ANALYTICAL

BIOCHEMISTRY

188,1%&l%?

(1990)

pOEV: A Xenopus Oocyte Protein Expression Vector Samuel L. Pfaff, Michael Department

Received

of Molecular

December

M. Tamkun,

Physiology

and William

and Biophysics,

L. Taylor

School of Medicine,

Vanderbilt

University,

Nashville,

Tennessee

37232

13,1989

We have constructed a Xenopus laevis oocyte expression vector, pOEV, which allows cloned DNA to be transcribed and translated directly in the oocyte. Since proteins translated in oocytes are post-translationally modified according to conserved eukaryotic signals, these cells offer a convenient system for performing structural and functional analyses of cloned genes. pOEV can be used for direct analysis of proteins encoded by cloned cDNAs without preparing mRNA in vitro, simplifying existing protocols for translating proteins in oocytes with a very high translational yield. Transcription of the vector in oocytes is driven by the promoter for the TFIIIA gene, which can generate l-2 ng (per oocyte within 2 days) of stable mRNA template for translation. The vector also contains SP6 and T7 promoters for in vitro transcription to make mRNA and hybridization probes. DNA clones encoding chloramphenicol acetyltransferase (CAT) were injected into oocyte germinal vesicles and CAT protein accumulated in the cell over a 2- to 4-day period. We found that the concentration of DNA injected affected protein yields; surprisingly relatively low concentrations in the range 25-50 pg DNA per oocyte gave maximum yields of CAT protein. When as little as 5 pg of pOEV DNA is injected we typically expressed 40 fmol of CAT protein per oocyte, after 4-day incubations. In addition, we have shown that this system is amenable to the expression of o 1990 Academic PAM, k. nuclear and membrane proteins.

The Xenopus Levis oocyte has become a popular vehicle for translating mRNA from a variety of sources ((l), reviewed in Ref. (2)). Not only do oocytes have the enormous endogenous translational capacity of 17 ng protein/h (3), but also this cell type offers a suitable background for studying many different proteins. Structural and functional analyses of proteins expressed in oocytes have been facilitated by the competence of oocytes to carry out post-translational processes such as proteolytic modifications, glycosylation, acetylation, subunit assembly, transport, and compartmentalization (2).

Oocytes have been used to study proteins normally expressed at very low levels in cells by injecting large samples of total cellular RNA and using sensitive assays to functionally detect expression. This approach has made it possible to study proteins without extensive purification, and for which antibody and nucleic acid probes are not available (for example, see Refs. (4-6)). Oocytes have been demonstrated to be capable of translating RNA transcribed in vitro (7), which has been a useful tool for expressing cDNA clones (8-10). In addition, cDNA clones have been isolated using sensitive assays to detect protein expression in oocytes; however, this screening technique is very tedious (11-15). In addition to the large translational capacity of oocytes, these cells are rich in transcriptional components as well, for example stage VI oocytes transcribe 2 ng poly(A) RNA per day (16). DNA injected into the germinal vesicle, containing the proper c&acting transcription elements, will be transcribed with high fidelity (reviewed in Ref. (17)). In vivo transcription and translation of DNA was first demonstrated in oocytes by expression of SV4O virion proteins from injected SV40 DNA (18). In theory, in vivo transcription of DNA would simplify existing protocols for expressing exogenous proteins in oocytes from cDNA clones or cDNA libraries because the intermediate step of in vitro transcription before injection would not be necessary. We have constructed an oocyte expression vector, pOEV, which is useful for the overexpression of proteins in oocytes. The pOEV vector is designed so that transcription of an inserted gene is driven by the promoter for the TFIIIA gene. The TFIIIA promoter is a natural choice for such a vector because the endogenous single copy gene is expressed at high levels in oocytes, producing 106-lo7 mRNA copies/oocyte (19). In addition, the positive elements controlling RNA polymerase II transcription of the TFIIIA gene have been well characterized (20-22). These studies defined the necessary promoter sequences for maximal vector transcription. The site of insertion of the gene to be expressed in pOEV is a polylinker which is adjacent to a SV40 small T antigen

192 All

Copyright 0 1990 rights of reproduction

0003~2697/90 $3.00 by Academic Press, Inc. in any form reserved.

CONSTRUCTION

splice site and a poly(A) addition plifies existing cDNA expression high translational yield.

MATERIALS

AND

METHODS

OF

Xenopus

OOCYTE

site. This vector simprotocols and gives a

PROTEIN

EXPRESSION

VECTOR

193

stock (1). DNA samples were injected in a 5-nl volume into the germinal vesicle and RNA samples were injected in a 40-nl volume into the cytoplasm, with a calibrated drawn capillary tube. Oocytes were not centrifuged prior to injection, since centrifugation is not necessary to inject a high percentage of germinal vesicles. Injection volumes were controlled with a Picospritzer II (General Valve Corp.). Typically 100 stage VI oocytes were injected with each sample and incubated at 18°C in incubation buffer (5 mM Hepes, pH 7.8, 82.5 mM NaCl, 2.5 mM KCl, 1 mM CaCl,, 1 mM MgCl*, 10 pg/ml streptomycin, and 10 yg/ml penicillin), and separate pools of five healthy oocytes were collected 1, 2, 3, and 4 days after injection for primer extension and CAT analysis.

Plasmid constructs. pOEV was constructed by inserting a Sac1 to BamHI fragment, from pCATF35 (20) spanning the -325 to +7 region of the TFIIIA promoter into the PuuII site of pGEM-1 (Promega). The Sac1 and BamHI sites were conserved, but the PuuII site was destroyed. An SV40 intron and poly(A) addition site were added to the pGEM-l/TFIIIA intermediate plasmid by Primer extension. After injection, pooled oocytes replacing the SphI-NarI region (base pairs 2433-2579 in Promega sequence) with an 800-bp MspI to KpnI fragwere incubated in lysis buffer (50 mM Tris-HCl, pH 7.8, 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid, ment of pSVl-cat (23). The restriction sites at the junctions between pGEM and SV40 sequences were de- 2% (w/v) sodium dodecyl sulfate, and 0.5 mg/ml proteinstroyed. pOEV is 3900 bp. ase K) for 40 min at room temperature. Lysed oocytes pOEV/CATl was constructed by insertion of the were extracted twice with phenol, twice with phenol/ chloramphenicol acetyltransferase coding sequences, chloroform (l/l), and twice with chloroform and the contained in the 804-bp Sau 3A fragment of pUC13 nucleic acids precipitated with ethanol. For each sample CAT3 (24), into the polylinker-BamHI site of partially 2.5 oocyte equivalents of nucleic acid were resuspended digested pOEV. in 30 ~1 hybridization buffer (10 mM Tris-HCl, pH 8, pOEV/a was constructed by replacing the short 250 mM KCl, and 1 mM ethylenediaminetetraacetic acid) BamHI-BamHI region of pOEV with a 3.6-kb BamHI with 2 X lo5 counts/min 5’-end-labeled oligonucleotide fragment encoding the a-subunit of the (Naf and Kf) (5’dCTCCATTTTAGCTTCCTT) complementary to a ATPase cDNA (25). pOEV/P was constructed by clon- portion of the CAT gene and annealed 1 h at 60°C. The ing a 2.0-kb EcoRI fragment of the P-subunit of the (Na+ annealed sample was cooled, and 80 ~1 of reverse tranand K+) ATPase cDNA (26) into the EcoRI site of scriptase buffer (20 mM Tris-HCl, pH 8.5,lO mM MgC&, pOEV. 10 mM dithiothreitol, 250 PM dCTP, 250 PM dGTP, 250 pOEV/myc was constructed by replacing the EcoRIPM dATP, 250 PM TTP, and 2.5 units AMV reverse Hind111 region of pOEV with the 1.6-kb EcoRI-Hind111 transcriptase [Promega]) was added followed by incubafragment of the murine c-myc cDNA containing the cod- tion at 37°C for 1 h. These samples were phenol exing exons of the gene (27). tracted, ethanol precipitated, and analyzed on a 6% deThe CAT, ATPase, and myc test constructs all con- naturing polyacrylamide gel. tain poly(A) signals provided by the SV40 sequences in CAT assay. At 1,2,3, and 4 days after injection, five the vector. It is possible that the poly(A) signal sequence pooled oocytes were homogenized in 200 ~1 of 250 mM AATAAA would be contained in some cDNA clones con- Tris-HCl, pH 8, and centrifuged 10 min at 4°C in a misidered for use in pOEV. We have not thoroughly tested crofuge (10,OOOg). Typically 10 ~1 of the supernatant the effect of multiple poly(A) signals in the vector; there(0.25 oocyte equivalent) was used in a standard CAT asfore, we recommend subcloning cDNAs into pOEV withsay (23). Purified chloramphenicol acetyltransferase out additional poly(A) signals. (Boehringer-Mannheim) was used to quantify CAT proInjections. Oocytes were isolated from adult female tein levels in oocytes. The purified CAT enzyme has a X. luevis and prepared for injection by standard proce- specific activity of 72,300 units/mg (from Boehringerdures (2,20). DNA samples were stored in TE (10 mM Mannheim); thus the levels of CAT protein in oocytes Tris-HCl, pH 7.8, 1 mM ethylenediaminetetraacetic were extrapolated from standard curves with the puriacid). RNA samples were freshly prepared by standard fied enzyme by doing assays in parallel. in vitro transcription protocols (28). RNA samples were Oocyte nuclei were injected Immune precipitations. resuspended in diethyl pyrocarbonate-treated water. with 50 pg of DNA template followed 12 h later by injecBefore injection samples were adjusted to 15 mM Tristion of 5 PC!1[35S]methionine, in a 20-m volume, into the HCl, pH 7.6, 88 mM NaCl, and 1 mM KC1 with a 10X cytoplasm. In addition, [35S]methionine was added to the incubation buffer, to a final concentration of 30 &i/ ml, at this time. One day after the labeling was initiated, 1 Abbreviation used: CAT, cbloramphenicol acetyltransferase. the oocytes were injected a second time with 5 &i [35S]-

194

PFAFF.

TAMKUN.

methionine and placed in fresh incubation buffer containing 30 pCi/ml [35S]methionine. After a 2- to 3-day labeling period, lo-30 healthy oocytes were pooled for immune precipitations. Oocytes expressing (Na+ and K+) ATPase were homogenized in 1 ml extraction buffer (1% (w/v) Triton X-100, 2 mM benzamidine, 5 mM Nethylmaleimide, 1 mg/ml bacitracin, and 20% (v/v) horse serum) and centrifuged 15 min at 10,OOOg. Chicken specific anti-o- or anti-P-subunit ATPase monoclonal antibody coupled to Sepharose beads was added to the supernatant. Affinity purification and analysis were performed as previously described (2526). Oocytes expressing myc were homogenized in 1 ml lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% (v/v) NonidetP40, and 1 mM phenylmethylsulfonyl fluoride), and centrifuged 5 min at 10,OOOg. The supernatant was adjusted to 0.5% (v/v) Nonidet-P40, 0.5% (w/v) sodium deoxycholate, and 0.1% (w/v) sodium dodecyl sulfate. Antibodies specific for myc were added to the supernatant and incubated overnight at 4°C. Following immune precipitation, samples were prepared by standard procedures and analyzed on sodium dodecyl sulfate-polyacrylamide gels (29).

AND

TAYLOR

mega sequence). Translation of pOEV transcripts must therefore initiate at ATG codons within sequences inserted into this vector. The level of mRNA transcribed from the DNA template pOEV/CAT was quantified over a 3-day period after injection into stage VI oocyte germinal vesicles. Within 1 day after injection the level of mRNA transcribed from pOEV/CAT was 0.8 ng mRNA per oocyte from 500 pg template (Fig. 2B). Over a 2- to 3-day period after injection the level of mRNA increased 87% from the l-day level, up to 1.5 ng mRNA (Fig. 2B). Since the X. laevis oocyte contains a large store of transcriptional components for oogenesis and embryo development, we analyzed the effect of varying the template copy number in the germinal vesicle on mRNA accumulation. In general, the level of mRNA transcribed from pOEV/CAT increased as the level of template in the germinal vesicle increased (Fig. 3A). The maximum level of mRNA was observed at 500 pg pOEV/CAT DNA per oocyte ( lo8 copies), and the mRNA yield was slightly reduced at 2.5-4.15 ng of template per oocyte. Translation of POE V Transcripts

The amount of CAT protein expressed from pOEV/ CAT was determined using a CAT enzyme assay (MatePOE V Transcription rials and Methods and Fig. 4A). We found that the level The positive elements controlling TFIIIA gene ex- of CAT protein accumulated in the oocyte over a 2-4 day pression in stage VI oocytes lie within the sequences period after pOEV/CAT was injected into the germinal -290 to - 1 relative to the transcription initiation site of vesicle of oocytes. CAT activity was detectable after 1 the gene (20-22). pOEV was constructed by inserting the day, and increased 5-lo-fold by 3-4 days after injection -325 to +7 region of the TFIIIA promoter into the when 5 pg pOEV/CAT DNA was injected (Figs. 4A pGEM-1 plasmid (Promega) upstream of the polylinker and B). region (Materials and Methods and Fig. 1). pOEV also In oocytes injected with at least 250 pg DNA the level had an 800-nucleotide region derived from SV40, con- of CAT transcript was high 1 day after injection and retaining a small T antigen intron and polyadenylation mained high over a 2- to 3-day period (see above). We signal sequence (23), cloned downstream of the polyexpected to see a direct correlation between the level of linker region. CAT mRNA in the oocyte and the level of CAT protein For use as a test gene we cloned the CAT coding se- expressed. Surprisingly, a wide range of DNA injections quences into the polylinker of pOEV, resulting in (ranging from 5 to 250 pg DNA per oocyte) gave similar pOEV/CAT (Materials and Methods). The pOEV/CAT total CAT protein levels at 3-4 days postinjection, but DNA was injected into stage VI oocyte germinal vesicles, the kinetics of protein accumulation differed within this range of DNA concentrations (Fig. 3B). In fact, high and total oocyte RNA was prepared 1,2, and 3 days later. An oligonucleotide complementary to CAT sequences DNA template concentrations such as 500 pg per oocyte was used to map the transcription initiation site of expressed less CAT protein than 5 pg template per oomRNA produced from pOEV/CAT by primer extension. cyte (Fig. 3B) despite greater CAT mRNA levels from The primer extension product of pOEV/CAT was 125 higher template concentrations (Fig. 3A). From renucleotides (Fig. 2A), demonstrating that transcription peated experiments we have found that maximum CAT initiated from pOEV/CAT at the same site used by the protein was expressed from 25 to 50 pg of template DNA endogenous TFIIIA gene (initiation site shown in at approximately 3 days postinjection, and this DNA Fig. 1). template concentration was tolerant of five fold variaSince the transcription initiation site from the TFIIIA tions with only modest affects on the total amount of promoter was maintained in pOEV, this vector ex- CAT protein expressed (Fig. 3B). presses transcripts with a 56-nucleotide leader before The absolute level of CAT protein expressed from the polylinker. This leader contains no ATG codons in pOEV/CAT in oocytes was estimated by comparing any reading frame to initiate translation (based on ProCAT enzyme levels to standard curves using purified RESULTS

CONSTRUCTION

OF

Xenopus

OOCYTE

PROTEIN

EXPRESSION

GEM 1 POLYLINKER

TFIIIA PROMOTER

195

VECTOR sv40

TFIIIA

TFIIIA

GGCTACXTG Promoter sac1 -TCGAGCTCGC

I

Transcription Start 17 Start Barn HI EC0 RI GCTTCTACGG GGATCCTGGC TTATCGAAAT TAATACGACT CRCTATAGGG AGACCGGRAT Tl Promoter

sma1

SamHI

CCGGGGRTCC

XbaI

Sal

I

TCTAGAGTCGCTGCAGCC

Psti

Hind111 CAAGCTTGTA

I

SP6 Start

TTCTATAGTG

TCACCTAAAT

SP6 Promoter

FIG. 1. Schematic of pOEV. TFIIIA, T7, and SP6 promoters

Line drawing are marked

of the pOEV vector, with partial sequence information listed. The by arrows, and some of the pertinent restriction sites are overlined.

CAT enzyme (Materials and Methods). In seven different experiments we found a lo-fold range in protein expression from different Xenopus oocytes using the same DNA samples. Yields of 40 fmol of CAT protein per oocyte 4 days after injection with 5 pg DNA were typical (Fig. 4). Variability in the translational capacity of different batches of Xenopus oocytes has been described by others (30) and probably accounts for the variability in CAT protein yields. As noted above, the best yields of CAT protein were achieved when 25-50 pg of DNA template was injected. Comparison of Translation Yield from POE V and in Vitro Prepared RNA The development of in vitro transcription systems using T7 or SP6 RNA polymerase has made it possible to inject large quantities (routinely lo-100 ng) of homogeneous RNA for translation in oocytes. Saturation of the oocyte’s translational capacity varies among transcripts; for example zein RNA reaches saturation at 10 ng RNA, whereas globin RNA does not reach saturation until at least 80 ng RNA has been injected (31). Both natural and synthetic RNA are degraded to varying degrees after injection, and the percentage of the injected sample that remains intact after 24 h declines as higher concentrations of RNA are injected. For example, when 1 ng rabbit globin mRNA was injected 0.5 ng is stable after 24 h, but when 80 ng of rabbit globin mRNA was injected only 1.7 ng was stable after 24 h (31,32). We detect l-2 ng mRNA expressed from 500 pg pOEV DNA l-2 days after injection (see above and Fig. 2B), which is approximately the

transcription

start

sites of the

same range of mRNA that is stable 1 day after injection of l-80 ng globin mRNA. Therefore, we expected protein yields from pOEV and injected mRNA to be nearly the same after a l- to S-day incubation. We confirmed this by comparing CAT protein levels expressed from 2 ng capped poly(A) tailed CAT mRNA and 500 pg pOEV/ CAT DNA (data not shown). Injection of higher levels of capped poly(A) mRNA may increase CAT protein expression, but we did not test this. Depending on the reaction conditions during in vitro transcription, in vitro synthesized RNA may differ from natural RNA in several regards. For example, most natural cellular mRNAs are capped at the 5’-end and polyadenylated at the 3’-end. Analysis of the effect of capping and polyadenylation on RNA stability and translation efficiency has shown that both modifications increase the stability and translation efficiency of RNAs in oocytes, but neither structure is absolutely required for translation of RNA in oocytes (7,32,35,36). RNA transcribed in vitro can be capped by including the m7G(5’)ppp(5’)G cap analog in the reaction mix (33), but we observed a significant reduction in the yield of RNA prepared in vitro when the cap analog was included in the reaction (data not shown). Polyadenylation of the 3’end requires a separate reaction with poly(A) polymerase unless poly(dA/dT) is encoded in the template. However, long poly(dA/dT) tracts in plasmids are unstable and difficult to maintain in bacteria (34). Because of the extra steps involved in polyadenylating in vitro transcripts it is still common to inject transcripts lacking a poly(A) tail into oocytes for translation, despite reduced protein yields from this substrate.

196

PFAFF, Day

1

Day

2

Day

3

56

34

12

TAMKUN,

.125

al S ::

1.6

0

1.2

; P

1.0

iit

0.8 0.6

E

0.4

nt

AND

TAYLOR

than those from pOEV DNA (Fig. 5). However, CAT protein accumulated over a 3-day period in oocytes injected with pOEV/CAT, while the level of CAT protein expressed from the RNA template remained constant, at the l-day level, over this period. The DNA template gave a six-fold greater yield of CAT protein than the RNA sample 4 days after injection (Fig. 5). In an attempt to maximize protein expression we synthesized RNA in vitro using the SP6 promoter of pOEV/ CAT and injected oocytes with both 80 ng pOEV/CAT RNA and 250 pg pOEV/CAT DNA in the cytoplasm and germinal vesicle, respectively. We found that these oocytes expressed higher levels of CAT at l-day postinjection than either DNA or RNA alone, and the CAT protein accumulated over a 4-day period (Fig. 5). By injecting RNA and DNA templates encoding CAT there was an additive affect on protein expression at 1 day, but

1.4

0.2 0.0 1

2

3

Days After Injection FIG. 2. mRNA analysis and quantification. (A) Primer extension analysis of mRNA transcribed from pOEV/CAT. Lanes 1, 3, and 5 had 50 pg DNA injected; and lanes 2,4, and 6 had 500 pg DNA injected. RNA was purified 1 day after injection, lanes 1 and 2; 2 days after injection, lanes 3 and 4, and 3 days after injection, lanes 5 and 6. Labeled DNA fragments were included as standards, and the 125-nucleotide position is marked. (B) The amount of CAT mRNA per oocyte was determined by scintillation counting the bands from the primer extensions shown in (A) and extrapolating from a standard curve. The standard curve was prepared using RNA transcribed by SP6 polymerase from pOEV/CAT, which was quantified spectrophotometrically, and primer extensions were then done in parallel with the samples in (A) but not shown. Samples injected with 50 pg of pOEV/CAT are denoted by I and samples injected with 500 pg of pOEV/CAT are denoted by W

B

5

25

5

25

50 250 pg DNA InjecIed

500 per Oocyte

25M)

4150

2500

4150

100000 I SOMO 4

0 3

70000

ZE

50000

E 0

4owo

it 30

60000

30000

Since it is difficult to determine the percentage of transcripts that are capped during in vitro transcription with the m7G(5’)ppp(B)G cap analog, and variations in capping could cause variation among experiments, we chose to compare protein expression from pOEV and RNA lacking 5’- and 3’-modifications. Although this RNA is not as stable or translated as efficiently as capped polyadenylated RNA in oocytes, it does reproducibly express moderate levels of protein and is an easy reference substrate to synthesize in large quantities. We compared the translational yield from 250 pg pOEV/ CAT DNA and from 80 ng CAT RNA prepared in vitro, using the SP6 promoter in pOEV/CAT. One day after injection the CAT protein yields from RNA were greater

s 8

20000 10w0 50

250

po DNA lnjecled

500 per Oocyte

FIG. 3. Effect of pOEV concentration on mRNA and protein expression. (A) Increasing amounts of pOEV/CAT were injected into oocytes, and RNA was purified at 1 day (I), 2 days ( (I ) after injection. 2.5 oocyte eq of RNA was used sion, and bands were cut from the gel and quantified by scintillation counting. (B) The same batch of oocytes as in (A) were used, except that CAT extracts were prepared at 1 day (I), 2 days ( ), 3 days ), and 4 days ( FZZ ) after injection. 0.25 oocyte equivalent of ex( tract was used for CAT assay, the acetylated forms of [i4C]chloramphenicol were resolved by TLC, and spots were cut out and quantified by scintillation counting.

CONSTRUCTION

OF

Xenopus

OOCYTE

PROTEIN

EXPRESSION

197

VECTOR

60000

/C

250wDNA

1

4

4

acetylated chl.

T ? 2 h ii 5 I ? P,

50000 40000 30000 -

non-acetylated .chl.

0

1

2

3

4

Days After Injection

FIG. 5. Comparison of translation yield from pOEV and in vitro prepared RNA. 250 pg pOEV/CAT DNA or 80 ng of in vitro RNA from the pOEV/CAT template or both were injected into oocytes as indicated, and CAT extracts were prepared at 1,2,3, and 4 days after injection. 0.25 oocyte equivalent was used in the CAT assay, acetylated [i4C]chloramphenicol was resolved by TLC, and bands were cut and quantified by scintillation counting.

J 40 m

i

1

2 Days

After

3

4

[35S]methionine, and immune precipitations were done with antibodies specific for either the CY- or the P-subunit. The o-subunit was immune precipitated by an (Yspecific monoclonal antibody (not shown). Both the (Yand the P-subunit copurified when a species specific monoclonal antibody specific for the P-subunit was used (lane 1, Fig. 6A). The P-specific antibody specifically precipitated the P-subunit and the a-subunit, indicating

Injection

FIG. 4. Quantification of CAT protein expression from pOEV. (A) 5 pg pOEV/CAT DNA was injected into oocytes, and CAT extracts were prepared 1,2,3, and 4 days after injection. 0.25 oocyte equivalent was used in the CAT assay, and analyzed by TLC. (B) The amount of CAT protein per oocyte was determined by scintillation counting the acetylated [i4C]chloramphenicol bands in (A), and extrapolating from a standard curve, made with increasing concentrations of purified CAT enzyme, done in parallel with the samples in (A) but not shown (Materials and Methods).

A

B 1

2

1

2

we did not observe an additive affect with longer incubations. Therefore this experimental protocol only served to alter the kinetics of protein accumulation. Additional Protein

Examples Expression

of POE V-Directed

In addition to expressing CAT protein using pOEV we have tested the universal application of the vector by expressing several different proteins. We inserted the CDNAs encoding the (Y- and B-subunit of the avian (Na+ and K+) ATPase into pOEV (25,26), resulting in pOEV/ LYandpOEV//? (Materials and Methods). These two constructs were coinjected into oocytes and labeled with

FIG. 6. Na/K ATPase cu/&subunit expression and myc expression from pOEV. (A) Lane 1, pOEV/ol and pOEV//3 DNA were coinjected into oocytes and the oocytes were labeled 3 days in [?‘S]methionine. Immune precipitation was done with a B-subunit specific antibody (Materials and Methods). Lane 2 is the uninjected control. (B) Lane 1, oocytes were injected with pOEV/myc and labeled 3 days in [%]methionine before immune precipitation with a myc antibody (Materials and Methods). Lane 2 is the uninjected control.

198

PFAFF,

TAMKUN,

that the two proteins had assembled with each other in the oocyte (this result is identical to that observed in Ref. (25)). Oocytes injected with only [35S]methionine generated no CX-or P-subunit bands (lane 2, Fig. 6A). The P-subunit appeared as two bands in the gel; the upper diffuse band represented the fully glycosylated form. The lower band migrated where a partially glycosylated P-subunit would be expected (25). We conclude that pOEV is suitable for the expression of multisubunit membrane proteins. We also expressed the murine c-myc nuclear protein in oocytes by cloning the cDNA into pOEV (Materials and Methods). Although we did not test the localization of the protein, it was expressed at levels easily detected by immune precipitation (Fig. 6B). DISCUSSION

We have constructed an oocyte expression vector, termed pOEV, which has several convenient features. The plasmid is derived from pGEM-1 (Promega), and contains the TFIIIA promoter and SV40 signals for splicing and polyadenylation. The TFIIIA promoter is extremely active in oocytes, demonstrated by the 106lo7 TFIIIA transcripts per oocyte. This amount is equivalent to approximately l-10 pg mRNA per cell encoded by this single copy gene (19). pOEV is a relatively small plasmid at 3900 bp and contains a polylinker with many unique restriction sites to facilitate cloning. There are no ATG codons in any reading frame between the TFIIIA transcription start site and the polylinker; therefore, translation of genes cloned into the polylinker can only be initiated within cloned sequences. The TFIIIA promoter does not interrupt the T7 or SP6 RNA polymerase promoters in the plasmid, allowing the vector to be used to express RNA in vitro for translation or for hybridization probes. Analysis of mRNA encoded by pOEV shows that transcription initiation is very precise and conserves the same start site as the endogenous TFIIIA gene. Under optimal conditions pOEV can express 0.8 ng mRNA per oocyte in 1 day. This rate of transcription is nearly 50% of an uninjected oocyte’s normal rate of mRNA synthesis. The high transcription rate of pOEV is probably due to the efficient promoter and high copy number of template which can be injected. In order to optimize protein expression from pOEV, we tested a wide variety of template concentrations in oocytes. We found that the concentration of DNA injected did affect protein yields, and we recommend trying several DNA concentrations when optimizing expression from this vector. We found that 25-50 pg DNA injected in a 5-nl volume per oocyte gave a good translational yield of CAT, and that this DNA concentration can vary by five-fold with very little effect on the level of protein expressed after 3 days. Yields of 40 fmol CAT protein were typical after 2- to 4-day incubations.

AND

TAYLOR

When we tested the application of pOEV for the expression of membrane proteins we were able to detect the expression of the (Y- and P-subunit of the avian Na+ and K+ ATPase by immune precipitation. The coprecipitation of both proteins with a P-specific monoclonal antibody indicated that these subunits are assembling into a higher order structure, which is an example of the complex biochemical interactions afforded by oocyte protein expression. Recently we have detected functional voltage-gated K+ channels expressed from pOEV using voltage clamp analysis of oocytes (manuscript in preparation). pOEV expression of the myc nuclear protein was also tested in oocytes, and the vector was quite suitable for this use. Protein expression from pOEV does not appear to be limited by the subcellular localization of proteins. A caveat to using pOEV for oocyte expression was the saturation of protein translation at lower template concentrations than required for the saturation of transcription. We do not presently understand at what step saturation occurs in the oocyte, since 250 and 2500 pg of DNA per oocyte give similar mRNA levels in the oocyte, but 250 pg DNA gave five times more CAT protein than 2500 pg (Fig. 3). Primer extension analysis of mRNA used here did not determine whether the transcript was spliced, polyadenylated, or otherwise processed, therefore, the structure or subcellular localization of the RNA produced from 250 pg DNA may be different than that produced from 2500 pg DNA. This phenomenon does not appear to be unique to the TFIIIA promoter or pOEV plasmid, since we found that the pSV2-cat plasmid (23) also exhibited inefficient CAT mRNA translation from high copy numbers of DNA template (data not shown). Others have also noted this discrepancy with different constructs (30). Since only DNA samples are necessary for pOEV expression, the vector simplifies the handling and storage of samples, compared to the extra precautions that are necessary when working with RNA. By transcribing mRNA for translation in duo, pOEV simplifies the expression of cloned cDNA in oocytes because it does not require the intermediate step of in vitro transcription before injection. This system will help simplify sample preparation for structural and functional analyses of proteins expressed in oocytes and will provide a faster means of cDNA “expression cloning” using oocytes if pOEV is used as a vector to construct cDNA libraries. The pOEV vector and other test constructs are available upon request. ACKNOWLEDGMENTS Steve Hann (Vanderbilt University) greatly helped with the myc protein analysis, Richard Harland (U.C. Berkeley) kindly provided several CAT clones, and Walter Smithwick provided technical assistance. We appreciate the help and critical comments of Robert Hall, Tony Weil, and Roland Stein. This work was supported by NIH

CONSTRUCTION Grants GM39234 spectively. S.L.P. DK07563.

and GM4132, was supported

awarded by a NIH

OF

Xenopus

to W.L.T. postdoctoral

OOCYTE

and M.M.T. retraining grant,

PROTEIN

EXPRESSION

16. Dolecki,

G. J., and Smith,

17. Gurdon,

J. B., and

18. De Robertis,

19. Ginsberg,

1. Gurdon, J. B., Lane, C. D., Woodland, (1971) Nature (London) 233,177-182.

2. Colman,

A. (1984) Approach (Hames, IRL, Oxford.

J. D., Wasserman, 89,159-167.

W. J., and

4. Methfessel,

C., Witzemann, V., S., and Sakmann, B. (1986)

5. Houamed, D.A.,

K. M., Bilbe, G., Smart, Barnard, E. A., and Richards,

310,318-321. 6. Hediger, M. A., Ikeda, Wright,

E. M. (1987)

Eur.

Smith,

Takahashi, Pfluegers

A Practical pp. 271-302,

L. D. (1982)

Dew.

T., Mishina, Arch. 407,577-588.

M.,

T. G., Constanti, A., Brown, B. M. (1984) Nature (LondonJ

Coady, M., Gundersen, C. B., and Proc. Natl. Acad. Sci. USA 84,2634-2637.

Biophys.

D. A. (1984)

10. Noguchi, S., Mishina, FEBS Lett. 225,27-32.

Nucleic

Acids

Res.

12,

J.

B., Noda,

M., and Numa,

14,131-138. M. M. (1988)

15,

Reu. Genet.

M. A., Coady, Nature (London)

D. M. (1988)

Kawamura,

M. J., Ikeda, 330,379-381.

King,

J. E. (1977)

B. O., and Roeder,

26.

J. Biol.

Chem.

Cell 12,175182. R. G. (1984)

Cell 39,

263,4347-4354.

Takeyasu, K., Tamkun, M. M., D. M. (1987) J. Biol. Chem. 262, (1983)

Siegel, N. R., and 10,733-10,740.

O., Cory, S., Gerondakis, EMBO J. 2,2375-2383.

Acud.

Sci. USA

S., Webb,

Fambrough,

E., and Adams,

J. M.

D. A., Krieg, P. A., Rebagliati, M. R., Maniatis, T., Zinn, K., and Green, M. R. (1984) Nucleic Acids Res. 12,7035-7056.

29. Laemmli, M.,

E. M., and Mertz, A. M.,

28. Melton, Proc. N&Z. M.,

and Numa,

S. (1987)

11. Julius, D., MacDermott, A. B., Axel, R., and Jessell, T. M. (1988) Science 24 1,558-564. 12. Masu, Y., Nakayama, K., Tamaki, H., Harada, Y., Kuno, M., and Nakanishi (1987) Nature (London) 329,836-838. P., Naito, T., Bergstedt-Lindqist, S., Azuma, 13. Noma, Y., Sideras, C., Severinson, E., Tanabe, T., Kinashi, T., Matsuda, F., Yaoita, Y., and Honjo, T. (1986) Nature (London) 319,640-646. (1987)

69,217-236.

Annu.

20. Hall, R. K., and Taylor, W. L. (1989) Mol. Cell. Biol. 9,5003-5011. 21. Matsumoto, Y., and Korn, L. J. (1988) Nucleic Acids Res. 16, 3801-3814. 22. Scotto, K. W., Kaulen, H., and Roeder, R. G. (1989) Genes Deu. 3,651-662. 23. Gorman, C. M., Moffat, L. F., and Howard, B. H. (1982) Mol. Cell. Biol. 2,1044-1051. 24. Minth, C. D., Andrews, P. C., and Dixon, J. E. (1986) J. Biol. Chem. 261,11,974-11,979. 25. Takeyasu, K., Tamkun, M. M., Renaud, K. J., and Fambrough,

27. Bernard, C., Sakmann,

9. Buller, A. L., and White, 85,8717-8721.

14. Hediger,

Deu. Biol.

D. A. (1981)

479-489.

G.

T.,

7. Krieg, P. A., and Melton, 7057-7070. 8. Stuhmer, W., Methfessel, S. (1987)

H. R., and Marbaix,

in Transcription and Translation: B. D., and Higgins, S. J., Eds.),

3. Richter,

Numa,

L. D. (1979)

Melton,

189-218.

REFERENCES

Biol.

199

VECTOR

T. S., and

Wright,

E. M.

15. Lubbert, H., Hoffman, B. J., Snutch, T. P., van Dyke, T., Levine, A. J., Hartig, P. R., Lester, H. A., and Davidson, N. (1987) Proc. Natl. Acad. Sci. USA 84,4332-4336.

30. Colman, Approach Oxford. 31. Richter,

32. Drummond, AcidsRes.

33. Konarska,

U. K. (1970) A. (1984) (Hames,

Nature

(London)

227,680-685.

in Transcription and Translation: A Practical B. D., and Higgins, S. J., Eds.), pp. 49-69, IRL,

J. D., and Smith,

L. D. (1981)

D. R., Armstrong,

Cell 27,183-191.

J., and Colman,

A. (1985)

Nucleic

13,7375-7394. M. M.,

Padgett,

R. A., and Sharp,

P. A. (1984)

Cell 38,

731-736.

34. Harland, 35. Furuichi, (London)

R., and Misher,

L. (1988)

Y., LaFiandra, 266,235-239.

A., and

36. Soreq, H., Sagar, A. D., and Sehgal, Sci. USA

78,1741-1745.

102,837-852.

Deuelopment Shatkin,

A. J. (1977)

P. B. (1981)

Proc.

Natl.

Nature Acad.