion is pyruvate. [(14)C]pyruvate uptake exhibited the same pattern of functional properties evidenced for L-lactate. Kinetic parameters were calculated for both monocarboxylates, and a higher affinity for pyruvate was revealed. Various inhibitors of monocarboxylate transporters reduced significantly pyruvate uptake. These studies demonstrate that Xenopus laevis oocytes possess a monocarboxylate transport system that shares some functional features with the members of the mammalian monocarboxylate cotransporters family, but, in the meanwhile, exhibits some particular properties, mainly concerning cation specificity.     

Photocrosslinking of proteins to maternal mRNA in Xenopus oocytes

Ultraviolet irradiation was used to covalently crosslink poly(A) RNA and associated proteins in Xenopus oocytes and reticulocytes. Each cell type contained similar as well as unique crosslinked proteins. The somatic cells contained a single 78-kDa 3' poly(A) tract binding protein while oocyte poly(A), however, was bound by this protein and at least three additional proteins. Based on the mass of poly(A) RNA, oocytes in their earliest stages of growth contained crosslinked proteins that were generally more prevalent than in fully grown oocytes. An investigation of possible messenger RNA-specific proteins was undertaken by a series of RNA injection experiments. Two radiolabeled SP6-derived mRNAs were injected into oocytes; the first, globin mRNA, assembled into polysomes, while the second, a maternal mRNA termed G10, entered a nontranslating ribonucleoprotein compartment. Following the induction of oocyte maturation, additional globin mRNA was recruited onto polysomes while G10 mRNA remained a nontranslating mRNP. The proteins that can be crosslinked to these injected mRNAs were detected by 32P nucleotide transfer. Each mRNA associated with shared as well as unique proteins, some of which were detected only in mature oocytes. The possible function of these proteins is discussed.     

Tetrabutylammonium induces a voltage-dependent block of kainate current in Xenopus oocytes injected with rat brain mRNA.

Following injection of rat striatal and cerebrellar mRNA, Xenopus oocytes were voltage clamped and current responses to the excitatory amino acid receptor agonist, kainate, were recorded. This nonspecific cationic current is carried principally by Na+ and K+ and reverses polarity at a membrane potential of approximately -5 mV. When the membrane potential was voltage clamped to -60 mV, bath-applied tetrabutylammonium (0.1-30 mM) produced a rapid, concentration dependent and reversible block of kainate-induced inward current with an IC50 of 1.3 mM. Tetraalkylammonium derivatives having shorter chains (methyl, ethyl, and propyl) were relatively ineffective blockers. Longer alkyl chain derivatives (pentyl, hexyl, and heptyl) were more potent than tetrabutylammonium but limited in their usefulness by their toxicity. The antagonism of kainate-induced current by tetrabutylammonium displayed apparently uncompetitive kinetics, in contrast with the competitive antagonism by gamma-D-glutamylaminomethylsulfonate. The block by tetrabutylammonium was strongly voltage dependent; an e-fold change in IC50 was observed for a 27 mV change in holding potential. Replacement of the Na+ in the medium with a more permeant cation (NH4+), a less permeant cation (tetramethylammonium), or an uncharged solute (mannitol) had little effect on the block of kainate-induced current by tetrabutylammonium. The rates of association and dissociation of tetrabutylammonium with the kainate receptor-channel are clearly rapid. These observations suggest that tetrabutylammonium enters and blocks the kainate receptor-associated cation selective channel. Tetrabutylammonium appears to traverse 80-90% of the membrane electrical field to reach a relatively low-affinity binding site that may simply be a narrowing of the channel.     

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.     

Gonadotropin-releasing hormone receptor expression in Xenopus oocytes

The rodent GnRH receptor was characterized in Xenopus oocytes injected with RNA isolated from rat pituitary and from a gonadotrope cell line, alpha T3, derived from a transgenic mouse. Three to 4 days after 150-200 ng RNA injection, 93% of the oocytes, which were recorded by voltage clamp, responded to 10(-7) M GnRH. The mean inward currents obtained after RNA injection were 620 +/- 88 nA (n = 22) with pituitary RNA and 1415 +/- 598 (n = 4) with alpha T3 RNA. The threshold GnRH concentration able to evoke the dose dependent current after pituitary RNA injection was 3 x 10(-9) M GnRH. The GnRH receptor response of the oocyte was antagonized by [D-Phe2,6,Pro3] GnRH and [N-Ac-D-Na](2)1, D-alpha D-Me, pCl-Phe2, D-Arg6, D-Ala10-NH2]GnRH and could be elicited by D-Ser(But)6,Pro9-N-ethylamide GnRH (buserelin). The reversal potential of the GnRH generated current as determined by voltage-ramp was -22.5 +/- 1.0 mV (n = 7) and -25.6 +/- 3.3 mV (n = 3) in pituitary and cell line RNA-injected oocytes respectively, consistent with the chloride reversal potential. The GnRH receptor response was virtually eliminated by intracellular EGTA injection but was unaffected by ligand application in calcium-free perfusate. The GnRH-evoked response is mimicked by intracellular injection of inositol 1,4,5-trisphosphate. To determine the size of the GnRH receptor mRNA, alpha T3 RNA was size fractionated through a sucrose gradient. The maximal GnRH response was induced by a fraction larger than the 28S ribosomal peak. Thus we find that oocytes injected with RNA from an appropriate source develop an electrophysiological response to GnRH which is dependent on intracellular calcium mobilization, is independent of extracellular calcium, and may be mediated by inositol 1,4,5-trisphosphate.     

Translation of mRNA for human lymphotoxin in microinjected Xenopus oocytes

Synthesis and secretion of biologically active human lymphotoxin (LT) can be detected in Xenopus laevis oocytes following their inoculation with poly(A+) RNA from human stimulated peripheral blood lymphocytes, but not in oocytes inoculated with RNA from unstimulated lymphocytes or from fibroblastoid cells. In size-fractionating mRNA of stimulated lymphocytes most LT activity is found to be coded for by RNA with an approximate sedimentation value of 19 S.     

Translation of human interleukin 2 mRNA in Xenopus laevis oocytes

Poly(A)-positive mRNA extracted from tonsillar mononuclear cells stimulated with phytohemagglutinin-M and 12-o-tetradecanoyl phorbol 13-acetate was successfully translated into biologically active interleukin 2 (IL-2) in Xenopus laevis oocytes, and secreted into the incubation medium. In control experiments, the extract of oocytes injected with either poly(A)-negative RNA or buffer did not show any IL-2 activity. By sucrose density gradient centrifugation analysis, IL-2 mRNA was found as a single peak corresponding to a sedimentation coefficient of 10-11S.     

Effect of lipid hydroperoxide on Xenopus oocytes and on neurotransmitter receptors synthesized in Xenopus oocytes injected with exogenous mRNA

The effect of 13-L-hydroperoxylinoleic acid (LOOH) on both Xenopus oocytes and neurotransmitter receptors synthesized in the oocytes was studied by electrophysiological and ion flux measurement. Addition of LOOH to the incubation mixture of the oocytes raised the membrane potential and decreased the membrane resistance of the oocytes. These effects of LOOH on the oocytes were reversed within a few hours by incubation with frog Ringer solution. Addition of LOOH also caused an increase of Li+ and 45Ca2+ uptake into the oocytes. However, production of alkoxy radicals by the addition of FeCl2 to the incubation mixture containing LOOH did not accelerate the damage to the oocytes by LOOH. So essential toxicity is caused possibly by an increase in the membrane permeability resulting from disturbance of the lipid bilayer arrangement, not from production of active alkoxy radicals during decomposition of LOOH. Nicotinic acetylcholine and gamma-aminobutyric acid receptors were synthesized in Xenopus oocytes by injecting mRNA prepared from Electrophorus electricus electroplax and rat brain. LOOH noncompetitively inhibited the function of these receptors and also increased the rate of desensitization of the receptors.     

Expression of amino acid transport systems in Xenopus oocytes injected with mRNA of rat small intestine and kidney

Xenopus and Cynops oocytes were injected with exogenous mRNA prepared from rat small intestine and kidney and their electrical responses to amino acids were measured by both the current clamped and the voltage clamped methods. Oocytes injected with mRNA of rat small intestine showed a depolarization response to several neutral and basic amino acids, and almost no response to acidic amino acids. The responses to amino acids increased with incubation time after injection of mRNA, and followed Michaelis-Menten type kinetics. The responses were dependent on both Na+ concentration and membrane potential, and were inactivated by a sulfhydryl reagent, 5,5-dithiobis(2-nitrobenzoate). These results are interpreted as due to the expression of Na+/amino acid cotransporter(s) in oocytes injected with rat small intestine mRNA. On the other hand, the oocyte injected with rat kidney mRNA showed a hyperpolarization response to neutral amino acids, a depolarization response to basic ones, and almost no response to acidic ones in frog Ringer solution. These responses were independent of Na+ concentration and followed Michaelis-Menten type kinetics. These amino acid response characteristics in oocytes injected with rat kidney mRNA are interpreted as due to the expression of facilitated diffusion carrier protein(s) (uniporter) of amino acids in the oocyte.     

Glycophorin A facilitates the expression of human band 3-mediated anion transport in Xenopus oocytes.

The effects of human red cell glycophorin A (GPA) on the expression of the human erythrocyte anion transporter (band 3, AE1) has been examined in Xenopus oocytes. The coexpression of GPA with band 3 increased stilbene disulfonate-sensitive chloride transport into the oocytes. The effect of GPA was particularly noticeable at low band 3 concentrations and less marked at high band 3 cRNA concentrations. The enhancement of chloride transport was specific to GPA and was not observed when either glycophorin B or glycophorin C was coexpressed with band 3. Immunoprecipitations of whole oocyte homogenates showed the amount of band 3 synthesized was not affected by GPA at subsaturating cRNA concentrations. More band 3 was detected at the oocyte surface by immunoprecipitation when GPA was also expressed. Chymotrypsin treatment of intact oocytes was also used to assess surface band 3 and greater cleavage of band 3 by chymotrypsin was observed when GPA was present. Band 3 synthesis and assembly into canine pancreatic microsomes in the reticulocyte cell-free translation system was not altered by cotranslation of GPA. We suggest that GPA facilitates the translocation of band 3 to the plasma membrane at some point during band 3 biosynthesis in Xenopus oocytes. However, GPA is not essential for the expression of band 3 in red cells, since GPA-deficient individuals have apparently normal levels of band 3. Other GPA-independent mechanisms must also allow translocation of band 3 to the surface membrane in erythroid cells and oocytes. GPA may affect the rate of accumulation of band 3 at the cell surface, rather than the final level in the plasma membrane.     

On the existence of polyadenylated histone mRNA in Xenopus laevis oocytes

In a variety of systems, histone mRNA has been shown to lack poly(A) (Adesnik and Darnell, 1972;Grunstein et al., 1973). We have found, however, that in Xenopus laevis oocytes, poly(A)-containing mRNA codes for histones, in a wheat germ cell-free system, based on the following criteria: first, co-migration with authentic X. laevis oocyte histones on polyacrylamide gels; second, no detectable incorporation of tryptophan; third, differential incorporation of lysine and methionine into histone fraction H2A; fourth, resistance of histone fraction H2A to cleavage with cyanogen bromide; and fifth, correspondence of tryptic peptide maps of partially purified cell-free products with authentic X. laevis oocyte histone. RNA which directs the synthesis of histones in the cell-free system is retained on oligo(dT)-cellulose, even after denaturation in 80% DMSO at 70°C, thereby demonstrating the covalent attachment of polyadenylic acid sequences to the mRNA. Poly(A)^? RNA (7S–14S fraction) was also found to code for histones using the same criteria. We discuss the significance of the finding that X. laevis oocytes contain two classes of histone mRNA as well as the potential developmental implications of this observation.     

Export of mRNA from microinjected nuclei of Xenopus laevis oocytes

Export of mRNA from the nucleus to the cytoplasm was studied in mature Xenopus laevis oocytes. In vitro transcribed, capped 32P-labeled mRNA was microinjected into nuclei, and its appearance in the cytoplasm measured by counting radioactivity or by RNA extraction and gel electrophoresis. Both for a 5.0-kb transferrin receptor mRNA and a 2.0-kb 4F2 antigen heavy chain mRNA we found saturable transport with an apparent Km of 3.6 x 10(8) molecules per oocyte nucleus. Under non-saturating conditions the half-time for mRNA export from the nucleus was approximately 2 min at 20 degrees C. At higher concentrations of injected mRNA this half-time was prolonged, and the maximal transport rate was reached at approximately 1.6 x 10(8) molecules/min. mRNA transport showed properties of an energy-dependent mechanism, since it was inhibited at 4 degrees C or by ATP depletion. Co-injection of the cap dinucleotide m7GpppG blocked the export effectively, suggesting a role for the cap in this process. The export was also inhibited by the pre-injection of wheat germ agglutinin. The effect of the lectin was specific and abolished by co-injection of N-acetylglucosamine. Finally, we found significant competitive inhibition in mRNA export by the presence of tRNA. Our results suggest that mRNA transport is a facilitated process which may share common steps with tRNA transport. Preliminary gel retardation experiments show that injected mRNA associates with endogenous nuclear proteins and suggest an exchange of some of the bound components during the transport to the cytoplasm.