Expression in Xenopusoocytes Alan J. Kingsman* ', Martin Braddock*, Andrew Thorburn *P, Alistair Chambers* and Susan M. Kingsman* *Department of Biochemistry, Oxford, UK, tDepartment of Virology, British Bio-technology Ltd, Oxford, UK. PRegulatory Biology Laboratory, Salk Institute, La Jolla, San Diego, CA 92138-9216, USA. Current Opinion in Biotechnology1990, 1:82-85
Introduction
(many days) as non-integrated and non-replicating circular molecules.
Unlike most of the other systems reviewed in this volume, the oocyte of the frog Xenopus laevis will never be used as a production source for biological products. The oocyte system is, however, an extremely powerful analytical tool because of the ease with which functional macromolecules can be microin[ected into the cell. The system has been reviewed recently by Heikkila [1 o-]. Oocytes, and those from Xenopus are no exception, are unlike somatic cells. They are large, about 100 000 times the volume of a somatic cell, and they are arrested in prophase of the first meiotic division. They are extremely active in transcription and translation, possessing sufficient ribosomes and RNA polymerases to take the developing embryo through to the 30 000 cell stage. However, they are not capable of DNA replication. Oocytes can be induced to pass through the first meiotic division (maturation) by treatment with progesterone in vitro. During this process the nuclear membrane breaks down and the cell then arrests in meiotic metaphase II. If the mature egg is then fertilized (activated) it continues through meiosis and an embryo is formed [2]. The oocyte is about 1.0-1.5mm in diameter. This fact alone makes it reasonably easy to microinject DNA, RNA or protein into either the nucleus or the cytoplasm. Furthermore, multiple injections can be carried out that are separated in both space (nucleus or cytoplasm) and/or time [3]. Xenopua oocytes have been used widely in the analysis of gene expression yet while studies of Xenopus genes in this more or less homologous system have yielded important results the use of the oocyte system to express heterologous, usually mammalian, pol II genes has given the more important contribution to biology and biotechnology. In most cases, the oocyte accurately intitiates and terminates transcription, splices primary transcripts and translates the message [4-8]. In some cases, specific ffanscription factors may be required (see below). A further feature of this system is that injected plasmid DNA can be maintained for long periods
RNA can also be injected into oocytes and translated. This was first shown using RNAs synthesized in vivo [9], but more recently the use of SP6- or T7-driven RNAs made in vitro [10] has made the oocyte system even more versatile (see below). The ability of oocytes to translate injected RNA was, in fact, used to isolate the first human czinterferon clone using hybrid-selected fractionated RNA [11]. In the past year, the Xenopua oocyte system has contfibuted to studies in the areas of cell biology, receptor research, mammalian gene expression and AIDS research, which we will review in turn.
Oocytes and cell biology The analysis of secretion pathways in eukaryotic cells is central to making biological products using the techniques of genetic engineering. The dissection of these pathways in yeast has been facilitated by the isolation of a range of sec mutants and their subsequent biochemical analysis. The same genetic approach cannot be used in mammalian cells. Recently, it has been shown that the Golgi apparatus from rat can be partially reconstituted in Xenopus oocytes [12,13]. This system has important implications for analysing secretion in higher eukaryotes as it opens the way to manipulating the components of the Golgi apparatus before injection into oocytes. This system has now been taken one step further, in that Golgi components, labelled in vitro with 3H-sialic acid, have been shown to affect the formation of stacked saccules and to migrate into the Xenopus secretory pathway [14 oo]. In addition, it has been shown that transfer to the oocyte surface is inhibited by vinblastine, demonstrating that the process is microtubule dependent. Continued refinement of this system offers huge potential to manipulate the secretion of biological products by animal cells.
Abbreviations
AIDS~acquired immunodeficiencysyndrome;CAT--chloramphenicolacetyltransferase; HIV--human immunodeficiencyvirus;PCR--polymerasechain reaction. 82
C) Current Biology Ltd ISSN 0958-1669
Expressionin XenopusoocytesKingsman, Oocytes, receptors and channels Oocytes have been used widely in the study of receptor/ligand interactions since the demonstration that the Torpedo acetylcholine receptor could assemble and bind ligand in the oocyte membrane after microinjection of its mRNA [15]. For example, coexpression of SP6 RNAs encoding the cz and [3 subunits of the bovine 0t-aminobutyric acid receptor resulted in the assembly of a functional receptor and ion channel with full pharmacological properties [16]. The past year has seen other examples of this strategy, ranging from the analysis of yeast mating pheremone receptors to the study of K+-channels [17 °'-20 °.,21 "-23 ", 24o.]. In yeast, diploids are formed by mating between the two different haploid mating cell types, a and oz. Both haploids secrete mating factors that interact with the cells of the opposite mating type to prepare for conjugation [25]. Genetic and biochemical evidence has suggested that the product of the STE2 gene of yeast is the cell-surface receptor for cz-factor [26,27] and that this receptor operates via a G-protein [28], probably the product of the GPAI gene. Expression of T7 STE2 RNAs in oocytes has now demonstrated that the STE2 product is the only product required for a-factor binding and that with or without coexpression of GPAI RNA the STE2-encoded receptor does not alter the membrane conductance of oocytes either in the presence or absence of a-factor [17 °°]. This shows that the a-factor receptor, like other G-proteincoupled receptors, does not act through an intrinsic or ligand-gated ion channel. This result now opens the way for a detailed structure-function analysis that will draw on the joint potential of the Xenopus oocyte and yeast genetic systems. It will provide a powerful model for other G-protein-coupled receptors. Data from oocyte microinjection experiments have also thrown light on the diversity of K + -channels found in excitable cells. Three recent papers [18 °°-20 o.] report similar approaches. Cloned cDNA for various rat K + -channel proteins [29] are available, together with variants of the Shaker gene of Drosophila, which is known to encode a K + -channel protein. When expressed alone these individual proteins assemble into ion channels that differ with respect to their kinetics, single-channel conductance, pharmacology and sensitivity to voltage and second messengers. In the past, data have been reported that indicated that these channels were multimeric structures. Coexpression of different channel proteins by microinjection of RNAs synthesized in vitro has now confirmed this notion and, in addition, shown that heteromultimeric channels, with properties intermediate between the homomultimeric structures, can be formed. These observations have lead to the suggestion that at least some of the diversity of K + -channels may arise from the in vivo assembly of a spectrum of heteromultimeric channels. Other recent applications of the oocyte system to receptor research include the analysis of cotranslational modifications of the complement C3b/C4b receptor [21 °], the demonstration that mercuric ions are potent n o n - c o m -
Braddock, Thorburn et a/.
petitive inhibitors of brain kainate receptors [22 o], and a structure-function study of glycine analogues that activate the glycine recognition site of the N-methyl-D-aspartate receptor [23 °]. A further development of the use of oocytes in identifying receptor cDNAs came from Meyerhof and Richter [24 °']. It is common to identify the presence of a receptor in a mRNA population by injecting the mRNA into oocytes and then assaying for the receptor on the cell surface. The next stage is usually to clone the gene(s) for that receptor. Positive identification of a putative cDNA or polymerase chain reaction (PCR) fragment usually requires expression of that clone. This, in turn, demands that the clone is full length and engineered into an expression vector. A faster and more convenient route would be to take DNA fragments and hybrid arrest the in vivo expression of the receptor mRNA in the mRNA pool. Unfortunately, this strategy has met with only limited success. The altemative approach described by Meyerhof and Richter [24°°] is to hybridize the putative DNA clone to the mRNA pool and then use RNaseH to selectively remove the mRNA that has hybridized. Clones are identified by their ability to remove the receptor mRNA from the pool. This technique may expedite the cloning of receptor genes in many systems, including expression oocytes. Taken together, these papers illustrate the contribution that the Xenopus oocyte system is making to both the isolation and analysis of receptors. Furthermore, the expression and function of receptors on the oocyte surface will prove most valuable in the search for agonists and antagonists.
Oocytes and gene expression Microinjection of oocytes with plasmids containing defined transcription units has contributed substantially to our understanding of the control of gene expression. A recent paper [30 *] on the adenovirus EIA transactivator illustrates this point well. It has been shown previously [31] that promoters, such as the adenovims E3 promoter or the promoter from the HSP70 gene, that are normally activated by EIA are also activated when both EIA and the promoter are coinjected into Xenopus oocytes. Richter and colleagues also showed that this activation was prevented by inhibitors of protein synthesis, implying that some other protein, that was either induced by EIA or which turned over rapidly, was required for EIAmediated activation. This group has now identified this protein by gel-shift assays with oocyte extracts, a new in vivo photo cross-linking technique and immunoselection of biotinylated oligonucleotides. They have shown that a 75 kD protein that binds to the mammalian transcription factor, ATF, recognition sequence in vivo is essential for EIA activation in oocytes. Treatment with cycloheximide prevented in vivo binding of this factor but, interestingly, EIA had no effect. This implies that EIA modifies the activity of an oocyte transcription factor without affecting its binding.
83
84
Expression systems
Oocytes and AIDS
Acknowledgements
A key feature of the acquired immunodeficiency syndrome (AIDS) virus, human immunodeficiency virus (HIV), that distinguishes it from many other viruses is the complex set of regulatory systems that it uses to control gene expression. Amongst these is the TAT/TAR system, which is virus specific and a prime target for antiviral agents. TAT is an 86-amino-acid protein that activates expression of the virus. It acts through a target sequence called TAR, which is located in the first 60 nucleotides of the transcription unit of the virus. It is present, therefore, in both the DNA of the provirus and in all the viral transcripts. The presence of the TAR region in both DNA and RNA raises the different issue of whether activation is at the level of transcription or at some post-transcriptional level.
The work in this laboratory is funded by the Medical Research Council, Science and Engineering Research Council, British Biotechnology Ltd and Glaxo plc.
This problem has dominated the TAT/TAR literature, which contains papers supporting all possible mechanisms of TAT action. We [32"'] have recently been able to address this issue with Xenopus oocytes, using the fact that both DNA and RNA targets can be coinjected with purified TAT protein to ask whether TAT acts on DNA or on RNA. We showed that while TAT activates the expression of a chloramphenicol acetyltransferase (CAT) reporter gene from a DNA target, it can also activate an SP6 RNA as long as it contains the TAR sequence. This demonstrated directly that TAT can activate the translation of RNA, lending weight to the notion that TAT acts at the post-transcriptional level. Another interesting feature of this experiment was that TAT activation of RNA could only occur when both the RNA and TAT were injected into the nucleus of the oocyte. No activation was seen when the RNA was injected into the cytoplasm. This is in sharp contrast to other RNA injection experiments and suggests that the TAR sequence has some inhibitory effect on translation unless TAT has 'activated' the RNA in the nucleus. This inhibition of translation by TAR in oocytes has been seen before [33], although the effect of TAT was not tested. Taken together these data demonstrate that TAT programmes TAR-containing RNA in the nucleus for subsequent translation in the cytoplasm and suggests that RNA molecules must get onto an 'expression pathway' in the nucleus if they are to be efficiently translated. It is possible that H1V RNA fails to access such a pathway and that the TAT/TAR interaction rescues it. It remains to be seen why H1V needs this activity. This oocyte assay for post-transcriptional activation of HIV expression by TAT opens the way to a more detailed analysis of the mechanism of TAT action and also provides an assay for anti-TAT antivirals.
Annotated references and recommended reading • ••
1. ••
HEIKKILA JJ: Use of X e n o p u s oocytes to study t h e expression of cloned genes and translation of mRNA. Biotecb Adv 1989, 7:47 59. A short, but very good contemporary review of the technology associated with the oocyte system. 2.
GURDONJB, MELTON DA: Gene transfer in amphibian eggs and oocytes. A n n u Rev Genet 1981, 15:189-218
3.
GURDONJB, WICKENS MP: The u s e of X e n o p u s oocytes for the expression of cloned genes . Methods Enzymol 1983, 101:370-386
4.
DEROBERTISEM, MERTZJE: Hybridization of cytoplasmic and total RNA of synchronized HeLa cells 12 h o u r s after thymidine release. Cell 1977, 12:172 182.
5.
MERTZJE, GURDON JB: Purified DNAs are transcribed after microinjection into X e n o p u s oocytes. Proc Natl Acad Sci USA 1977, 74:1502 1506.
6.
ETKIN LD, MAXSONRE: T h e synthesis of authentic sea u r c h i n transcriptional and translational products by sea u r c h i n histone genes injected into X e n o p u s laevis oocytes . Dev Biol 1980, 75:13-25.
7.
BIENZ M, PELHAM HRB: Expression of a Drosophila heatshock protein in X e n o p u s oocytes: conserved and divergent regulatory signals. EMBO J 1982, 1:1583-1588.
8.
VOELLMYR, RUNGGERD Transcription of a Drosophila heat shock is heat-induced in X e n o p u s oocytes. Proc Natl Acad Sci USA 1982, 79:1776-1780.
9.
GURDONJB, LANECD, WOODLANDHR, MARBAIXG: Use of frog eggs and oocytes for the study of m e s s e n g e r RNA and its translation in living cells. Nature 1971, 233:177-182.
10.
KRIEGPA, MELTON DA: Functional mRNA are p r o d u c e d by SP6 in vitro transcription of cloned cDNA. Nucl Acids Res 1984, 12:7057-7070.
11.
NAGATAS, TAIRA H, HALLA, JOHNSRUD L, STREUH M, ECS"ODI J, BOLL W, CANTELLK, WElSSMANN C: Synthesis in E. coli of a polypeptide with h u m a n leukocyte interferon activity. Nature 1980, 284:316-320.
12.
PAIEMENTJ: Morphology and endoplasmic reticulum and golgi e l e m e n t s following microinjection of rat liver micros o m e s into X e n o p u s laevis oocyte cytoplasm Exp Cell Res 1986, 166:510-518.
13.
PAmMENTJ, KAN FWK, LANOIXJ, BLAINM: Cytochemical analysis of the reconstitution of endoplasmic reticulum after microinjeetion of rat liver m i c r o s o m e s into Xenopus oocytes. J Histochem Cytochem 1988, 36:1263-1273.
Conclusion In summary, the past few years have not seen great breakthroughs in the technology we use, rather a refining of the techniques in order to apply them to new areas of study.
Of interest Of outstanding interest
14. ••
PAIEMENTJ, JOLICOEURM, FAZELA, BERGERONJJM: Reconstruction of the Golgi apparatus after microinjection of rat liver Golgi fragments into X e n o p u s oocytes. J Cell Biol 1989, 108:1257-1269. A very interesting example of the emerging technology of manipulating in vitro organelles assembled from components.
Expression in Xenopusoocytes Kingsman, Braddock, Thorburn eta/. 15.
SUMIKAWAK, HOUGHTON M, EMTAGEJS, RICHARDSBM, BARNARD EA: Active multi-subunit ACh receptor assembled by translation of heterologous mRNA in Xenopus oocytes. Nature 1981, 292:862 864.
16.
SCHOFIELDPR, DARHSONMG, FUJITA N, BURT DR, STEPHENSON FA, RODRIGUEZ H, RHEE LM, RAMACHANDRANJ, REALE V, GLENCORSETA, SEEBURGPH, BARNARDEA: Sequence and functional expression of the GABAA receptor s h o w s a llgandgated r e c e p t o r super-family. Nature 1987, 328:221-227.
Yu L, BLtlMER KJ, DAVIDSONN, LESTER HA, THORNERJ: Eunctional expression of t h e yeast-factor r e c e p t o r in Xenopus oocytes. J Biol Chem 1989, 264:29 = 0847-20850. A very good example of the fusion of two very powerful technologies, oocyte injection and yeast genetics, to show the function of a receptor.
24. •,
MEYERHOFD, RICHTER D: Identification of G protein-coupied receptors by RNAsH-mediated hybrid depletion using Xenopus laevis oocytes as expression system. FEBS Lett 1990, 266:19~194. An interesting new approach to identifying receptor genes from PCRamplified fragments. 25.
HERSKOWITZI: Life cycle of the budding yeast Saccharomyces cerevisiae. Microbiol Rev 1988, 52:536-553.
26.
BLUMEI~KJ, RENEKE JE, THORNER J: T h e STE2 gene produ c t os the ligand-binding c o m p o n e n t of t h e c~-factor rec e p t o r of Saccharomyces cerevisiae. J Biol Cbem 1988, 263:10836-10842.
27.
MARSH L, HERSKOWITZ I: STE2 protein of Saccharomyces kluyveri is a m e m b e r of t h e rhodopsin/~-adrenergic rec e p t o r family and is responsible for recognition of the p e p t i d e ligand alpha factor. Proc Natl Acad Sci USA 1988, 85:3855-3859.
28.
JAHNG KY, FERGUSONJ, REED ST: Mutations in a g e n e encoding the alpha subunit of Saccharomyces cerevisiae G p r o t e i n indicate a role in mating p h e r o m o n e signaling. Mol Cell Biol 1988, 8:2484-2493.
29.
JAN LY, JAN YN: Voltage-sensitive ion channels. Cell 1989, 56:13~25.
17. ••
18.
RUPPERSBERGJP, SCHROTERKH, SAKMANB, STOCKERM, SEWING S, PONGS O: H e t e r o m u h i m e r i c channels formed by rat brain potassium c h a n n e l proteins. Nature 1990, 343:535-537. Describes the assembly of different K + -channel proteins to form hybrid channels. •
•
19. ••
ISACOFFEY, JAN YN, JAN LY: Evidence for t h e formation of h e t e r o m n l t i m e r i c potassium channels in Xenopus oocytes. Nature 1990, 345:530-534. This paper is similar to [18 ••] and draws the same conclusions. 20. ••
CHRISTIEMJ, NORTH RA, OSBORNE PB, DOUGLASSJ, ADELMANJP: heteropolymeric potassium channels e x p r e s s e d in X e n o p u s oocytes from cloned subunits. Neuron 1990, 2:405-411. This paper is similar to [18 "° ] and [19 °° ] and draws the same conclusions. 21. •
KUMARV, EARRIEST, SW1ERKOZJ, ATK!NSONJP: Translation of t h e h u m a n C 3 b / C 4 b receptor mRNA in a cell-free system and by X e n o p u s oocytes. Biochemistry 1989, 28:4040-4046. Demonstrates the simple use of oocytes as a convenient translation sys tem. OMBACHJA, GUNDERSEN CB: Mercuric ions are p o t e n t noncompetitive antagonists of h u m a n brain kainate receptors e x p r e s s e d in Xenopus oocytes. Mol Pbarmacol 1989, 36:582-588. Gives a good example of the use of oocytes in the pharmacological analysis of receptor antagonists.
30. •
RICHTER JD: In vivo p h o t o crosslinking reveals that transcription factor binding to the mammalian ATF recognition s e q u e n c e is required for EIA-induced transactivation in injected Xenopus laevis oocytes. Nucl Acids Res 1989, 17:4503-4516. This study adds to the body of evidence illustrating that E1A acts by modifying a transcription factor; the study involves a new cross-linking technique that is used after oocyte microinjection. 31.
22. ,
23. •
MCBAINCJ, KLECKNERNW, WYRICKS, DINGLEDINE R: Structural r e q u i r e m e n t s for activation of t h e glycine coagonist site of N-methyl-D-aspartate receptors e x p r e s s e d in Xenopus oocytes. Mol Pharmacol 1989, 36:556-565. Provides a good example of the use of oocytes in the pharmacological analysis of receptor agonists.
RICHTERJD, HURST HC, JONES NC: Adenovirus E1A requires synthesis of the cellular proteins to establish a stable transcription c o m p l e x in injected Xenopus laevis oocytes. Mol Cell Biol 1987, 7:3049-3056.
32. •
BRADDOCKM, CHAMBERSA, WILSON W, ESNOUF MP, ADAMSSE, KINGSMANAJ, K1NGSMANSM: HIV-1 TAT 'activates' presynthesized RNA in t h e n u c l e u s Cell 1989, 58:269-279. This paper makes the most of both DNA, RNA and protein injection to throw light on a controversial subject in AIDS research. 33.
PARKIN NT, COHEN EA, DARVEAU A, ROSEN C, HASELTINE
W, SONENBERG Iq: Mutational analysis of t h e 5'non-coding region of h u m a n immunodeficiency virus type 1: effects of secondary structure on translation. EMBO J 1988, 7:2831-2837.
85
Introduction
(many days) as non-integrated and non-replicating circular molecules.
Unlike most of the other systems reviewed in this volume, the oocyte of the frog Xenopus laevis will never be used as a production source for biological products. The oocyte system is, however, an extremely powerful analytical tool because of the ease with which functional macromolecules can be microin[ected into the cell. The system has been reviewed recently by Heikkila [1 o-]. Oocytes, and those from Xenopus are no exception, are unlike somatic cells. They are large, about 100 000 times the volume of a somatic cell, and they are arrested in prophase of the first meiotic division. They are extremely active in transcription and translation, possessing sufficient ribosomes and RNA polymerases to take the developing embryo through to the 30 000 cell stage. However, they are not capable of DNA replication. Oocytes can be induced to pass through the first meiotic division (maturation) by treatment with progesterone in vitro. During this process the nuclear membrane breaks down and the cell then arrests in meiotic metaphase II. If the mature egg is then fertilized (activated) it continues through meiosis and an embryo is formed [2]. The oocyte is about 1.0-1.5mm in diameter. This fact alone makes it reasonably easy to microinject DNA, RNA or protein into either the nucleus or the cytoplasm. Furthermore, multiple injections can be carried out that are separated in both space (nucleus or cytoplasm) and/or time [3]. Xenopua oocytes have been used widely in the analysis of gene expression yet while studies of Xenopus genes in this more or less homologous system have yielded important results the use of the oocyte system to express heterologous, usually mammalian, pol II genes has given the more important contribution to biology and biotechnology. In most cases, the oocyte accurately intitiates and terminates transcription, splices primary transcripts and translates the message [4-8]. In some cases, specific ffanscription factors may be required (see below). A further feature of this system is that injected plasmid DNA can be maintained for long periods
RNA can also be injected into oocytes and translated. This was first shown using RNAs synthesized in vivo [9], but more recently the use of SP6- or T7-driven RNAs made in vitro [10] has made the oocyte system even more versatile (see below). The ability of oocytes to translate injected RNA was, in fact, used to isolate the first human czinterferon clone using hybrid-selected fractionated RNA [11]. In the past year, the Xenopua oocyte system has contfibuted to studies in the areas of cell biology, receptor research, mammalian gene expression and AIDS research, which we will review in turn.
Oocytes and cell biology The analysis of secretion pathways in eukaryotic cells is central to making biological products using the techniques of genetic engineering. The dissection of these pathways in yeast has been facilitated by the isolation of a range of sec mutants and their subsequent biochemical analysis. The same genetic approach cannot be used in mammalian cells. Recently, it has been shown that the Golgi apparatus from rat can be partially reconstituted in Xenopus oocytes [12,13]. This system has important implications for analysing secretion in higher eukaryotes as it opens the way to manipulating the components of the Golgi apparatus before injection into oocytes. This system has now been taken one step further, in that Golgi components, labelled in vitro with 3H-sialic acid, have been shown to affect the formation of stacked saccules and to migrate into the Xenopus secretory pathway [14 oo]. In addition, it has been shown that transfer to the oocyte surface is inhibited by vinblastine, demonstrating that the process is microtubule dependent. Continued refinement of this system offers huge potential to manipulate the secretion of biological products by animal cells.
Abbreviations
AIDS~acquired immunodeficiencysyndrome;CAT--chloramphenicolacetyltransferase; HIV--human immunodeficiencyvirus;PCR--polymerasechain reaction. 82
C) Current Biology Ltd ISSN 0958-1669
Expressionin XenopusoocytesKingsman, Oocytes, receptors and channels Oocytes have been used widely in the study of receptor/ligand interactions since the demonstration that the Torpedo acetylcholine receptor could assemble and bind ligand in the oocyte membrane after microinjection of its mRNA [15]. For example, coexpression of SP6 RNAs encoding the cz and [3 subunits of the bovine 0t-aminobutyric acid receptor resulted in the assembly of a functional receptor and ion channel with full pharmacological properties [16]. The past year has seen other examples of this strategy, ranging from the analysis of yeast mating pheremone receptors to the study of K+-channels [17 °'-20 °.,21 "-23 ", 24o.]. In yeast, diploids are formed by mating between the two different haploid mating cell types, a and oz. Both haploids secrete mating factors that interact with the cells of the opposite mating type to prepare for conjugation [25]. Genetic and biochemical evidence has suggested that the product of the STE2 gene of yeast is the cell-surface receptor for cz-factor [26,27] and that this receptor operates via a G-protein [28], probably the product of the GPAI gene. Expression of T7 STE2 RNAs in oocytes has now demonstrated that the STE2 product is the only product required for a-factor binding and that with or without coexpression of GPAI RNA the STE2-encoded receptor does not alter the membrane conductance of oocytes either in the presence or absence of a-factor [17 °°]. This shows that the a-factor receptor, like other G-proteincoupled receptors, does not act through an intrinsic or ligand-gated ion channel. This result now opens the way for a detailed structure-function analysis that will draw on the joint potential of the Xenopus oocyte and yeast genetic systems. It will provide a powerful model for other G-protein-coupled receptors. Data from oocyte microinjection experiments have also thrown light on the diversity of K + -channels found in excitable cells. Three recent papers [18 °°-20 o.] report similar approaches. Cloned cDNA for various rat K + -channel proteins [29] are available, together with variants of the Shaker gene of Drosophila, which is known to encode a K + -channel protein. When expressed alone these individual proteins assemble into ion channels that differ with respect to their kinetics, single-channel conductance, pharmacology and sensitivity to voltage and second messengers. In the past, data have been reported that indicated that these channels were multimeric structures. Coexpression of different channel proteins by microinjection of RNAs synthesized in vitro has now confirmed this notion and, in addition, shown that heteromultimeric channels, with properties intermediate between the homomultimeric structures, can be formed. These observations have lead to the suggestion that at least some of the diversity of K + -channels may arise from the in vivo assembly of a spectrum of heteromultimeric channels. Other recent applications of the oocyte system to receptor research include the analysis of cotranslational modifications of the complement C3b/C4b receptor [21 °], the demonstration that mercuric ions are potent n o n - c o m -
Braddock, Thorburn et a/.
petitive inhibitors of brain kainate receptors [22 o], and a structure-function study of glycine analogues that activate the glycine recognition site of the N-methyl-D-aspartate receptor [23 °]. A further development of the use of oocytes in identifying receptor cDNAs came from Meyerhof and Richter [24 °']. It is common to identify the presence of a receptor in a mRNA population by injecting the mRNA into oocytes and then assaying for the receptor on the cell surface. The next stage is usually to clone the gene(s) for that receptor. Positive identification of a putative cDNA or polymerase chain reaction (PCR) fragment usually requires expression of that clone. This, in turn, demands that the clone is full length and engineered into an expression vector. A faster and more convenient route would be to take DNA fragments and hybrid arrest the in vivo expression of the receptor mRNA in the mRNA pool. Unfortunately, this strategy has met with only limited success. The altemative approach described by Meyerhof and Richter [24°°] is to hybridize the putative DNA clone to the mRNA pool and then use RNaseH to selectively remove the mRNA that has hybridized. Clones are identified by their ability to remove the receptor mRNA from the pool. This technique may expedite the cloning of receptor genes in many systems, including expression oocytes. Taken together, these papers illustrate the contribution that the Xenopus oocyte system is making to both the isolation and analysis of receptors. Furthermore, the expression and function of receptors on the oocyte surface will prove most valuable in the search for agonists and antagonists.
Oocytes and gene expression Microinjection of oocytes with plasmids containing defined transcription units has contributed substantially to our understanding of the control of gene expression. A recent paper [30 *] on the adenovirus EIA transactivator illustrates this point well. It has been shown previously [31] that promoters, such as the adenovims E3 promoter or the promoter from the HSP70 gene, that are normally activated by EIA are also activated when both EIA and the promoter are coinjected into Xenopus oocytes. Richter and colleagues also showed that this activation was prevented by inhibitors of protein synthesis, implying that some other protein, that was either induced by EIA or which turned over rapidly, was required for EIAmediated activation. This group has now identified this protein by gel-shift assays with oocyte extracts, a new in vivo photo cross-linking technique and immunoselection of biotinylated oligonucleotides. They have shown that a 75 kD protein that binds to the mammalian transcription factor, ATF, recognition sequence in vivo is essential for EIA activation in oocytes. Treatment with cycloheximide prevented in vivo binding of this factor but, interestingly, EIA had no effect. This implies that EIA modifies the activity of an oocyte transcription factor without affecting its binding.
83
84
Expression systems
Oocytes and AIDS
Acknowledgements
A key feature of the acquired immunodeficiency syndrome (AIDS) virus, human immunodeficiency virus (HIV), that distinguishes it from many other viruses is the complex set of regulatory systems that it uses to control gene expression. Amongst these is the TAT/TAR system, which is virus specific and a prime target for antiviral agents. TAT is an 86-amino-acid protein that activates expression of the virus. It acts through a target sequence called TAR, which is located in the first 60 nucleotides of the transcription unit of the virus. It is present, therefore, in both the DNA of the provirus and in all the viral transcripts. The presence of the TAR region in both DNA and RNA raises the different issue of whether activation is at the level of transcription or at some post-transcriptional level.
The work in this laboratory is funded by the Medical Research Council, Science and Engineering Research Council, British Biotechnology Ltd and Glaxo plc.
This problem has dominated the TAT/TAR literature, which contains papers supporting all possible mechanisms of TAT action. We [32"'] have recently been able to address this issue with Xenopus oocytes, using the fact that both DNA and RNA targets can be coinjected with purified TAT protein to ask whether TAT acts on DNA or on RNA. We showed that while TAT activates the expression of a chloramphenicol acetyltransferase (CAT) reporter gene from a DNA target, it can also activate an SP6 RNA as long as it contains the TAR sequence. This demonstrated directly that TAT can activate the translation of RNA, lending weight to the notion that TAT acts at the post-transcriptional level. Another interesting feature of this experiment was that TAT activation of RNA could only occur when both the RNA and TAT were injected into the nucleus of the oocyte. No activation was seen when the RNA was injected into the cytoplasm. This is in sharp contrast to other RNA injection experiments and suggests that the TAR sequence has some inhibitory effect on translation unless TAT has 'activated' the RNA in the nucleus. This inhibition of translation by TAR in oocytes has been seen before [33], although the effect of TAT was not tested. Taken together these data demonstrate that TAT programmes TAR-containing RNA in the nucleus for subsequent translation in the cytoplasm and suggests that RNA molecules must get onto an 'expression pathway' in the nucleus if they are to be efficiently translated. It is possible that H1V RNA fails to access such a pathway and that the TAT/TAR interaction rescues it. It remains to be seen why H1V needs this activity. This oocyte assay for post-transcriptional activation of HIV expression by TAT opens the way to a more detailed analysis of the mechanism of TAT action and also provides an assay for anti-TAT antivirals.
Annotated references and recommended reading • ••
1. ••
HEIKKILA JJ: Use of X e n o p u s oocytes to study t h e expression of cloned genes and translation of mRNA. Biotecb Adv 1989, 7:47 59. A short, but very good contemporary review of the technology associated with the oocyte system. 2.
GURDONJB, MELTON DA: Gene transfer in amphibian eggs and oocytes. A n n u Rev Genet 1981, 15:189-218
3.
GURDONJB, WICKENS MP: The u s e of X e n o p u s oocytes for the expression of cloned genes . Methods Enzymol 1983, 101:370-386
4.
DEROBERTISEM, MERTZJE: Hybridization of cytoplasmic and total RNA of synchronized HeLa cells 12 h o u r s after thymidine release. Cell 1977, 12:172 182.
5.
MERTZJE, GURDON JB: Purified DNAs are transcribed after microinjection into X e n o p u s oocytes. Proc Natl Acad Sci USA 1977, 74:1502 1506.
6.
ETKIN LD, MAXSONRE: T h e synthesis of authentic sea u r c h i n transcriptional and translational products by sea u r c h i n histone genes injected into X e n o p u s laevis oocytes . Dev Biol 1980, 75:13-25.
7.
BIENZ M, PELHAM HRB: Expression of a Drosophila heatshock protein in X e n o p u s oocytes: conserved and divergent regulatory signals. EMBO J 1982, 1:1583-1588.
8.
VOELLMYR, RUNGGERD Transcription of a Drosophila heat shock is heat-induced in X e n o p u s oocytes. Proc Natl Acad Sci USA 1982, 79:1776-1780.
9.
GURDONJB, LANECD, WOODLANDHR, MARBAIXG: Use of frog eggs and oocytes for the study of m e s s e n g e r RNA and its translation in living cells. Nature 1971, 233:177-182.
10.
KRIEGPA, MELTON DA: Functional mRNA are p r o d u c e d by SP6 in vitro transcription of cloned cDNA. Nucl Acids Res 1984, 12:7057-7070.
11.
NAGATAS, TAIRA H, HALLA, JOHNSRUD L, STREUH M, ECS"ODI J, BOLL W, CANTELLK, WElSSMANN C: Synthesis in E. coli of a polypeptide with h u m a n leukocyte interferon activity. Nature 1980, 284:316-320.
12.
PAIEMENTJ: Morphology and endoplasmic reticulum and golgi e l e m e n t s following microinjection of rat liver micros o m e s into X e n o p u s laevis oocyte cytoplasm Exp Cell Res 1986, 166:510-518.
13.
PAmMENTJ, KAN FWK, LANOIXJ, BLAINM: Cytochemical analysis of the reconstitution of endoplasmic reticulum after microinjeetion of rat liver m i c r o s o m e s into Xenopus oocytes. J Histochem Cytochem 1988, 36:1263-1273.
Conclusion In summary, the past few years have not seen great breakthroughs in the technology we use, rather a refining of the techniques in order to apply them to new areas of study.
Of interest Of outstanding interest
14. ••
PAIEMENTJ, JOLICOEURM, FAZELA, BERGERONJJM: Reconstruction of the Golgi apparatus after microinjection of rat liver Golgi fragments into X e n o p u s oocytes. J Cell Biol 1989, 108:1257-1269. A very interesting example of the emerging technology of manipulating in vitro organelles assembled from components.
Expression in Xenopusoocytes Kingsman, Braddock, Thorburn eta/. 15.
SUMIKAWAK, HOUGHTON M, EMTAGEJS, RICHARDSBM, BARNARD EA: Active multi-subunit ACh receptor assembled by translation of heterologous mRNA in Xenopus oocytes. Nature 1981, 292:862 864.
16.
SCHOFIELDPR, DARHSONMG, FUJITA N, BURT DR, STEPHENSON FA, RODRIGUEZ H, RHEE LM, RAMACHANDRANJ, REALE V, GLENCORSETA, SEEBURGPH, BARNARDEA: Sequence and functional expression of the GABAA receptor s h o w s a llgandgated r e c e p t o r super-family. Nature 1987, 328:221-227.
Yu L, BLtlMER KJ, DAVIDSONN, LESTER HA, THORNERJ: Eunctional expression of t h e yeast-factor r e c e p t o r in Xenopus oocytes. J Biol Chem 1989, 264:29 = 0847-20850. A very good example of the fusion of two very powerful technologies, oocyte injection and yeast genetics, to show the function of a receptor.
24. •,
MEYERHOFD, RICHTER D: Identification of G protein-coupied receptors by RNAsH-mediated hybrid depletion using Xenopus laevis oocytes as expression system. FEBS Lett 1990, 266:19~194. An interesting new approach to identifying receptor genes from PCRamplified fragments. 25.
HERSKOWITZI: Life cycle of the budding yeast Saccharomyces cerevisiae. Microbiol Rev 1988, 52:536-553.
26.
BLUMEI~KJ, RENEKE JE, THORNER J: T h e STE2 gene produ c t os the ligand-binding c o m p o n e n t of t h e c~-factor rec e p t o r of Saccharomyces cerevisiae. J Biol Cbem 1988, 263:10836-10842.
27.
MARSH L, HERSKOWITZ I: STE2 protein of Saccharomyces kluyveri is a m e m b e r of t h e rhodopsin/~-adrenergic rec e p t o r family and is responsible for recognition of the p e p t i d e ligand alpha factor. Proc Natl Acad Sci USA 1988, 85:3855-3859.
28.
JAHNG KY, FERGUSONJ, REED ST: Mutations in a g e n e encoding the alpha subunit of Saccharomyces cerevisiae G p r o t e i n indicate a role in mating p h e r o m o n e signaling. Mol Cell Biol 1988, 8:2484-2493.
29.
JAN LY, JAN YN: Voltage-sensitive ion channels. Cell 1989, 56:13~25.
17. ••
18.
RUPPERSBERGJP, SCHROTERKH, SAKMANB, STOCKERM, SEWING S, PONGS O: H e t e r o m u h i m e r i c channels formed by rat brain potassium c h a n n e l proteins. Nature 1990, 343:535-537. Describes the assembly of different K + -channel proteins to form hybrid channels. •
•
19. ••
ISACOFFEY, JAN YN, JAN LY: Evidence for t h e formation of h e t e r o m n l t i m e r i c potassium channels in Xenopus oocytes. Nature 1990, 345:530-534. This paper is similar to [18 ••] and draws the same conclusions. 20. ••
CHRISTIEMJ, NORTH RA, OSBORNE PB, DOUGLASSJ, ADELMANJP: heteropolymeric potassium channels e x p r e s s e d in X e n o p u s oocytes from cloned subunits. Neuron 1990, 2:405-411. This paper is similar to [18 "° ] and [19 °° ] and draws the same conclusions. 21. •
KUMARV, EARRIEST, SW1ERKOZJ, ATK!NSONJP: Translation of t h e h u m a n C 3 b / C 4 b receptor mRNA in a cell-free system and by X e n o p u s oocytes. Biochemistry 1989, 28:4040-4046. Demonstrates the simple use of oocytes as a convenient translation sys tem. OMBACHJA, GUNDERSEN CB: Mercuric ions are p o t e n t noncompetitive antagonists of h u m a n brain kainate receptors e x p r e s s e d in Xenopus oocytes. Mol Pbarmacol 1989, 36:582-588. Gives a good example of the use of oocytes in the pharmacological analysis of receptor antagonists.
30. •
RICHTER JD: In vivo p h o t o crosslinking reveals that transcription factor binding to the mammalian ATF recognition s e q u e n c e is required for EIA-induced transactivation in injected Xenopus laevis oocytes. Nucl Acids Res 1989, 17:4503-4516. This study adds to the body of evidence illustrating that E1A acts by modifying a transcription factor; the study involves a new cross-linking technique that is used after oocyte microinjection. 31.
22. ,
23. •
MCBAINCJ, KLECKNERNW, WYRICKS, DINGLEDINE R: Structural r e q u i r e m e n t s for activation of t h e glycine coagonist site of N-methyl-D-aspartate receptors e x p r e s s e d in Xenopus oocytes. Mol Pharmacol 1989, 36:556-565. Provides a good example of the use of oocytes in the pharmacological analysis of receptor agonists.
RICHTERJD, HURST HC, JONES NC: Adenovirus E1A requires synthesis of the cellular proteins to establish a stable transcription c o m p l e x in injected Xenopus laevis oocytes. Mol Cell Biol 1987, 7:3049-3056.
32. •
BRADDOCKM, CHAMBERSA, WILSON W, ESNOUF MP, ADAMSSE, KINGSMANAJ, K1NGSMANSM: HIV-1 TAT 'activates' presynthesized RNA in t h e n u c l e u s Cell 1989, 58:269-279. This paper makes the most of both DNA, RNA and protein injection to throw light on a controversial subject in AIDS research. 33.
PARKIN NT, COHEN EA, DARVEAU A, ROSEN C, HASELTINE
W, SONENBERG Iq: Mutational analysis of t h e 5'non-coding region of h u m a n immunodeficiency virus type 1: effects of secondary structure on translation. EMBO J 1988, 7:2831-2837.
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