Xenopus oocytes as a heterologous expression system for plant proteins

TheXenopus oocyte is a robust and convenient system for the transient expression of many different animal proteins and it has recently been demonstrated that oocytes can also translate, process, and target plant proteins. This expression system can also be used to clone genes, characterize function, and study posttranslational processing of proteins. Here we describe the methodology for the expression of plant proteins, in particular membrane proteins, inXenopus oocytes.     

5-羟色胺转运体基因异体表达模型的建立

目的:探讨建立5-羟色胺转运体(5-HTT)基因异体表达模型的可行性,且为进一步探讨5-羟色胺重摄取的动力过程和条件以及其功能的调控机制奠定基础.方法:利用体外转录cRNA技术将克隆至pOTV中的5-HTT的cDNA转录、合成5HTT的cRNA,通过显微注射技术将该cRNA注入成熟雌性非洲爪蟾卵母细胞的胞质中,使其表达以建立5-羟色胺转运体(SERT)的异体表达模型,并用电压钳技术检测其转运功能.结果:爪蟾卵母细胞可被用做5-HTT的异体表达模型,其转运功能呈浓度依赖性并具饱和现象,转运过程可被特异阻断剂Desipramine阻断.结论:非洲爪蟾卵母细胞可作为5-羟色胺等单胺类神经递质转运体的异体表达系统,为进一步研究转运体蛋白的功能和调控提供了有效工具.     

Translational control by cytoplasmic polyadenylation in Xenopus oocytes

Elongation of the poly(A) tails of specific mRNAs in the cytoplasm is a crucial regulatory step in oogenesis and early development of many animal species. The best studied example is the regulation of translation by cytoplasmic polyadenylation elements (CPEs) in the 3' untranslated region of mRNAs involved in Xenopus oocyte maturation. In this review we discuss the mechanism of translational control by the CPE binding protein (CPEB) in Xenopus oocytes as follows: Finally we discuss some of the remaining questions regarding the mechanisms of translational regulation by cytoplasmic polyadenylation and give our view on where our knowledge is likely to be expanded in the near future.     

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.     

Microtubule assembly in cytoplasmic extracts of Xenopus oocytes and eggs

We have investigated the differences in microtubule assembly in cytoplasm from Xenopus oocytes and eggs in vitro. Extracts of activated eggs could be prepared that assembled extensive microtubule networks in vitro using Tetrahymena axonemes or mammalian centrosomes as nucleation centers. Assembly occurred predominantly from the plus-end of the microtubule with a rate constant of 2 microns.min-1.microM-1 (57 s-1.microM-1). At the in vivo tubulin concentration, this corresponds to the extraordinarily high rate of 40-50 microns.min-1. Microtubule disassembly rates in these extracts were -4.5 microns.min-1 (128 s-1) at the plus-end and -6.9 microns.min-1 (196 s-1) at the minus-end. The critical concentration for plus-end microtubule assembly was 0.4 microM. These extracts also promoted the plus-end assembly of microtubules from bovine brain tubulin, suggesting the presence of an assembly promoting factor in the egg. In contrast to activated eggs, assembly was never observed in extracts prepared from oocytes, even at tubulin concentrations as high as 20 microM. Addition of oocyte extract to egg extracts or to purified brain tubulin inhibited microtubule assembly. These results suggest that there is a plus-end-specific inhibitor of microtubule assembly in the oocyte and a plus-end-specific promoter of assembly in the eggs. These factors may serve to regulate microtubule assembly during early development in Xenopus.     

Regulation of simian virus 40 gene expression in Xenopus laevis oocytes.

Expression of the simian virus 40 (SV40) early and late regions was examined in Xenopus laevis oocytes microinjected with viral DNA. In contrast to the situation in monkey cells, both late-strand-specific (L-strand) RNA and early-strand-specific (E-strand) RNA could be detected as early as 2 h after injection. At all time points tested thereafter, L-strand RNA was synthesized in excess over E-strand RNA. Significantly greater quantities of L-strand, relative to E-strand, RNA were detected over a 100-fold range of DNA concentrations injected. Analysis of the subcellular distribution of [35S]methionine-labeled viral proteins revealed that while the majority of the VP-1 and all detectable small t antigen were found in the oocyte cytoplasm, most of the large T antigen was located in the oocyte nucleus. The presence of the large T antigen in the nucleus led us to investigate whether this viral product influences the relative synthesis of late or early RNA in the oocyte as it does in infected monkey cells. Microinjection of either mutant C6 SV40 DNA, which encodes a large T antigen unable to bind specifically to viral regulatory sequences, or deleted viral DNA lacking part of the large T antigen coding sequences yielded ratios of L-strand to E-strand RNA that were similar to those observed with wild-type SV40 DNA. Taken together, these observations suggest that the regulation of SV40 RNA synthesis in X. laevis oocytes occurs by a fundamentally different mechanism than that observed in infected monkey cells. This notion was further supported by the observation that the major 5' ends of L-strand RNA synthesized in oocytes were different from those detected in infected cells. Furthermore, only a subset of those L-strand RNAs were polyadenylated.     

The Mos pathway regulates cytoplasmic polyadenylation in Xenopus oocytes.

Cytoplasmic polyadenylation controls the translation of several maternal mRNAs during Xenopus oocyte maturation and requires two sequences in the 3' untranslated region (UTR), the U-rich cytoplasmic polyadenylation element (CPE), and the hexanucleotide AAUAAA. c-mos mRNA is polyadenylated and translated soon after the induction of maturation, and this protein kinase is necessary for a kinase cascade culminating in cdc2 kinase (MPF) activation. Other mRNAs are polyadenylated later, around the time of cdc2 kinase activation. To determine whether there is a hierarchy in the cytoplasmic polyadenylation of maternal mRNAs, we ablated c-mos mRNA with an antisense oligonucleotide. This prevented histone B4 and cyclin A1 and B1 mRNA polyadenylation, indicating that the polyadenylation of these mRNAs is Mos dependent. To investigate a possible role of cdc2 kinase in this process, cyclin B was injected into oocytes lacking c-mos mRNA. cdc2 kinase was activated, but mitogen-activated protein kinase was not. However, polyadenylation of cyclin B1 and histone B4 mRNA was still observed. This demonstrates that cdc2 kinase can induce cytoplasmic polyadenylation in the absence of Mos. Our data further indicate that although phosphorylation of the CPE binding protein may be involved in the induction of Mos-dependent polyadenylation, it is not required for Mos-independent polyadenylation. We characterized the elements conferring Mos dependence (Mos response elements) in the histone B4 and cyclin B1 mRNAs by mutational analysis. For histone B4 mRNA, the Mos response elements were in the coding region or 5' UTR. For cyclin B1 mRNA, the main Mos response element was a CPE that overlaps with the AAUAAA hexanucleotide. This indicates that the position of the CPE can have a profound influence on the timing of cytoplasmic polyadenylation.     

Orthopoxvirus gene expression in Xenopus laevis oocytes: a component of the virion is needed for late gene expression.

We have examined the feasibility of using Xenopus laevis oocytes microinjected with rabbit poxvirus as a system to study poxvirus gene expression. The injection of either intact virus or subviral cores resulted in accurate synthesis of viral proteins. This expression was dependent on the multiplicity of injected virus, with the optimal injected dose being equivalent to approximately 300 PFU per oocyte. Extensive viral gene expression including late viral protein synthesis was observed when intact virions were microinjected into the oocyte. However, the injection of subviral cores resulted in only early protein synthesis. When oocytes were injected with a mixture of subviral cores and the nonionic detergent-soluble fraction was removed from virus during the preparation of cores, both early and late viral proteins were synthesized. Therefore, the detergent-soluble fraction appears to contain a factor(s) required for the transition from early to late gene expression.     

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.     

Cationic oligonucleotides can mediate specific inhibition of gene expression in Xenopus oocytes.

Base-specific hydrogen bonding between an oligonucleotide and the purines in the major groove of a DNA duplex provide an approach to selective inhibition of gene expression. Oligonucleotide-mediated triplex formation in vivo may be enhanced by a number of different chemical modifications. We have previously described an in vitro analysis of triplex formation using oligonucleotides containing internucleoside phosphate linkages modified with the cation N , N -diethyl-ethylenediamine (DEED). When compared with unmodified oligonucleotides of identical base composition, DEED-modified oligonucleotides were better able to form DNA triplexes under conditions that approximate the pH, magnesium and potassium levels found in vivo . Here we report the ability of DEED-modified oligonucleotides to inhibit the expression of plasmid DNA injected into Xenopus oocytes. Inhibition is specific to plasmids containing a triplex formation target and sensitive to sequence alteration in the triplex forming target site. Inhibition of gene expression was nearly complete when oligonucleotide and plasmid were mixed together prior to injection. Inhibition was partial when oligonucleotide was injected first and not evident when plasmid was injected and allowed to form chromatin prior to oligonucleotide injection. Thus, access to DNA is a determining factor in effective triplex inhibition of gene expression.     

CIRP2, a major cytoplasmic RNA-binding protein in Xenopus oocytes

In an attempt to isolate mRNA-binding proteins we fractionated Xenopus oocyte lysate by oligo(dT)–cellulose chromatography. A 20 kDa protein was the major component of the eluate. cDNA cloning revealed that this protein is a Xenopus homolog of the cold-inducible RNA-binding protein (CIRP) which was originally identified in mammalian cells as a protein that is overexpressed upon a temperature downshift. This Xenopus protein, termed here xCIRP2, is highly expressed in ovary, testis and brain in adult Xenopus tissues. In oocytes it is predominantly localized in the cytoplasm. By biochemical fractionation we provide evidence that xCIRP2 is associated with ribosomes, suggesting that it participates in translational regulation in oocytes. Microinjection of labeled mRNA into oocytes followed by UV cross-linking of the oocyte lysate led to identification of two major RNA-binding activities. Immunoprecipitation of the RNA-binding proteins demonstrated that one is xCIRP2 and that the other contains FRGY2. FRGY2, which is one of the principal constituents of mRNA storage particles involved in translational masking of maternal mRNA, has an RNA-binding domain conserved to those of bacterial cold shock proteins. Possible implications of the highly abundant expression in oocytes of cold shock RNA-binding proteins of both eukaryotic and prokaryotic types are discussed.     

Maturation-specific deadenylation in Xenopus oocytes requires nuclear and cytoplasmic factors.

During the meiotic maturation of Xenopus oocytes, maternal mRNAs that lack a cytoplasmic polyadenylation element are deadenylated and translationally inactivated. In this report, we have characterized the regulation of poly(A) removal during maturation. Deadenylation in vivo is detected only after germinal vesicle breakdown and does not require de novo protein synthesis. Enucleated oocytes do not deadenylate either endogenous or microinjected RNAs upon maturation, indicating that a nuclear component is required for poly(A) removal. Whole cell extracts prepared from both immature and mature oocytes deadenylate exogenous RNA substrates in vitro. Deadenylation activity is not detected in isolated nuclear or cytoplasmic extracts obtained from immature oocytes, but is reconstituted when these fractions are combined in vitro. These results indicate that the factors required for deadenylation activity are present in immature oocytes, but that poly(A) removal is prevented by the sequestration of one or more of these components within the nucleus. Maturation-specific deadenylation of maternal mRNAs occurs upon the release of nuclear factors into the cytoplasm at germinal vesicle breakdown.     

Go G-proteins mediate rapid heterologous desensitization of G-protein coupled receptors in Xenopus oocytes

We have shown previously that responses to lysophosphatidic acid (LPA) in Xenopus oocytes exhibit pronounced rapid homologous desensitization mediated by Go family of G-proteins (Itzhaki-Van Ham et al., 2004, J Cell Physiol, 200: 125-133). The present study was aimed at examining the involvement of Go G-proteins in rapid heterologous desensitization of native and expressed G-protein-coupled receptors in Xenopus oocytes. Threshold stimulation of the native lysophosphatidic acid receptors (LPA-Rs) induced about 50% rapid desensitization of responses evoked by stimulation of either native trypsin or expressed M1-muscarinic cholinergic receptors (M1-Rs). Similarly, threshold stimulation of expressed M1-Rs or thyrotropin-releasing hormone receptors induced 40% rapid desensitization of responses to LPA. Inactivation of all Gi/o G-proteins with pertussis toxin (PTX) completely abolished rapid heterologous desensitization in all protocols. Depletion of either Galphao or Galphao1 by antisense oligodeoxynucleotides targeted at either member of the Galphao family decreased or completely abolished rapid heterologous desensitization. Expression of two dominant negative mutants of the human Galphao family, highly homologous to oocyte Galphao species, either decreased or virtually abolished rapid desensitization. Homologous and heterologous desensitizations of the LPA response were non-additive and proceeded, apparently, via the same pathway. We conclude that Go G-proteins mediate both homologous and heterologous rapid desensitization of responses mediated by G-protein-coupled receptors (GPCRs) coupled to the phosphoinositide phospholipase C-inositol 1,4,5-trisphosphate-Ca(2+) (PI-PLC-InsP(3)-Ca(2+)) pathway in Xenopus oocytes.     

Xenopus oocytes as an expression system for plant transporters

The Xenopus oocyte provides a powerful system for the expression and characterisation of plant membrane proteins. Many different types of plant membrane proteins have been expressed and characterised using this system. As there are already several general reviews on the methodology for oocyte expression of channel proteins, we have summarised the particular advantages and disadvantages of using the system for the characterisation of plant cotransporter proteins. As an example of how the system can be used to identify transporters, we describe evidence for a low affinity nitrate transporter in oocytes injected with poly(A) RNA extracted from nitrate-induced barley roots. Furthermore, we describe evidence that the expression of some transporters in oocytes can modify the properties of endogenous membrane proteins. We conclude that although care must be taken in the interpretation of results and in choosing appropriate controls for experiments, oocyte expression is an excellent tool which will have an important role in characterising plant membrane proteins.