Gene expression using Xenopusoocytes Joel D. Richter Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts, USA
Xenopus oocytes have been used as a surrogate genetic system for the study of many facets of gene expression. Some recent salient examples where injected oocytes proved to be indispensable for the analysis of transcription, translation, RNA/protein transport, and ion channel formation are described. Current Opinion in Biotechnology 1991, 2:698-703
Introduction In 1971, Gurdon and colleagues published a paper [1] that would have major ramifications for the fields of molecular, cellular, and developmental biology and, more recently, the neurosciences as well. In those few pages, they described experiments that showed that Xenopus laevis oocytes would translate injected rabbit globin mRN& This demonstrated not only a lack of species specificity at the translational level, but also suggested that oocytes could be used to examine the c/selements in mRNA that regulate protein synthesis. Moreover, this idea that oocytes could be used as a surrogate genetic system, was taken up by others who showed that oocytes would also transcribe injected DNA [2-4], utilize injected membranes for translation at the rough endoplasmic reticulum [5] and correctly process and secrete heterologous proteins into the surrounding medium [6]. Thus, the injected Xenopus oocyte, an easily manipulated in vivo sys tern, could be used as a bioassay for virtually all aspects of eukaryotic gene expression. This review will discuss some recent examples where injected Xenopus oocytes have been particularly useful for the study of gene expression.
amino acids, or by the enzymatic activity of the protein. An example of the latter assay is illustrated in Fig. 1. Oocyte nuclei were injected with 0.1 ng of supercoiled plasmid DNA encoding the chloramphenicol acetyltransferase (CAT) gene that was driven by the adenovirus E3 promoter. Adenovirus EIA, which normally activates the E3 promoter following infection of mammalian cells by the virus, was expressed in Escherichia coli and injected into the cytoplasm of some of these oocytes. Oocytes receiving EIA in addition to DNA exhibited CAT levels about eight-fold greater than those injected with DNA alone. Thus, oocytes contain all the machinery necessary for EIA to enter the nucleus and stimulate transcription (see below).
Injected Xenopus oocytes as a surrogate genetic system Table 1 lists some recent examples where the injected Xenopus oocyte has been used as a surrogate in vivo genetic system. This catalog is not meant to be exhaustive, but rather to point out the different facets of gene expression that have been studied using Xenopua oocytes.
The oocyte as an in vivo assay system
Transcriptional control
Owing to its large size (1.2-1.4 mm in diameter), the fully grown Xenopus oocyte (stage VI according to the classification scheme of Dumont [7]) is a rather facile biochemical tool. Up to 100 nl of solution containing RNA (at an initial concentration of about I mg/ml) may be injected into the cytoplasm. In addition, the nucleus may be injected with 5-10 nl of solution containing DNA (at an initial concentration of about 0.1 mg/ml). Following incubation, the protein products derived from these injected molecules may be assessed in a number of different ways, such as by the incorporation of radiolabeled
As stated above, adenovims EIA is a potent activator of transcription of viral, and in some cases, cellular genes. However, E1A does not bind specific promoter sequences, which implies that cellular factors might mediate EIA-induced transcription. Indeed, just such a role has been suggested for the cellular protein ATF [8]. In Xenopus oocytes, gel retardation and DNA injection studies demonstrated that a factor required for E1A-induced transcription was present and bound the ATF-recognition sequence [9]. To estimate the size of the protein, a 36 nucleotide single-stranded oligonucleotide spanning the ATF-recognition sequence was used as a template
Abbreviations AChR--acetylcholine receptor; CAT--chloramphenicol acetyltransferase; CPE~cytoplasmic polyadenylation element; IL-2--interleukin 2; NLSs--nuclear localization signals;U snRNPs--uridine rich small nuclear ribonuclease particles; U snRNAs---uridine rich small nuclear RNAs.
698
(~) Current Biology Ltd ISSN 0958-1669
Gene expression using Xenopusoocytes Richter
(a)
(b) % Chloramphenicol acetylated
/
75
~i E3+CAT '~
.•
100 "1
CAT
~l
(~J
so
/"
E1A ~ A A A
25
E3/CAT
E3/CAT and E1A
Fig. 1. Analysis of E1A-induced transcription in injected Xenopus oocytes. (a) Depicts the injection of plasmid DNA encoding bacterial chloramphenicol acetyltransferase (CAT), which is under the control of the adenovirus E3 promoter, into the nucleus and the injection of E. coil-expressed adenovirus EIA protein into the cytoplasm. E1A is then transported to the nucleus where it activates E3/CAT gene transcription. The resulting CAT RNA is polyadenylated, transported to the cytoplasm, and translated into enzymatically active CAT. (b) Shows the relative amounts of CAT activity produced from oocytes injected with E3/CAT DNA only or E3/CAT DNA plus EIA. CAT activity is determined by the percentage of 14C-chloramphenicol that is acetylated when mixed with acetyl coenzyme A and extracts prepared from injected oocytes.
for second strand synthesis by reverse transcriptase. In this case, however, second strand synthesis took place in the presence of 32p-dCTP and the photoactivatable crosslinking reagent BrdUTP. The oligonucleotide was then injected into the oocyte nucleus (sometimes called the germinal vesicle). After incubation the transparent germinal vesicles were manually isolated, irradiated with 302 nm light, and prepared for sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE) and autoradiography. A single protein species of 66 kD was crosslinked to the injected DN& indicating that this protein recognizes the ATF-binding site in vivo. Thus, injected Xenopus oocytes can be used to examine the in vivo interactions of specific DNA sequences and their binding proteins.
ulated primary T-lymphocytes or mitogenically stimulated T-lymphocytes. Using an S-1 nuclease protectionassay for IL-2 transcription, Mouzaki et al. found basal levels of IL-2 RNA in oocytes injected with DNA only. This basal IL-2 transcription, however, was silenced when oocytes were also injected with protein from unstimulated Tlymphocytes. This suggests that unstimulated T-lymphocytes contain a specific IL-2 gene repressor. When these 'repressed' oocytes were injected yet again with protein from stimulated T-lymphocytes, Ib2 transcription increased dramatically. Thus, these investigators suggest that IL-2 transcription in T-lymphocytes is under dual control; repression in unstimulated cells and stimulation in mitogenically activated cells.
Mouzaki et al. [10 °] have also used injected oocytes to study transactivation of the lymphokine interleukin2 (IL-2) gene. IL-2 transcription is normally activated in T-lymphocytes, but only after they have been stimulated by antigens or mitogens. Based on this observation, oocytes were injected with the IL-2 gene and, in some instances, with protein isolated from either unstim-
Translational control Although several older investigations used injected Xen(> pus oocytes to examine translational control, the recent advent of bacteriophage RNA polymerase (SP6, T7 and T3) to synthesize pure mRNA in vitro has allowed manipulation of the sequences of the injected mRNA in ways that hitherto had not been possible. For example, con-
699
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Expressionsystems Table 1. Analysis of gene expression using Xenopus oocytes.
Process studied I Transcriptional control (a) analysis of EIA transactivation (b) factors required for IL-2 expression RNA processing/translational control (a) cis elements responsible for cytoplasmic polyadeny]ation (b) RNA deadenylation (c) RNA secondary structure RNA/RNP transport/degradation (a) snRNP nuclear transport (b) transport of ribosomal subunits (c) transport of 5S rRNA (d) RNA degradation signals IV Protein processing/degradation (a) processing of influenza haemagglutin (b) degradation signal of adenovirus E1A v Cloning and analysis of membrane proteins (a) potassium channel (b) N-methyI-D-aspartate channel (c) assembly of acetylcholine receptor (d) clustering of acetylchoine receptor
References
cific c,Xelements were required for deadenylation, rather, it appeared to occur along a default pathway. That is, RNAs were either polyadenylated during maturation under the direction of a CPE, or, if they contained no CPE, were deadenylated by default [16",17"].
[9] [10]
[12,13,14",15"'] [16",17"] [29]
[20",21",22,23"] [30] [31] [32]
[33] [34]
[25",26",27"35'',36",37-39] [4O] [411 [28..]
sider the relationship between cytoplasmic polyadenylation and translational control. Oocytes normally contain several mRNAs that are translationally dormant, but their entry into polysomes occurs when their poly(A) tails are elongated during oocyte maturation, the point when these cells re-enter meiosis in preparation for fertilization (for review see Richter [ 11 ] ). To examine the regulation of polyadenytation, and hence translation, radiolabeled RNA (synthesized in vitro by bacteriophage RNA polymerases in the presence of radioactive nucleotide) was injected into oocytes. These oocytes were then induced to mature with progesterone, the natural stimulus of maturation. An analysis of the RNA following its extraction and resolution by gel electrophoresis and autoradiography revealed that it was polyadenylated in a similar manner to its endogenous counterpart. A further set of injection experiments with RNAs containing various mutant sequences in their 3' untranslated regions revealed that two cis elements controlled cytoplasmic polyadenylation: the near-ubiquitous nuclear cleavage/polyadenylation sequence AAUAAA and an upstream uracil-rich element termed the cytoplasmic polyadenylation element (CPE) [ 12,13,14--,15..]. Moreover, polyadenylation was shown to control translation because mutations in the cis elements that abrogated polyadenylation also prevented translation [12,15.-]. In contrast to those mRNAs that are translated only when they undergo poly(A) elongation during maturation, other mRNAs that are normally translated in oocytes are deadenylated during this time and concomitantly leave polysomes. To delineate the c~ signals that control deadenylation, radiolabeled RNA injection experiments similar to those described for poly(A) elongation were performed. The results showed that surprisIngly, no spe-
Oocytes as a model system to study protein and snRNP transport Since 1970, injected Xenopus oocytes have been used to study protein translocation across the nuclear membrane [18]. Partly because of work using oocytes, it is now clear that proteins contain discrete sequences, called nuclear localization signals (NLSs), that are essential for their facilitated import into the nucleus. However, recent studies using injected oocytes to study nuclear import of U-snRNPs (uridine rich small nuclear ribonucleoprotein particles) suggest that the process of nuclear localization is more complex than previously thought. For example, although U-snRNAs are synthesized in the nucleus, they form complexes with proteins in the cytoplasm, and then the complexes enter the nucleus. Experiments using injected Xenopus oocytes have shown that RNA/protein complex formation is a prerequisite for nuclear localization because neither the U snRNAs nor the snRNP proteins alone enter the nucleus [19]. It now appears that one critical determinant for snRNP nuclear import is the 5' terminal trimethylguanosine cap present on most U snRNAs. Thus, radiolabeled U1 snRNA (synthesized in vitro) injected into the oocyte cytoplasm is transported to the nucleus when it contains the normal trimethylguanosine cap, but not when it is either devoid of the cap or contains a chemical derivative of the cap [20".,21.-]. Receptors associated with nuclear pores appear to be the means by which proteins enter the nucleus. This was inferred from oocyte injection experiments in which NLS-bovine serum albumin conjugates competed with each other for nuclear entry [22]. Recent oocyte injection experiments [23"], however, show that although the NI£s from SV40 large T-antigen and nucleoplasmin compete with each other for nuclear entry, they do not compete with U2 snRNP, which suggests that at least this snRNP might utilize unique nuclear pore receptors. In the same vein, wheat germ agglutinin, which has been used to distinguish different pathways for nuclear import, prevents nuclear localization of most proteins with a 'conventional NLS', but has no effect on the nuclear import of U1 and U5 snRNPs [24.]. Thus, injected oocytes have proven to be indispensable for the study of protein and snRNP nuclear tralficking.
cDNA cloning on the basis of protein function: the injected oocyte as an assay system One of the major problems in cell and molecular biology is the isolation of cDNA clones coding for specific pro-
Gene expression using Xenopus oocytes Richter 701 teins of interest. Once a cDNA clone has been isolated the investigator may derive the primary sequence of the protein, make inferences as to the nature of its three dimensional structure, and overexpress the protein to obtain sufl]cient quantities for biochemical analysis. However, the isolation of a desired cDNA d o n e usually requires some knowledge of the protein, such as a partial amino acid sequence, or an antibody to the protein. Often one only knows the function of the protein, which precludes the use of limited sequence information or antibodies. Fortunately, the injected Xenopus ooc~e has proven very useful for screening mRNAs (to be used to synthesize cDNA clones) on the basis of the function of the encoded protein. This has been exploited with great success in studies on the nature of ion channel proteins. For example, oocytes injected with mRNA derived from a tissue expressing relatively high amounts of ion channel protein more often than not synthesize and position the encoded protein in the membrane so that it may be detected by the very sensitive assays of voltage clamping or ligand binding. After mRNA purification and cDNA synthesis, the desired clone is obtained. Recently, studies of channel function have also used injected oocytes. All voltage-dependent potassium channels, for example, do not exhibit identical kinetics or pharmacologic responses to drugs. Because these channels are composed of several subunits, it has been suggested that formation of heteromultimeric channels might give rise to unique characteristics not seen in homomultimeric channels. Three recent studies [25°,26.,27 °] have used injected oocytes to assess whether this is the case. cDNA clones for various K + channel subunits from rat brain or Drosophila were transcribed in vitrowith bacteriophage RNA polymerases and injected into oocytes. Following incubation, the oocytes were voltage-clamped and their K + currents were measured. Because channels composed of identical subunits (homomultimer) would have unique K + currents depending upon the voltage at which the oocyte was clamped, altered K + currents could result from the formation of channels formed from different subunits (heteromultimer) [26°]. In addition, the sensitivity of the channels to blocking agents such as dendrotoxin and tetraethylammonium would also be different in channels composed of identical versus non-identical subunits [25"]. The aggregate data [25",26,,27.] indicate that indeed heteromultimeric K + channels are formed in injected oocytes, and it has been suggested that this could be the basis for the diversity of channel behaviour observed in different tissues. The oocyte not only inserts, usually correctly, channel and other cell surface proteins into its plasma membrane, but it can also cluster those proteins in response to a specific stimulus. Consider, for example, the case of the acetylcholine receptor (AChR). This receptor is an integral membrane protein normally clustered in the postsynaptic membrane of the neuromuscular junction. In theory the receptor should be able to diffuse within the plane of the membrane, but it is actively aggregated and prevented from diffusing, seemingly by an associated
peripheral membrane protein with a molecular weight of 43 kD. Froehner and colleagues [28 .°] examined the function of this 43 kD protein in oocytes. Following injection of mRNA for the AChR into oocytes, AChR protein (detected by its binding to bungarotoxin) was uniformly distributed over the surface of the oocyte. When mRNA encoding the 43 kid protein was co-injected with AChR mRNA, however, AChR formed discrete clusters reminiscent of those found in the postsynaptic membrane. Therefore, because the 43 kD protein induces AChR clustering in oocytes, one would infer that it almost certainly performs the same function in the postsynaptic membrane.
Conclusions We have discussed some recent examples where Xen~ pus oocytes have been used to examine gene expression. Investigators in many fields have taken advantage of their ability to correctly transcribe genes, translate mRNAs, and process proteins [29-34]. Oocytes have also been used to study negative regulation of gene expression [10o], and the functions of particular sequences of a protein [35°°,36,]. Similarly, the fact that AChRs expressed in oocytes were not clustered, led to the establishment of the 43 kD protein as a clustering factor. Thus, the Xenopus oocyte, once the exclusive domain of the developmental biologist, has now become a widely used laboratory tool.
References and recommended reading Papers of special interest, published within the annual period of review, have been highlighted as: . of interest ** of outstanding interest 1.
GURDONJB: Use of Frog Eggs and Oocytes for the Study of Messenger RNA and its Translation in Living Cells. Nature 1971, 233:177-182.
2.
COLMAN A: Transcription of DNAs of Known Sequence After Injection into the Eggs and Oocytes of Xenopus laevi~ Eur J Biochem 1975, 57:85-96.
3.
MERTZJ, GURIX)NJB: Purified DNAs are Transcribed After Microinjection into Xenopus Oocytes. Proc Natl Acad Sci USA 1977, 74:1502-1506.
4.
DEROBERTISEM, MERTZ J: Coupled Transcription-Translation of DNA Injected into Xenopus Oocytes. Cell 1977, 12:175-182.
5.
RICHTERJD, SMITH LD: Differential Capacity for Translation and Lack of Competition Between mRNAs that Segregate to Free and Membrane-Bound Polysomes. Cell 1981, 27:183-192.
6.
COI2aANA, MORSERJ: Export of Proteins from Oocytes of Xenopus laevis. Cell 1979, 17:517-526.
7.
DUMONTJ: Oogenesis in Xenopus laevis (Daudin). I. Stages of Oocyte Development in Laboratory Maintained Animals. J Morph 1972, 136:153-180.
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Expressionsystems 8.
9.
LEE KAW, HAl T-Y, SIVA RAMAN L, THIMMAPPAYAB, HURST HC, JONES NC, GREEN MR: A Cellular Protein, Activating Transcription Factor, Activates Transcription of Multiple E1A-Inducible Adenovirus Early Promoters. Proc Natl Acad Sci USA 1987, 84:8355-8359. RICHTER JD: In Vivo Photocrosslinking Reveals that Transcription Factor Binding to t h e Mammalian ATF Recognition Sequence is Required for E1A-Induced Transactivation in Injected Xenopus Oocytes. Nucl Acids Res 1989, 17:4503-4516.
10. •
MOUZAK1A, WEIL R, MUSTER L, RUNGGER D: Silencing and tran~Activation of t h e Mouse IL-2 Gene in Xenopus Oocytes by Proteins from Resting and Mitogen-Induced Primary T-Lymphocytes. EMBO J 1991, 10:139°/1406. injected oocytes are used to show that protein factors isolated from Tlymphocytes can both repress and activate interleukin-2 transcription. 11.
RICHTERJD: Translational Control During Early Development. Bioessays 1991, 13:179~183.
12.
MCGREW LL, DWORKIN-RASTL E, DWORKIN MB, RICHTER JD: Poly(A) Elongation During X e n o p u s Oocyte Maturation is Required for Translational R e c r u i t m e n t and is Mediated by a Short Sequence Element. Genes Dev 1989, 3:803-815.
13.
Fox CA~ SHEETS MD, WICKENS MP: Poly(A) Addition During Maturation of Frog Oocytes: Distinct Nuclear and Cytoplasmic Activities and Regulation by t h e Sequence UUUUUAU. Genes Dev 1989, 3:2151-2162.
14. ••
MCGREWLL, RICHTERJD: Translational Control by Cytoplasmic Polyadenylation During Xenopus Oocyte Maturation: Characterization of a s and trans Elements and Regulation of Cyclin/MPF. EMBO J 1990, 9:3743-3751. This study uses injected oocytes to define, in detail, the cytoplasmic polyadenytation element. In addition, it shows that injected cydin mRNA and maturation promoting factor both stimulate polyadenylation. 15. ••
PARISJ, RICHTERJD: Maturation-Specific Polyadenylation and Translational Control: Diversity of Cytoplasmic Polyadenylation Elements, Influence o f Poly(A) Tail Size, and Formation of Stable Polyadenyfation Complexes. Mol Cell Biol 1990, 10:5634-5645. This study shows that cytoplasmic polyadenylation is required for the translation of certain RNAs and that different cytoplasmic polyadenylation elements confer unique poly(A) tail lengths. In addition, an analysis of the factors that interact with the CPE is presented. 16. •
VAmqUMSM, WORMINGTON MW: Deadenylation of Maternal mRNAs During Xe~opus Oocyte Maturation does not Require Specific cis S e q u e n c e s : a Default Mechanism for Translational Control. Genes Dev 1990, 12b:2278-2286. This paper demonstrates that deadenylarion during oocyte maturation occurs via a default pathway. 17. •
FOX CA, WICKENSM: Poly(A) Removal During Oocyte Maturation: a Default Reaction Selectively Prevented by Specific Sequences in t h e 3' UTR of Certain Maternal mRNAs. Genes Dev 1990, 12b:2287-2298. An analysis of cytoplasmic deadenylarion in injected oocytes. 18.
GURDONJB: Nuclear Translation and the Control of Gene Activity in Animal Development. Proc R Soc Lond [Biol] 1970, 176:303-314.
19.
MATFAJ IW, DEROBERTIS EM: Nuclear Segregation of U2 snRNA Requires Binding of Specific snRNP Proteins. Cell 1985, 40:111-118.
HAMM J, DARZYNKIEWlCZ E, TAHARA SM, MATFAJ IW: T h e Trimethylguanosine Cap Structure of U1 snRNA is a Comp o n e n t of Bipartite Nuclear Targeting Signal. Cell 1990, 62:569-577. injected oocytes are used as an assay system to show that the 5' terminal cap is an important component of the U snRNP nuclear localization
apparatus. This study also suggests that nuclear import mechanisms are more diverse than previously thought. 21. ..
FISCHERU, LUHRMANNR: An Essential Signaling Role for the m3G Cap in the Transport of U1 snRNP to t h e Nucleus. Science 1990, 249:786-789. Injected Xenopus oocytes are used to s h o w that the cap structure unique to U snRNAs comprises a portion of a nuclear localization signal. 22.
GOLDFARBDS, GARIEPYJ, SCHOOLNIK G, KORNBERG RD: Synthetic Peptides as Nuclear Localization Signals. Nature 1986, 322:641-444.
23. •
MICHAUD N, GOLDFARB DS: Multiple Pathways in Nuclear Transport: t h e import of U2 snRNP Occurs by a Novel Kinetic Pathway. J Cell Biol 1991, 112:215-223. Injection of various substances that signal nuclear localization sequences into Xenopus oocytes is used to show that nuclear import of specific proteins and U2 snRNP probably require different nuclear pore receptors. 24.
FISCHER U, DARYZNKIEWICZ E, TAHARA SM, DATHAN NA, LUHRMANNR, MATFAJIW: Diversity in the Signals Required for Nuclear Accumulation of U snRNPs and Variety in t h e Pathways of Nuclear Transport. J Cell Biol 1991, 113:705-714. Using injected oocytes, these investigators show that wheat germ agglutinin inhibits nuclear import of U6 snRNP but not of U1 or U5 snRNPs. This underscores the remarkable complexity of the nuclear localization apparatus. •
25. •
CHRISTIEMJ, NORTH RA, OSBORNE PB, DOUGLASSJ, ADELMANJP: Heteropolymeric Potassium Channels Expressed in X e n ~ p u s Oocytes from Cloned Subunits. Neuron 1990, 2:405-411. An attempt to detemaine whether heteromulrimeric channels are formed when pure mRNAs encoding three different K + channel subunits are injected into oocytes. The paper demonstrates that channels with different sensitivities to tetraethylammonium are produced if combinations of mRNAs are injected, but not w h e n a single mRNA is used. It is suggested that these different sensitivities are the result of the formarion of heteromulrimeric channels. 26. •
ISACOFFEY, JAN YN, JAN LY: Evidence for the Formation of Heteromultimeric Potassium Channels in Xenopus Oocytes. Nature 1990, 345:530-534. Messenger RNAs encoding different K + channel subunits were injected into oocytes. Because the electrical properties of the channels resulting from mixed mRNA injections differed from those obtained with uniform mRNA injections, it is suggested that these must have resulted from the formation of a heteromulrimeric chatmel. In addition, the formation of a functional channel composed of subunits from rat and Drosophila is shown. 27.
RUPPERSBERGJP, SCHROTER KH, SAKMANN B, STOCKER M, SEWINGS, PONGS O: Heteromultimeric Channels Formed by Rat Brain Potassium-Channel Proteins. Nature 1990, 345:535-537. After showing the formation of a heteromulrimeric K + channel in mRNA-injected oocytes, these authors suggest that this could be the basis for the functional diversity of K + channels in the central nervous system. •
28. •.
FROeHNeRSC, LUETJECW, SCOTLANDPB, PATRICKJ: The Postsynaptic 43K Protein Clusters Muscle Nicotinic Acetylcholine Receptors in Xenopus Oocytes. Neuron 1990, 5:403-410. Using mRNA-injected oocytes, Froehner and colleagues demonstrate that a 43 kD protein is responsible for the clustering of AChRs. 29.
Fu L, YE R, BROWDER LW, JOHNSTON RN: Translational Potentiation of Messenger RNA w i t h Secondary Structure in Xenopus~ Science 1991, 251:807-810.
30.
BATAILLEN, HELSER T, FRIED HM: Cytoplasmic Transport of Ribosomal Subunits Microinjected into t h e Xenopus laevis Oocyte Nucleus: a Generalized, Facilitated Process. J Cell Biol 1990, 111:1571-1582.
31.
GUDATU, BAKKEN AH, PmLER T: Protein-Mediated Nuclear Export of RN& 5S rRNA Containing Small RNPs in Xenopus Oocytes. Cell 1990, 60:619-628.
20. ••
Gene expression using Xenopusoocytes Richter 32.
BROWNBD, HARLANDRM: Endonucleolytic Cleavage of a Maternal H o m e o Box mRNA in Xenopus Oocytes. Genes Dev 1990, 4:1925-1935.
Injected oocytes are used to identify a region of the Drosophila shaker K + channel that might be involved in the formation of the ion conducting pore.
33.
CEPaOTrlA, COLMAN& Trimer Formation Determines t h e Rate of Influenza Virus Haemagglutinin Transport in t h e Early Stages of Secretion in Xenopus Oocytes. J Cell Biol 1990, 111:409-420.
37.
KORENG, LIMAN ER, LOGOTHETIS DE, NADAL-GINARDB, HESS P: Gating Mechanism of a Cloned Potassium Channel Expressed in Frog Oocytes and Mammalian Cells. Neuron 1990, 2:39-51.
34.
SIMONR, RiCHTER JD: The Degradation Sequence of Ade-
38.
PHIL1PSONLH, HICE RE, SCHAEFER K, LAMENDOLAJ, BELL GI, NELSON DJ, STEINER DF: Sequence and Functional Expression in Xenopus Oocytes of a H u m a n Insulinoma and Islet Potassium Channel. Proc Natl Acad Sci USA 1991, 88:53-57.
39.
HARTMANNHA, KIRSCH GE, De,EWE JA, TAGLIALATELAM, JOHO RH, BROWNAM: Exchange of C o n d u c t i o n Pathways Between Two Related K + Channels. Science 1991, 251:942-944.
40.
RASSENDRENF-A, LORYP, PIN J-p, NARGEOTJ: Zinc has Opposite Effects on NMDA and non-NMDA Receptors Expressed in Xenopus Oocytcs. Neuron 1990, 4:733-740.
41.
SAEDIMS, CONROY WG, taNDSTROMJ: Assembly of Torpedo Acetylcholine Receptors in Xenopus Oocytes. J Cell Biol 1991, 112:1007-1015.
novirus E1A Consists of the Amino-Terminal Tetrapeptide Met-Arg-His-lle. Mol Cell Biol 1990, 10:5609-5615. 35. ..
PAPAZIANDM, TIMPE LC, JAN YN, JAN LY: Alteration of VoltageD e p e n d e n c e of Shaker Potassium Channel by Mutation in t h e $4 Sequence. Nature 1991, 349:305-310. A sequence called $4, which is conserved among several voltagedependent ion channels (K +, Ca 2+, Na+), has been proposed as a voltage sensor. To determine whether this could be the case, K + channel mRNAs containing mutations in the $4 sequence were constructed and injected into oocytes. Analysis of the electrical properties of the resulting channels suggests that this sequence is indeed important for voltage-dependent activation but not for ion selectivity. 36. •
YELLENG, JURMAN ME, ABRAMSONT, MACKINNON R: Mutations Affecting Internal TEA Blockade Identify the Probable Pore-Forming Region of a K + Channel. Science 1991, 251:939-942.
JD Richter, Worcester Foundation for Experimental Biology, 222 Maple Avenue, Shrewsbury, Massachusetts 01545, US&
703
Xenopus oocytes have been used as a surrogate genetic system for the study of many facets of gene expression. Some recent salient examples where injected oocytes proved to be indispensable for the analysis of transcription, translation, RNA/protein transport, and ion channel formation are described. Current Opinion in Biotechnology 1991, 2:698-703
Introduction In 1971, Gurdon and colleagues published a paper [1] that would have major ramifications for the fields of molecular, cellular, and developmental biology and, more recently, the neurosciences as well. In those few pages, they described experiments that showed that Xenopus laevis oocytes would translate injected rabbit globin mRN& This demonstrated not only a lack of species specificity at the translational level, but also suggested that oocytes could be used to examine the c/selements in mRNA that regulate protein synthesis. Moreover, this idea that oocytes could be used as a surrogate genetic system, was taken up by others who showed that oocytes would also transcribe injected DNA [2-4], utilize injected membranes for translation at the rough endoplasmic reticulum [5] and correctly process and secrete heterologous proteins into the surrounding medium [6]. Thus, the injected Xenopus oocyte, an easily manipulated in vivo sys tern, could be used as a bioassay for virtually all aspects of eukaryotic gene expression. This review will discuss some recent examples where injected Xenopus oocytes have been particularly useful for the study of gene expression.
amino acids, or by the enzymatic activity of the protein. An example of the latter assay is illustrated in Fig. 1. Oocyte nuclei were injected with 0.1 ng of supercoiled plasmid DNA encoding the chloramphenicol acetyltransferase (CAT) gene that was driven by the adenovirus E3 promoter. Adenovirus EIA, which normally activates the E3 promoter following infection of mammalian cells by the virus, was expressed in Escherichia coli and injected into the cytoplasm of some of these oocytes. Oocytes receiving EIA in addition to DNA exhibited CAT levels about eight-fold greater than those injected with DNA alone. Thus, oocytes contain all the machinery necessary for EIA to enter the nucleus and stimulate transcription (see below).
Injected Xenopus oocytes as a surrogate genetic system Table 1 lists some recent examples where the injected Xenopus oocyte has been used as a surrogate in vivo genetic system. This catalog is not meant to be exhaustive, but rather to point out the different facets of gene expression that have been studied using Xenopua oocytes.
The oocyte as an in vivo assay system
Transcriptional control
Owing to its large size (1.2-1.4 mm in diameter), the fully grown Xenopus oocyte (stage VI according to the classification scheme of Dumont [7]) is a rather facile biochemical tool. Up to 100 nl of solution containing RNA (at an initial concentration of about I mg/ml) may be injected into the cytoplasm. In addition, the nucleus may be injected with 5-10 nl of solution containing DNA (at an initial concentration of about 0.1 mg/ml). Following incubation, the protein products derived from these injected molecules may be assessed in a number of different ways, such as by the incorporation of radiolabeled
As stated above, adenovims EIA is a potent activator of transcription of viral, and in some cases, cellular genes. However, E1A does not bind specific promoter sequences, which implies that cellular factors might mediate EIA-induced transcription. Indeed, just such a role has been suggested for the cellular protein ATF [8]. In Xenopus oocytes, gel retardation and DNA injection studies demonstrated that a factor required for E1A-induced transcription was present and bound the ATF-recognition sequence [9]. To estimate the size of the protein, a 36 nucleotide single-stranded oligonucleotide spanning the ATF-recognition sequence was used as a template
Abbreviations AChR--acetylcholine receptor; CAT--chloramphenicol acetyltransferase; CPE~cytoplasmic polyadenylation element; IL-2--interleukin 2; NLSs--nuclear localization signals;U snRNPs--uridine rich small nuclear ribonuclease particles; U snRNAs---uridine rich small nuclear RNAs.
698
(~) Current Biology Ltd ISSN 0958-1669
Gene expression using Xenopusoocytes Richter
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(b) % Chloramphenicol acetylated
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.•
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E3/CAT
E3/CAT and E1A
Fig. 1. Analysis of E1A-induced transcription in injected Xenopus oocytes. (a) Depicts the injection of plasmid DNA encoding bacterial chloramphenicol acetyltransferase (CAT), which is under the control of the adenovirus E3 promoter, into the nucleus and the injection of E. coil-expressed adenovirus EIA protein into the cytoplasm. E1A is then transported to the nucleus where it activates E3/CAT gene transcription. The resulting CAT RNA is polyadenylated, transported to the cytoplasm, and translated into enzymatically active CAT. (b) Shows the relative amounts of CAT activity produced from oocytes injected with E3/CAT DNA only or E3/CAT DNA plus EIA. CAT activity is determined by the percentage of 14C-chloramphenicol that is acetylated when mixed with acetyl coenzyme A and extracts prepared from injected oocytes.
for second strand synthesis by reverse transcriptase. In this case, however, second strand synthesis took place in the presence of 32p-dCTP and the photoactivatable crosslinking reagent BrdUTP. The oligonucleotide was then injected into the oocyte nucleus (sometimes called the germinal vesicle). After incubation the transparent germinal vesicles were manually isolated, irradiated with 302 nm light, and prepared for sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE) and autoradiography. A single protein species of 66 kD was crosslinked to the injected DN& indicating that this protein recognizes the ATF-binding site in vivo. Thus, injected Xenopus oocytes can be used to examine the in vivo interactions of specific DNA sequences and their binding proteins.
ulated primary T-lymphocytes or mitogenically stimulated T-lymphocytes. Using an S-1 nuclease protectionassay for IL-2 transcription, Mouzaki et al. found basal levels of IL-2 RNA in oocytes injected with DNA only. This basal IL-2 transcription, however, was silenced when oocytes were also injected with protein from unstimulated Tlymphocytes. This suggests that unstimulated T-lymphocytes contain a specific IL-2 gene repressor. When these 'repressed' oocytes were injected yet again with protein from stimulated T-lymphocytes, Ib2 transcription increased dramatically. Thus, these investigators suggest that IL-2 transcription in T-lymphocytes is under dual control; repression in unstimulated cells and stimulation in mitogenically activated cells.
Mouzaki et al. [10 °] have also used injected oocytes to study transactivation of the lymphokine interleukin2 (IL-2) gene. IL-2 transcription is normally activated in T-lymphocytes, but only after they have been stimulated by antigens or mitogens. Based on this observation, oocytes were injected with the IL-2 gene and, in some instances, with protein isolated from either unstim-
Translational control Although several older investigations used injected Xen(> pus oocytes to examine translational control, the recent advent of bacteriophage RNA polymerase (SP6, T7 and T3) to synthesize pure mRNA in vitro has allowed manipulation of the sequences of the injected mRNA in ways that hitherto had not been possible. For example, con-
699
700
Expressionsystems Table 1. Analysis of gene expression using Xenopus oocytes.
Process studied I Transcriptional control (a) analysis of EIA transactivation (b) factors required for IL-2 expression RNA processing/translational control (a) cis elements responsible for cytoplasmic polyadeny]ation (b) RNA deadenylation (c) RNA secondary structure RNA/RNP transport/degradation (a) snRNP nuclear transport (b) transport of ribosomal subunits (c) transport of 5S rRNA (d) RNA degradation signals IV Protein processing/degradation (a) processing of influenza haemagglutin (b) degradation signal of adenovirus E1A v Cloning and analysis of membrane proteins (a) potassium channel (b) N-methyI-D-aspartate channel (c) assembly of acetylcholine receptor (d) clustering of acetylchoine receptor
References
cific c,Xelements were required for deadenylation, rather, it appeared to occur along a default pathway. That is, RNAs were either polyadenylated during maturation under the direction of a CPE, or, if they contained no CPE, were deadenylated by default [16",17"].
[9] [10]
[12,13,14",15"'] [16",17"] [29]
[20",21",22,23"] [30] [31] [32]
[33] [34]
[25",26",27"35'',36",37-39] [4O] [411 [28..]
sider the relationship between cytoplasmic polyadenylation and translational control. Oocytes normally contain several mRNAs that are translationally dormant, but their entry into polysomes occurs when their poly(A) tails are elongated during oocyte maturation, the point when these cells re-enter meiosis in preparation for fertilization (for review see Richter [ 11 ] ). To examine the regulation of polyadenytation, and hence translation, radiolabeled RNA (synthesized in vitro by bacteriophage RNA polymerases in the presence of radioactive nucleotide) was injected into oocytes. These oocytes were then induced to mature with progesterone, the natural stimulus of maturation. An analysis of the RNA following its extraction and resolution by gel electrophoresis and autoradiography revealed that it was polyadenylated in a similar manner to its endogenous counterpart. A further set of injection experiments with RNAs containing various mutant sequences in their 3' untranslated regions revealed that two cis elements controlled cytoplasmic polyadenylation: the near-ubiquitous nuclear cleavage/polyadenylation sequence AAUAAA and an upstream uracil-rich element termed the cytoplasmic polyadenylation element (CPE) [ 12,13,14--,15..]. Moreover, polyadenylation was shown to control translation because mutations in the cis elements that abrogated polyadenylation also prevented translation [12,15.-]. In contrast to those mRNAs that are translated only when they undergo poly(A) elongation during maturation, other mRNAs that are normally translated in oocytes are deadenylated during this time and concomitantly leave polysomes. To delineate the c~ signals that control deadenylation, radiolabeled RNA injection experiments similar to those described for poly(A) elongation were performed. The results showed that surprisIngly, no spe-
Oocytes as a model system to study protein and snRNP transport Since 1970, injected Xenopus oocytes have been used to study protein translocation across the nuclear membrane [18]. Partly because of work using oocytes, it is now clear that proteins contain discrete sequences, called nuclear localization signals (NLSs), that are essential for their facilitated import into the nucleus. However, recent studies using injected oocytes to study nuclear import of U-snRNPs (uridine rich small nuclear ribonucleoprotein particles) suggest that the process of nuclear localization is more complex than previously thought. For example, although U-snRNAs are synthesized in the nucleus, they form complexes with proteins in the cytoplasm, and then the complexes enter the nucleus. Experiments using injected Xenopus oocytes have shown that RNA/protein complex formation is a prerequisite for nuclear localization because neither the U snRNAs nor the snRNP proteins alone enter the nucleus [19]. It now appears that one critical determinant for snRNP nuclear import is the 5' terminal trimethylguanosine cap present on most U snRNAs. Thus, radiolabeled U1 snRNA (synthesized in vitro) injected into the oocyte cytoplasm is transported to the nucleus when it contains the normal trimethylguanosine cap, but not when it is either devoid of the cap or contains a chemical derivative of the cap [20".,21.-]. Receptors associated with nuclear pores appear to be the means by which proteins enter the nucleus. This was inferred from oocyte injection experiments in which NLS-bovine serum albumin conjugates competed with each other for nuclear entry [22]. Recent oocyte injection experiments [23"], however, show that although the NI£s from SV40 large T-antigen and nucleoplasmin compete with each other for nuclear entry, they do not compete with U2 snRNP, which suggests that at least this snRNP might utilize unique nuclear pore receptors. In the same vein, wheat germ agglutinin, which has been used to distinguish different pathways for nuclear import, prevents nuclear localization of most proteins with a 'conventional NLS', but has no effect on the nuclear import of U1 and U5 snRNPs [24.]. Thus, injected oocytes have proven to be indispensable for the study of protein and snRNP nuclear tralficking.
cDNA cloning on the basis of protein function: the injected oocyte as an assay system One of the major problems in cell and molecular biology is the isolation of cDNA clones coding for specific pro-
Gene expression using Xenopus oocytes Richter 701 teins of interest. Once a cDNA clone has been isolated the investigator may derive the primary sequence of the protein, make inferences as to the nature of its three dimensional structure, and overexpress the protein to obtain sufl]cient quantities for biochemical analysis. However, the isolation of a desired cDNA d o n e usually requires some knowledge of the protein, such as a partial amino acid sequence, or an antibody to the protein. Often one only knows the function of the protein, which precludes the use of limited sequence information or antibodies. Fortunately, the injected Xenopus ooc~e has proven very useful for screening mRNAs (to be used to synthesize cDNA clones) on the basis of the function of the encoded protein. This has been exploited with great success in studies on the nature of ion channel proteins. For example, oocytes injected with mRNA derived from a tissue expressing relatively high amounts of ion channel protein more often than not synthesize and position the encoded protein in the membrane so that it may be detected by the very sensitive assays of voltage clamping or ligand binding. After mRNA purification and cDNA synthesis, the desired clone is obtained. Recently, studies of channel function have also used injected oocytes. All voltage-dependent potassium channels, for example, do not exhibit identical kinetics or pharmacologic responses to drugs. Because these channels are composed of several subunits, it has been suggested that formation of heteromultimeric channels might give rise to unique characteristics not seen in homomultimeric channels. Three recent studies [25°,26.,27 °] have used injected oocytes to assess whether this is the case. cDNA clones for various K + channel subunits from rat brain or Drosophila were transcribed in vitrowith bacteriophage RNA polymerases and injected into oocytes. Following incubation, the oocytes were voltage-clamped and their K + currents were measured. Because channels composed of identical subunits (homomultimer) would have unique K + currents depending upon the voltage at which the oocyte was clamped, altered K + currents could result from the formation of channels formed from different subunits (heteromultimer) [26°]. In addition, the sensitivity of the channels to blocking agents such as dendrotoxin and tetraethylammonium would also be different in channels composed of identical versus non-identical subunits [25"]. The aggregate data [25",26,,27.] indicate that indeed heteromultimeric K + channels are formed in injected oocytes, and it has been suggested that this could be the basis for the diversity of channel behaviour observed in different tissues. The oocyte not only inserts, usually correctly, channel and other cell surface proteins into its plasma membrane, but it can also cluster those proteins in response to a specific stimulus. Consider, for example, the case of the acetylcholine receptor (AChR). This receptor is an integral membrane protein normally clustered in the postsynaptic membrane of the neuromuscular junction. In theory the receptor should be able to diffuse within the plane of the membrane, but it is actively aggregated and prevented from diffusing, seemingly by an associated
peripheral membrane protein with a molecular weight of 43 kD. Froehner and colleagues [28 .°] examined the function of this 43 kD protein in oocytes. Following injection of mRNA for the AChR into oocytes, AChR protein (detected by its binding to bungarotoxin) was uniformly distributed over the surface of the oocyte. When mRNA encoding the 43 kid protein was co-injected with AChR mRNA, however, AChR formed discrete clusters reminiscent of those found in the postsynaptic membrane. Therefore, because the 43 kD protein induces AChR clustering in oocytes, one would infer that it almost certainly performs the same function in the postsynaptic membrane.
Conclusions We have discussed some recent examples where Xen~ pus oocytes have been used to examine gene expression. Investigators in many fields have taken advantage of their ability to correctly transcribe genes, translate mRNAs, and process proteins [29-34]. Oocytes have also been used to study negative regulation of gene expression [10o], and the functions of particular sequences of a protein [35°°,36,]. Similarly, the fact that AChRs expressed in oocytes were not clustered, led to the establishment of the 43 kD protein as a clustering factor. Thus, the Xenopus oocyte, once the exclusive domain of the developmental biologist, has now become a widely used laboratory tool.
References and recommended reading Papers of special interest, published within the annual period of review, have been highlighted as: . of interest ** of outstanding interest 1.
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MERTZJ, GURIX)NJB: Purified DNAs are Transcribed After Microinjection into Xenopus Oocytes. Proc Natl Acad Sci USA 1977, 74:1502-1506.
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DEROBERTISEM, MERTZ J: Coupled Transcription-Translation of DNA Injected into Xenopus Oocytes. Cell 1977, 12:175-182.
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RICHTERJD, SMITH LD: Differential Capacity for Translation and Lack of Competition Between mRNAs that Segregate to Free and Membrane-Bound Polysomes. Cell 1981, 27:183-192.
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DUMONTJ: Oogenesis in Xenopus laevis (Daudin). I. Stages of Oocyte Development in Laboratory Maintained Animals. J Morph 1972, 136:153-180.
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LEE KAW, HAl T-Y, SIVA RAMAN L, THIMMAPPAYAB, HURST HC, JONES NC, GREEN MR: A Cellular Protein, Activating Transcription Factor, Activates Transcription of Multiple E1A-Inducible Adenovirus Early Promoters. Proc Natl Acad Sci USA 1987, 84:8355-8359. RICHTER JD: In Vivo Photocrosslinking Reveals that Transcription Factor Binding to t h e Mammalian ATF Recognition Sequence is Required for E1A-Induced Transactivation in Injected Xenopus Oocytes. Nucl Acids Res 1989, 17:4503-4516.
10. •
MOUZAK1A, WEIL R, MUSTER L, RUNGGER D: Silencing and tran~Activation of t h e Mouse IL-2 Gene in Xenopus Oocytes by Proteins from Resting and Mitogen-Induced Primary T-Lymphocytes. EMBO J 1991, 10:139°/1406. injected oocytes are used to show that protein factors isolated from Tlymphocytes can both repress and activate interleukin-2 transcription. 11.
RICHTERJD: Translational Control During Early Development. Bioessays 1991, 13:179~183.
12.
MCGREW LL, DWORKIN-RASTL E, DWORKIN MB, RICHTER JD: Poly(A) Elongation During X e n o p u s Oocyte Maturation is Required for Translational R e c r u i t m e n t and is Mediated by a Short Sequence Element. Genes Dev 1989, 3:803-815.
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Fox CA~ SHEETS MD, WICKENS MP: Poly(A) Addition During Maturation of Frog Oocytes: Distinct Nuclear and Cytoplasmic Activities and Regulation by t h e Sequence UUUUUAU. Genes Dev 1989, 3:2151-2162.
14. ••
MCGREWLL, RICHTERJD: Translational Control by Cytoplasmic Polyadenylation During Xenopus Oocyte Maturation: Characterization of a s and trans Elements and Regulation of Cyclin/MPF. EMBO J 1990, 9:3743-3751. This study uses injected oocytes to define, in detail, the cytoplasmic polyadenytation element. In addition, it shows that injected cydin mRNA and maturation promoting factor both stimulate polyadenylation. 15. ••
PARISJ, RICHTERJD: Maturation-Specific Polyadenylation and Translational Control: Diversity of Cytoplasmic Polyadenylation Elements, Influence o f Poly(A) Tail Size, and Formation of Stable Polyadenyfation Complexes. Mol Cell Biol 1990, 10:5634-5645. This study shows that cytoplasmic polyadenylation is required for the translation of certain RNAs and that different cytoplasmic polyadenylation elements confer unique poly(A) tail lengths. In addition, an analysis of the factors that interact with the CPE is presented. 16. •
VAmqUMSM, WORMINGTON MW: Deadenylation of Maternal mRNAs During Xe~opus Oocyte Maturation does not Require Specific cis S e q u e n c e s : a Default Mechanism for Translational Control. Genes Dev 1990, 12b:2278-2286. This paper demonstrates that deadenylarion during oocyte maturation occurs via a default pathway. 17. •
FOX CA, WICKENSM: Poly(A) Removal During Oocyte Maturation: a Default Reaction Selectively Prevented by Specific Sequences in t h e 3' UTR of Certain Maternal mRNAs. Genes Dev 1990, 12b:2287-2298. An analysis of cytoplasmic deadenylarion in injected oocytes. 18.
GURDONJB: Nuclear Translation and the Control of Gene Activity in Animal Development. Proc R Soc Lond [Biol] 1970, 176:303-314.
19.
MATFAJ IW, DEROBERTIS EM: Nuclear Segregation of U2 snRNA Requires Binding of Specific snRNP Proteins. Cell 1985, 40:111-118.
HAMM J, DARZYNKIEWlCZ E, TAHARA SM, MATFAJ IW: T h e Trimethylguanosine Cap Structure of U1 snRNA is a Comp o n e n t of Bipartite Nuclear Targeting Signal. Cell 1990, 62:569-577. injected oocytes are used as an assay system to show that the 5' terminal cap is an important component of the U snRNP nuclear localization
apparatus. This study also suggests that nuclear import mechanisms are more diverse than previously thought. 21. ..
FISCHERU, LUHRMANNR: An Essential Signaling Role for the m3G Cap in the Transport of U1 snRNP to t h e Nucleus. Science 1990, 249:786-789. Injected Xenopus oocytes are used to s h o w that the cap structure unique to U snRNAs comprises a portion of a nuclear localization signal. 22.
GOLDFARBDS, GARIEPYJ, SCHOOLNIK G, KORNBERG RD: Synthetic Peptides as Nuclear Localization Signals. Nature 1986, 322:641-444.
23. •
MICHAUD N, GOLDFARB DS: Multiple Pathways in Nuclear Transport: t h e import of U2 snRNP Occurs by a Novel Kinetic Pathway. J Cell Biol 1991, 112:215-223. Injection of various substances that signal nuclear localization sequences into Xenopus oocytes is used to show that nuclear import of specific proteins and U2 snRNP probably require different nuclear pore receptors. 24.
FISCHER U, DARYZNKIEWICZ E, TAHARA SM, DATHAN NA, LUHRMANNR, MATFAJIW: Diversity in the Signals Required for Nuclear Accumulation of U snRNPs and Variety in t h e Pathways of Nuclear Transport. J Cell Biol 1991, 113:705-714. Using injected oocytes, these investigators show that wheat germ agglutinin inhibits nuclear import of U6 snRNP but not of U1 or U5 snRNPs. This underscores the remarkable complexity of the nuclear localization apparatus. •
25. •
CHRISTIEMJ, NORTH RA, OSBORNE PB, DOUGLASSJ, ADELMANJP: Heteropolymeric Potassium Channels Expressed in X e n ~ p u s Oocytes from Cloned Subunits. Neuron 1990, 2:405-411. An attempt to detemaine whether heteromulrimeric channels are formed when pure mRNAs encoding three different K + channel subunits are injected into oocytes. The paper demonstrates that channels with different sensitivities to tetraethylammonium are produced if combinations of mRNAs are injected, but not w h e n a single mRNA is used. It is suggested that these different sensitivities are the result of the formarion of heteromulrimeric channels. 26. •
ISACOFFEY, JAN YN, JAN LY: Evidence for the Formation of Heteromultimeric Potassium Channels in Xenopus Oocytes. Nature 1990, 345:530-534. Messenger RNAs encoding different K + channel subunits were injected into oocytes. Because the electrical properties of the channels resulting from mixed mRNA injections differed from those obtained with uniform mRNA injections, it is suggested that these must have resulted from the formation of a heteromulrimeric chatmel. In addition, the formation of a functional channel composed of subunits from rat and Drosophila is shown. 27.
RUPPERSBERGJP, SCHROTER KH, SAKMANN B, STOCKER M, SEWINGS, PONGS O: Heteromultimeric Channels Formed by Rat Brain Potassium-Channel Proteins. Nature 1990, 345:535-537. After showing the formation of a heteromulrimeric K + channel in mRNA-injected oocytes, these authors suggest that this could be the basis for the functional diversity of K + channels in the central nervous system. •
28. •.
FROeHNeRSC, LUETJECW, SCOTLANDPB, PATRICKJ: The Postsynaptic 43K Protein Clusters Muscle Nicotinic Acetylcholine Receptors in Xenopus Oocytes. Neuron 1990, 5:403-410. Using mRNA-injected oocytes, Froehner and colleagues demonstrate that a 43 kD protein is responsible for the clustering of AChRs. 29.
Fu L, YE R, BROWDER LW, JOHNSTON RN: Translational Potentiation of Messenger RNA w i t h Secondary Structure in Xenopus~ Science 1991, 251:807-810.
30.
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31.
GUDATU, BAKKEN AH, PmLER T: Protein-Mediated Nuclear Export of RN& 5S rRNA Containing Small RNPs in Xenopus Oocytes. Cell 1990, 60:619-628.
20. ••
Gene expression using Xenopusoocytes Richter 32.
BROWNBD, HARLANDRM: Endonucleolytic Cleavage of a Maternal H o m e o Box mRNA in Xenopus Oocytes. Genes Dev 1990, 4:1925-1935.
Injected oocytes are used to identify a region of the Drosophila shaker K + channel that might be involved in the formation of the ion conducting pore.
33.
CEPaOTrlA, COLMAN& Trimer Formation Determines t h e Rate of Influenza Virus Haemagglutinin Transport in t h e Early Stages of Secretion in Xenopus Oocytes. J Cell Biol 1990, 111:409-420.
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PHIL1PSONLH, HICE RE, SCHAEFER K, LAMENDOLAJ, BELL GI, NELSON DJ, STEINER DF: Sequence and Functional Expression in Xenopus Oocytes of a H u m a n Insulinoma and Islet Potassium Channel. Proc Natl Acad Sci USA 1991, 88:53-57.
39.
HARTMANNHA, KIRSCH GE, De,EWE JA, TAGLIALATELAM, JOHO RH, BROWNAM: Exchange of C o n d u c t i o n Pathways Between Two Related K + Channels. Science 1991, 251:942-944.
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SAEDIMS, CONROY WG, taNDSTROMJ: Assembly of Torpedo Acetylcholine Receptors in Xenopus Oocytes. J Cell Biol 1991, 112:1007-1015.
novirus E1A Consists of the Amino-Terminal Tetrapeptide Met-Arg-His-lle. Mol Cell Biol 1990, 10:5609-5615. 35. ..
PAPAZIANDM, TIMPE LC, JAN YN, JAN LY: Alteration of VoltageD e p e n d e n c e of Shaker Potassium Channel by Mutation in t h e $4 Sequence. Nature 1991, 349:305-310. A sequence called $4, which is conserved among several voltagedependent ion channels (K +, Ca 2+, Na+), has been proposed as a voltage sensor. To determine whether this could be the case, K + channel mRNAs containing mutations in the $4 sequence were constructed and injected into oocytes. Analysis of the electrical properties of the resulting channels suggests that this sequence is indeed important for voltage-dependent activation but not for ion selectivity. 36. •
YELLENG, JURMAN ME, ABRAMSONT, MACKINNON R: Mutations Affecting Internal TEA Blockade Identify the Probable Pore-Forming Region of a K + Channel. Science 1991, 251:939-942.
JD Richter, Worcester Foundation for Experimental Biology, 222 Maple Avenue, Shrewsbury, Massachusetts 01545, US&
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